Current Topics in Microbiology and Immunology

Jeffrey V. Ravetch
Falk Nimmerjahn Editors

Fc Mediated
Activity of
Antibodies
Structural and Functional Diversity
Current Topics in Microbiology
and Immunology

Volume 423

Series Editors
Rafi Ahmed
School of Medicine, Rollins Research Center, Emory University, Atlanta, GA,
USA
Shizuo Akira
Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan
Klaus Aktories
Faculty of Medicine, Institute of Experimental and Clinical Pharmacology and
Toxicology, University of Freiburg, Freiburg, Baden-Württemberg, Germany
Arturo Casadevall
W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns
Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
Richard W Compans
Department of Microbiology and Immunology, Emory University, Atlanta, GA,
USA
Jorge E Galan
Boyer Ctr. for Molecular Medicine, School of Medicine, Yale University, New
Haven, CT, USA
Adolfo Garcia-Sastre
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York,
NY, USA
Bernard Malissen
Parc Scientifique de Luminy, Centre d’Immunologie de Marseille-Luminy,
Marseille, France
Rino Rappuoli
GSK Vaccines, Siena, Italy
The review series Current Topics in Microbiology and Immunology provides a
synthesis of the latest research findings in the areas of molecular immunology,
bacteriology and virology. Each timely volume contains a wealth of information on
the featured subject. This review series is designed to provide access to up-to-date,
often previously unpublished information.
2018 Impact Factor: 3.153., 5-Year Impact Factor: 4.143

More information about this series at http://www.springer.com/series/82

Jeffrey V. Ravetch Falk Nimmerjahn
•

Editors

Fc Mediated Activity
of Antibodies
Structural and Functional Diversity
Responsible Series Editor: Rino Rappuoli

123
Editors
Jeffrey V. Ravetch Falk Nimmerjahn
Department of Leonard Wagner Laboratory Department of Biology
of Molecular Genetics and Immunology University of Erlangen-Nürnberg
Rockefeller University Erlangen, Bayern, Germany
New York, NY, USA

ISSN 0070-217X ISSN 2196-9965 (electronic)

Current Topics in Microbiology and Immunology
ISBN 978-3-030-31052-3 ISBN 978-3-030-31053-0 (eBook)
https://doi.org/10.1007/978-3-030-31053-0
© Springer Nature Switzerland AG 2019
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, expressed or implied, with respect to the material contained
herein or for any errors or omissions that may have been made. The publisher remains neutral with regard
to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

If one looks through the lists of novel drugs targeting cancers, autoimmune/chronic
inflammatory diseases, and infection, a molecule appearing very (if not most)
frequently on that list will be antibodies of the Immunoglobulin G (IgG) isotype.
Indeed, the much celebrated breakthrough in cancer immunotherapy is based on
intact antibodies or antibody fragments, such as bispecific T cell engaging anti-
bodies (BiTEs); and chimeric antigen receptors (CAR), which are antibody frag-
ments expressed on T cells. Thus, antibodies are the new and old kid on the block
for immunotherapy and surely will be in the focus of novel therapeutic approaches
for many years to come. An in depth understanding of how antibodies mediate their
activity is therefore of critical importance. Research of many groups over the last
three decades has provided clear evidence that IgG binding to cellular Fc-receptors
is essential for the activity of antibodies in vivo, emphasizing that the antibody
constant region is much more than a framework carrying the variable domain, but
rather is the essential—and actually not so constant—linker responsible for trig-
gering IgG effector functions.
In autoimmunity, IgG antibodies play several important roles. Firstly, as
autoantibodies they are drivers of autoimmune inflammation. Again, Fc-receptors
have been identified as central players responsible for triggering the
pro-inflammatory effects of autoantibodies. Second, IgG antibodies are used in the
form of monoclonal antibodies targeting key pro-inflammatory cytokines or
self-reactive immune cells. Finally, the infusion of polyclonal serum IgG prepa-
rations pooled from thousands of donors, also called intravenous IgG therapy
(IVIg), is efficiently able to suppress a wide variety of chronic and acute autoim-
mune diseases, prompting many groups to investigate its mechanism of action and
solve the mystery of IVIg activity. While many potential mechanisms of action of
IVIg have been proposed over the years, one completely unexpected finding was
that the sugar moiety attached to the IgG Fc-domain seemed to play a crucial role
for the anti-inflammatory activity of these polyclonal IgG preparations. More in
depth studies provided convincing evidence that especially IgG glycoforms car-
rying terminal sialic acid residues were responsible for the anti-inflammatory
activity and that a family of type II Fc-receptors was involved in this

v
vi Preface

anti-inflammatory pathway. A side effect of these observations was that it prompted

many groups to re-visit the impact of glycosylation on immunoglobulin activity
more broadly. This has led to the unexpected finding that not only IgG but also IgE
antibody activity is regulated through glycosylation. Of note, especially one of the
many sugar moieties attached to IgE was shown to be of critical importance for
productive binding of IgE to its Fc-receptor. Coming to IgG and infection, IgG
antibodies are essential to protect the host from infection or from the detrimental
activity of bacterial toxins. Again, Fc-receptors were shown to be of major
importance also for the activity of neutralizing antibodies, which were previously
thought to mediate their activity independently of Fc-receptors. But antibodies are
clearly not only beneficial in infection. Very recent studies have emphasized that
glycovariants of virus specific IgG antibodies lacking fucose residues may be
responsible for enhancing Dengue virus infection and pathology in hosts, also
known as antibody dependent enhancement of infection. As IgG glycovariants
lacking fucose modulate IgG binding to human FcgRIIIa (in humans) and FcgRIV
(in mice), this again pinpoints towards an important role of Fc-receptors in this
process. Finally, in a world of fading activity of antibiotics against bacteria more
successful vaccination strategies will be of major importance. Harnessing the host
through generating a most productive antibody response against microorganisms
will be key to cope with this global threat. Using antibodies in combination with the
antigens (that is: immune complexes) to boost immune responses is a very efficient
and in fact the most natural way of enhancing immunity. Again Fc-receptors present
on dendritic cells play a key role for such novel vaccination strategies.
In this book, we were able to bring together an exciting list of experts in the field
covering all of these hot topics of antibody and Fc-receptor research. Stylianos
Bournazos will look at Fc-receptors from an evolutionary point of view, while
Graziano and Engelhardt will discuss the role of Fc-receptors for immunotherapy of
cancer. The chapters of Beneduce and colleagues, Wang, and Shade and colleagues
will discuss the importance of IgG glycosylation for the pro- and anti-inflammatory
activities of IgG and IgE. Finally, the two chapters by Wen and Shi and Wieland
and Ahmed will focus on how Fc-receptors impact vaccination and infection.
Naturally the examples mentioned above are only a fraction of the research,
which is currently ongoing in the field of antibodies and Fc-receptors, but they
clearly highlight how broad the impact of research on Fc-mediated activities of
antibodies can be. We would like to thank all authors contributing to this volume
again for their excellent contributions and we hope that it will stimulate new
research ideas in this exciting area of immunology.

New York, USA Jeffrey V. Ravetch

Erlangen, Germany Falk Nimmerjahn
Contents

IgG Fc Receptors: Evolutionary Considerations . . . . . . . . . . . . . . . . . . . 1

Stylianos Bournazos
Role of FccRs in Antibody-Based Cancer Therapy . . . . . . . . . . . . . . . . 13
Robert F. Graziano and John J. Engelhardt
Anti-inflammatory Activity of IgG-Fc . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Christopher Beneduce, Elma Kurtagic and Carlos J. Bosques
IgG Fc Glycosylation in Human Immunity . . . . . . . . . . . . . . . . . . . . . . 63
Taia T. Wang
IgE Glycosylation in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . 77
Kai-Ting Shade, Michelle E. Conroy and Robert M. Anthony
Immune Complex Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Yu-mei Wen and Yan Shi
Fc Receptors in Antimicrobial Protection . . . . . . . . . . . . . . . . . . . . . . . . 119
Andreas Wieland and Rafi Ahmed

vii
IgG Fc Receptors: Evolutionary
Considerations

Stylianos Bournazos

Contents

1 The Emergence of IgG Antibodies ..................................................................................... 2

2 Evolution of FccR Genes in Mammalian Species ............................................................. 3
3 Inter-species Differences and Limitations for the Study of Human FccR
Function ............................................................................................................................... 7
4 Concluding Remarks ........................................................................................................... 10
References .................................................................................................................................. 10

Abstract Immunoglobulins (Ig), a critical component of the adaptive immune

system, are present in all jawed vertebrates and through sophisticated diversification
mechanisms are able to recognize antigens of almost infinite diversity. During
mammalian evolution, IgG has emerged as the predominant Ig isotype that is
elicited upon antigenic challenge, representing the most abundant isotype present in
circulation. Along with the IgG molecule, a family of specialized receptors has
evolved in mammalian species that specifically recognize the Fc domain of IgG.
These receptors, termed Fcc receptors (FccRs), are expressed on the surface of
effector leukocytes and upon crosslinking by the IgG Fc domain mediate diverse
immunomodulatory processes with profound impact on several aspects of innate
and adaptive immunity. FccRs share a high degree of sequence homology among
mammalian species and the ancestral locus, where the genes that encode for FccRs
are mapped, can be traced back early in mammalian evolution. FccRs also share a
number of common structural and functional properties among mammalian species
and utilize highly conserved motifs for transducing signals upon engagement.
Despite the high homology of FccRs in diverse mammalian species, human FccRs
exhibit unique features relating to the gene organization, expression pattern in the

S. Bournazos (&)
The Laboratory of Molecular Genetics and Immunology, The Rockefeller University,
1230 York Ave, New York, NY 10065, USA
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2019) 423: 1–11

https://doi.org/10.1007/82_2019_149
© Springer Nature Switzerland AG 2019
Published online: 10 February 2019
2 S. Bournazos

various leukocyte populations, as well as affinity for human IgGs. Such

inter-species differences in FccRs biology between humans and other mammalian
species represents a major limitation for the interpretation of in vivo studies on
human IgG function using conventional animal models.

1 The Emergence of IgG Antibodies

Complex defense mechanisms are ubiquitously found in all living organisms and
are critical for the survival of these organisms in an antigenic world. Immunity is
generally mediated by the early reactions of the innate immune system, followed by
the later responses of adaptive immunity, characterized by almost infinite diversity
and high specificity. Although immune mechanisms featuring the cardinal signs of
adaptive immunity, i.e. diversity and specificity, have been discovered in inverte-
brates and jawless vertebrates, adaptive immunity based on highly diversified
antigen-specific receptors is present only in jawed vertebrates. Indeed, key immune
components of adaptive immunity, including antigen receptors, such as
immunoglobulins (Ig) and T-cell receptors (TCR), as well as antigen-presenting
MHC molecules are locked in a coevolving unit that orchestrates clonal selection
and MHC-regulated processes during an adaptive immune response.
In addition to their common function, adaptive immune antigen receptors, such
as Ig and TCR, share structural similarities, as they both comprise domains of the
immunoglobulin superfamily (IgSF). IgSF domains are the building blocks for a
large number of molecules across several species of the animal kingdom and rep-
resent the most prevalent domain in immune defense molecules. Apart from antigen
receptors, IgSF domains are found in numerous receptors and molecules with a key
role in cell-cell interactions, cell adhesion, as well as immune cell signaling. Despite
the diverse functions of IgSF-containing molecules, these functions are somehow
related, as IgSF domains are almost exclusively involved in protein-ligand inter-
actions. The main structural feature of the IgSF domain is the stable shape of a b
barrel that comprises two interfacing b sheets that are typically linked by a disulfide
bond. Based on the domain constitution of their b strands and loops, IgSF domains
are classified into four major types: the variable (V), the two constant domains (C1
and C2), as well as an intermediate type, which shares common features with both
the C1 and C2 domains. Among the IgSF domains, the V domain type is the most
structurally flexible and complex, featuring more strands, which correspond to the
CDR2 region in Ig and TCRs. These unique structural features are critical for
mediating highly specific antigenic recognition, accommodating the high degree of
sequence diversity that characterizes antigen receptors, such as Ig and TCRs. The
final structure of these antigen receptors is achieved through a linear combination of
V and C-type IgSF domains. For example, a typical Ig molecule is a heterodimeric
structure that comprises two polypeptides of the heavy chain and two of the light
chain that are linked via several disulfide bonds forming a macromolecular
IgG Fc Receptors: Evolutionary Considerations 3

complex. Each light chain comprises a single V and a C1 IgSF domain, whereas the
Ig heavy chain is formed through the joining of the V-type domain with three-four
C1 IgSF domains; the exact number of C domains is variable among the different Ig
isotypes.
These unique structural features are highly conserved among all the different Ig
isotypes found in diverse species of jawed vertebrates. Indeed, IgM, which is
present in all jawed vertebrates, and is thought to represent the primordial Ig
isotype, consists of a heavy chain peptide with one V and four C1 domains, which
pairs with the light chain (one V and one C1 IgSF domain) through multiple
inter-chain disulfide bridges. Monomeric IgM subunits form pentamers or hexamers
in the majority of vertebrate species, apart from teleost fish, which form tetramers.
Comparative analysis of the Ig isotypes in the jawed vertebrates suggests that a
common precursor isotype that is related to IgM and can be traced at the inception
of adaptive immunity, gave rise to the various Ig isotypes in jawed vertebrates at
specific divergence points during evolution. These Ig isotypes have evolved under
constant selection pressure from the challenging antigenic conditions encountered
by the various vertebrate species. Indeed, the different Ig isotypes exhibit unique
structural and functional characteristics, as well as distinct expression patterns
among the various tissue compartments (intestinal, mucosal, serum etc.). For
mammalian species, IgG and IgE represent two Ig isotypes that are exclusively
found in mammals and mediate effector functions during an adaptive immune
response. These isotypes are thought to have emerged from a common,
structurally-related ancestor molecule, IgY, which is present in other vertebrate
classes, including birds, reptiles, and amphibians, and represents the most abundant,
secreted Ig isotype in these species.

2 Evolution of FccR Genes in Mammalian Species

Since the IgG isotype is exclusively found in mammals, receptors for IgG anti-
bodies only exist in mammalian species. These receptors, termed Fcc receptors
(FccRs), interact with the constant region of the IgG heavy chain heterodimer and
have co-evolved in mammalian species along with the emergence of the IgG iso-
type. For mammals, IgG represents the major Ig isotype in circulation and is
abundantly expressed during an adaptive immune response. By directly interacting
with IgG antibodies, FccRs are critical for mediating downstream effector activities
with tremendous impact on diverse innate and adaptive pathways. Indeed, despite
being a major component of adaptive immunity, IgG antibodies link the innate and
adaptive branches of immunity through specific interactions with the FccRs
expressed on the surface of effector leukocytes. Such interactions initiate diverse
signaling processes with pleiotropic immunomodulatory functions, which have
significant consequences for several aspects of innate and adaptive immunity. Such
functions include the regulation of innate leukocyte activation, the uptake of foreign
4 S. Bournazos

antigens via phagocytic mechanisms and their processing and presentation to MHC
molecules, the regulation of antigen-presenting cell function, the stimulation of
cytokine and chemokine synthesis, as well as the selection of high-affinity B-cells,
and the regulation of plasma cell survival and consequently IgG production
(Bournazos et al. 2017).
Given the diverse immunomodulatory consequences of FccR engagement by the
Fc domain of IgG antibodies, a number of FccRs have emerged during mammalian
evolution, each with distinct signaling activity and function, as well as unique
expression profile among the various leukocyte cell types. On the basis of their
intracellular signaling motifs and their capacity to induce stimulatory or inhibitory
signaling cascades, FccRs are broadly classified into activating or inhibitory
(Nimmerjahn and Ravetch 2005). The major determinant that controls the affinity of
an IgG molecule for the various FccR types is the intrinsic flexibility of the Fc
domain of IgG (Pincetic et al. 2014). Similar to the structural characteristics of all Ig
isotypes, IgG molecules comprise two heavy and two light chains that are linked
into a heterodimeric macromolecular complex through inter-chain disulfide bonds.
Each light chain comprises one V-type IgSF domain and a C1-type domain,
whereas each heavy chain consists of one V-type domain followed by three C1-type
domains, corresponding to the CH1–CH3 domains of the IgG molecule. The
characteristic Y structure of the IgG molecule arises from the presence of a hinge
domain that separates the Fab domain (CH1 and light chains) from the Fc domain,
which comprises the CH2 and CH3 domains. The FccR binding sites are mapped at
the Fc domain at two distinct positions; Type I FccRs engage the Fc domain at the
hinge-proximal region of the CH2 domain, whereas type II FccRs at the CH2–3
interface (Pincetic et al. 2014). FccR binding is accomplished through the char-
acteristic horseshoe-like conformation of the Fc domain, which is formed by the
tight association of the two CH3 domains at the C-terminal proximal region of the
IgG molecule, while the two CH2 domains remain spatially separated. This char-
acteristic tertiary structure of the Fc domain is achieved through the presence of an
N-linked glycan conjugated at the amino acid backbone of the CH2 domain of the
two heavy chains. The amino acid residue, where this glycan is conjugated is highly
conserved in all IgG subclasses from all mammalian species, highlighting the
importance of this glycan structure in the regulation of the Fc domain structure and
consequently in Fc-FccR interactions. This Fc-associated glycan resides within the
hydrophobic cleft formed by the two CH2 domains and its composition regulates
the conformational flexibility of the Fc domain and its capacity to interact with the
various type I and type II FccRs (Sondermann et al. 2013). Indeed, the presence of
this glycan structure is critical for maintaining the Fc domain structure permissive
for Fc-FccR interactions, as loss of this glycan either through enzymatic removal or
mutation of the amino acid residue where this structure is conjugated diminishes the
affinity of the IgG Fc domain for all FccR types (Albert et al. 2008; Lux et al.
2013).
Although the presence of the Fc-associated glycan structure is critical for
maintaining the conformational structure of the Fc domain, the precise composition
IgG Fc Receptors: Evolutionary Considerations 5

of the Fc glycan represents a key regulatory mechanism that controls the affinity of
the Fc domain for the various FccR classes. More specifically, the Fc-associated
glycan structure consists of a core biantennary heptasaccharide moiety, which can
be modified through the conjugation of additional saccharide units (fucose, galac-
tose, N-acetylglucosamine, sialic acid), thereby yielding numerous variants with
differential capacity to modulate Fc domain flexibility and consequently the affinity
for the various FccR types (Bournazos et al. 2017). Analysis of the Fc-associated
glycan structure in circulating IgG antibodies has previously revealed substantial
heterogeneity with specific Fc glycoforms becoming enriched upon infection and
vaccination, in metabolic disease, during pregnancy, as well as during the
remission/relapse phases of autoimmune disorders (Anthony et al. 2012; Nakagawa
et al. 2007; Scherer et al. 2010; Theodoratou et al. 2016; Wang et al. 2015). Apart
from the structure and composition of the Fc-associated glycan, differences in the
primary amino acid sequence between the various IgG subclasses represent an
additional level of regulation for Fc-FccR interactions. Although protein homology
between subclasses is typically over 95%, differences that exist at the hinge
proximal region of the CH2 domain, where the interface for Fc-FccR interactions is
mapped, account for the differential binding profile of the IgG subclasses for the
various FccR types. Such differences are evident in the IgG subclasses from all
mammalian species and along with the composition of the Fc-associated glycan,
represent the main determinants for regulating Fc-FccR interactions.
In all mammalian species, IgG molecules share highly conserved structural
characteristics that determine the capacity of the Fc domain to interact with the
various FccRs and regulate its affinity. As FccRs have co-evolved with IgG
molecules during the emergence of the mammalian class, they feature a number of
highly conserved properties that are fundamental for their ligand binding and sig-
naling activities. For example, type I FccRs can transduce either activating or
inhibitory signals upon engagement by the IgG Fc domain, which is accomplished
through specialized signaling motifs at their intracellular domains (Bournazos et al.
2017). Activating type I FccRs feature intracellular tyrosine activating motifs
(ITAMs) at their cytoplasmic domains, which transduce cell activating signals upon
receptor crosslinking by the Fc domains of IgG immune complexes. Such signaling
motifs are highly conserved among mammalian species and several innate and
adaptive immunoreceptors, including TCRs, depend on ITAM-mediated signaling
for their function. Activating type I FccR-mediated signaling is counterbalanced by
inhibitory FccRs, whose cytoplasmic domains comprise intracellular tyrosine
inhibition motifs (ITIMs) that associate with tyrosine phosphatases to inhibit kinase
activity. The balancing activity of ITAM- and ITIM-containing FccRs represents a
key homeostatic mechanism that controls IgG-mediated inflammation and limits
excessive or inappropriate cellular activation. Indeed, in nearly all effector leuko-
cytes, activating type I FccR expression is coupled with the expression of an
inhibitory FccR that regulates the signaling outcome of Fc-FccR interactions. In
addition to FccRs, paired co-expression of activating and inhibitory immunore-
ceptors is very commonly found in several immune pathways, representing a
6 S. Bournazos

fundamental mechanism for the control a number of processes of the innate and
adaptive immunity.
In addition to the intracellular motifs and the downstream components that are
required for the signal transduction of FccRs, the FccR ligand binding domains
share highly homologous features among mammalian species. All type I FccRs are
members of the IgSF and their extracellular, IgG-binding domain consists of two
(or three for the high-affinity FccRI) C2-type IgSF domains. On the contrary, type
II FccRs are not structurally related to type I FccRs, as their extracellular domain
belongs to the C-type lectin family of receptors. Consistent with the structural
differences in their extracellular, ligand-binding domains, type I and type II FccRs
engage the IgG Fc domain at distinct, non-overlapping regions; the hinge-proximal
region of CH2 serves as the binding site for type I FccRs, whereas type II FccRs
engage the Fc domain at the CH2–3 interface in a 2:1 (receptor:IgG) binding
stoichiometry (Sondermann et al. 2013). In humans, several genes, each with
multiple transcriptional isoforms, encode the different type I and type II FccRs
(Pincetic et al. 2014). Consistent with their common functional properties, members
of the type I and type II FccRs are mapped at specific loci on human chromosome 1
(1q23) and 19 (19p13), respectively. Clustering of the various human FccR genes at
specific genomic loci, along with the high degree of sequence homology between
members of the same FccR type, suggest that mammalian FccR genes have
emerged from a common ancestral precursor gene. Indeed, comparative analysis of
the type I FccR gene locus among different mammalian species shows a high
degree of sequence homology, indicative of the emergence of this locus very early
in mammalian evolutionary history (Fig. 1). This locus consists of three type I
FccR genes, corresponding to the human FCGR2A, FCGR3A, and FCGR2B genes.
The genomic organization of this locus is conserved among diverse mammalian
species, from very primitive mammals to primates, except for humans and chim-
panzees. Due to the high sequence similarity of the ancestral FCGR2A and
FCGR2B genes, sequential non-homologous recombination events that occurred
late in human and chimpanzee divergence from the common non-human primate
ancestor generated additional type I FccR genes (Qiu et al. 1990) (Fig. 2). These
genes are uniquely found in humans and chimpanzees and include FCGR2C and
FCGR3B, which encode for FccRIIc and FccRIIIb, respectively. FCGR3B origi-
nates from a gene duplication event of FCGR3A and both genes share a very high
degree of sequence homology. However, contrary to FccRIIIa, FccRIIIb is pro-
cessed as a GPI-anchored molecule and lacks an intracellular domain; an effect
attributed to the presence of a single nucleotide substitution (F203S) within the
FCGR3B coding sequence. This point mutation is mapped at the membrane
proximal region of the extracellular domain of FccRIIIb and creates a
post-translational modification signal sequence that processes FccRIIIb as a
GPI-anchored protein. Another type I FccR gene that is uniquely found in humans
and chimpanzees is FCGR2C, which is the result of the non-homologous recom-
bination between the FCGR2A and FCGR2B genes. FCGR2C is essentially a
IgG Fc Receptors: Evolutionary Considerations 7

Monotremes
Marsupials

Afrotheria Ancestral Locus

FCGR2A FCGR3 FCGR2B

Laurasiatheria

Mouse/Rat

Fcgr2b Fcgr4 Fcgr3

Rodents
Guinea Pig

Fcgr2a Fcgr3 Fcgr2b

Lagomorphs
Macaque
Non-human
primates FCGR2A FCGR3 FCGR2B
Humans Human

FCGR2A FCGR3A FCGR2C FCGR3B FCGR2B

Fig. 1 Evolutionary conservation of the FccR locus among mammalian species. The ancestral
FccR locus comprises three low-affinity FccR genes that encode two activating (FCGR2A and
FCGR3) and one inhibitory FccRs (FCGR2B). These FccRs feature two extracellular C2-type
IgSF domains that mediate the recognition of the IgG Fc domain. This ancestral locus shares a
common genomic structure and exhibits high sequence homology in diverse mammalian species,
including non-human primates, like rhesus macaques. However, in humans and chimpanzees, the
FccR locus features unique characteristics and comprises additional FccR genes (FCGR2C and
FCGR3B)

chimeric gene, which comprises the exons that encode the extracellular domain of
FccRIIb, whereas the transmembrane and intracellular domains originate from
FccRIIa.

3 Inter-species Differences and Limitations for the Study

of Human FccR Function

Differences in the FccR biology between humans and other mammalian species are
not limited to the unique organization of the human FccR locus and the presence of
additional FccR genes, like FCGR3B and FCGR2C, but extend to the FccR
structural characteristics and expression pattern among the various leukocyte cell
types. For example, in murine species, all activating FccRs require the FcR c-chain
for expression, assembly to the cell membrane, and signaling, whereas, in humans,
FccRIIa and FccRIIc expression and signaling is not dependent on FcR c-chain
expression, as the cytoplasmic domains of these FccRs contain ITAMs that can
sufficiently transduce signals upon receptor engagement. FccRIIIb, which is
exclusively expressed in humans, has a unique structure that is not found in any
8 S. Bournazos

Ancestral Fcγγ R Locus

FCGR2A FCGR3 FCGR2B

X Ancestral Fcγγ R Locus

FCGR2A FCGR3 FCGR2B

FCGR2A FCGR3A FCGR2C FCGR3B FCGR2B

Human Fc γ R Locus

Fig. 2 Emergence of the unique structure of the human FccR locus. The unique genomic
organization of the human FccR locus is attributed to sequential non-homologous recombination
events that occurred late in the divergence of humans and chimpanzees from their common
non-human primate ancestor. This process resulted in the emergence of two unique FccR genes:
FCGR2C and FCGR3B, which encode for FccRIIc and FccRIIIb, respectively. FccRIIc is a
chimeric receptor that comprises the extracellular domain of FccRIIb, whereas the transmembrane
and intracellular domains originate from FccRIIa. FccRIIIb shares high sequence homology with
FccRIIIa; however, FccRIIIb is processed as a GPI-anchored molecule, lacking an intracellular
domain

other FccR, as it is processed as a GPI-anchored protein, lacking a transmembrane

domain. As a consequence, FccRIIIb cannot transduce any signals following
crosslinking by IgG immune complexes; however, it is preferentially localized in
lipid rafts microdomains that are enriched in membrane-associated kinases, and
might utilize such signal transduction machinery to initiate intrinsic signals upon
receptor engagement (Zhou and Brown 1994; Unkeless et al. 1995). Additionally,
the FccR expression pattern differs dramatically between humans and other
mammalian species, including murine and non-human primates (Table 1). For
example, human neutrophils express FccRIIa, FccRIIb, and FccRIIIb, whereas
neutrophils from non-human primates express FccRI, FccRIIa, and FccRIIb.
Likewise, at steady state conditions, monocyte-derived dendritic cells from humans
express only FccRIIa and FccRIIb, whereas their murine counterparts express the
full array of murine FccRs, including FccRI, FccRIIb, FccRIII, and FccRIV.
These substantial inter-species differences in the FccR genomic organization,
structure, and function, as well as expression pattern, represent a major obstacle for
the study of human IgG antibody function in vivo using conventional experimental
animal model systems. Indeed, none of the commonly used experimental species,
including mice, guinea pigs, rats, and non-human primates, can mirror precisely the
structural diversity, IgG binding affinity, and the unique expression pattern for
human FccRs on human cells (Bournazos et al. 2015). Therefore, the use of such
strains provides limited information on the role of human IgG Fc-FccR interactions
in the in vivo function of antibodies and cannot guide the development and
pre-clinical evaluation of human IgG antibodies intended for clinical use in humans.
IgG Fc Receptors: Evolutionary Considerations 9

Table 1 FccR expression pattern in effector leukocytes among humans, murine and non-human
primate species

Such limitation also applies to studies aiming to evaluate the in vivo activity of
human-derived polyclonal IgG antibodies. More specifically, it is estimated that in
human circulation, over 103 different Fc domain variants exist, stemming from
differences in the amino acid sequence of the four IgG subclass and in the structure
and composition of the Fc-associated glycan. These Fc variants display differential
affinity for the various FccR types, thereby exhibiting variable immunomodulatory
activity (Bournazos et al. 2017). Such differences in the FccR affinity among the
human Fc domain variants present in circulation are specific for human FccRs and
do not extend to FccRs from other species. Therefore, any studies aiming either to
dissect the role of human-derived IgGs or to pre-clinically evaluate human IgG
antibody therapeutics, necessitate the use of species-matched IgG-FccR conditions.
To overcome this problem, during the past few years there have been efforts to
generate mouse strains that express all human FccRs in lieu of their mouse
counterparts. Such FccR humanized mouse model has been generated through the
specific deletion of the genes that encode the mouse FccRs and the introduction of
all human FccR genes as BAC transgenes under the control of their endogenous
promoters and regulatory elements (Smith et al. 2012). Characterization of the FccR
expression levels and distribution among the various leukocyte populations
revealed that FccR humanized mice exhibited an FccR expression pattern identical
to that of humans (Smith et al. 2012). More importantly, these mice faithfully
recapitulate the functional activity of human FccRs in vivo and have been used in
numerous studies to evaluate the in vivo activity of human therapeutic antibodies
against infectious and neoplastic disorders (Bournazos et al. 2017; Li et al. 2017;
Dahan et al. 2016; Georgoudaki et al. 2016; Lu et al. 2016; Dahan et al. 2015;
DiLillo and Ravetch 2015; Bournazos et al. 2014a, b, DiLillo et al. 2014; Li et al.
2014; Li and Ravetch 2013).
10 S. Bournazos

4 Concluding Remarks

Along with the emergence of the IgG molecule, as the major Ig isotype elicited
upon antigenic challenge, several FccRs have evolved in mammalian species to
interact with the Fc domain of IgG and mediate effector activity by directly linking
IgGs to effector leukocytes. Even among diverse mammalian species, FccRs are
highly conserved and share a number of structural and functional features necessary
for their ligand binding and signaling activities. Despite the high degree of
sequence homology between FccRs from different mammalian species, human
FccRs exhibit unique features related to the expression pattern among the various
leukocyte cell types, as well as to the affinity for the different IgG Fc domain
variants. These substantial inter-species differences represent major limitations for
the accurate evaluation of human IgG function in vivo, as none of the conventional
animal models can replicate the unique structural and functional diversity of human
FccRs. In-depth understanding of the evolutionary history of the FccR-encoding
genes, along with the thorough characterization of the unique features of FccR of
different mammalian species is essential for the design and interpretation of studies
on IgG function in mammalian strains that are commonly used as disease models.

References

Albert H, Collin M, Dudziak D, Ravetch JV, Nimmerjahn F (2008) In vivo enzymatic modulation
of IgG glycosylation inhibits autoimmune disease in an IgG subclass-dependent manner. Proc
Natl Acad Sci U S A 105:15005–15009
Anthony RM, Wermeling F, Ravetch JV (2012) Novel roles for the IgG Fc glycan. Ann N Y Acad
Sci 1253:170–180
Bournazos S, Chow SK, Abboud N, Casadevall A, Ravetch JV (2014a) Human IgG Fc domain
engineering enhances antitoxin neutralizing antibody activity. J Clin Invest 124:725–729
Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV (2014b) Broadly
neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell
158:1243–1253
Bournazos S, DiLillo DJ, Ravetch JV (2015) The role of Fc-FccR interactions in IgG-mediated
microbial neutralization. J Exp Med 212:1361–1369
Bournazos S, Wang TT, Dahan R, Maamary J, Ravetch JV (2017) Signaling by antibodies: recent
progress. Annu Rev Immunol 35:285–311
Dahan R, Sega E, Engelhardt J, Selby M, Korman AJ, Ravetch JV (2015) FccRs modulate the
anti-tumor activity of antibodies targeting the PD-1/PD-L1 axis. Cancer Cell 28:285–295
Dahan R, Barnhart BC, Li F, Yamniuk AP, Korman AJ, Ravetch JV (2016) Therapeutic activity of
agonistic, human anti-CD40 monoclonal antibodies requires selective FccR engagement.
Cancer Cell 29:820–831
DiLillo DJ, Ravetch JV (2015) Differential Fc-receptor engagement drives an anti-tumor vaccinal
effect. Cell 161:1035–1045
DiLillo DJ, Tan GS, Palese P, Ravetch JV (2014) Broadly neutralizing hemagglutinin
stalk-specific antibodies require FccR interactions for protection against influenza virus
in vivo. Nat Med 20:143–151
IgG Fc Receptors: Evolutionary Considerations 11

Georgoudaki AM, Prokopec KE, Boura VF, Hellqvist E, Sohn S, Ostling J, Dahan R, Harris RA,
Rantalainen M, Klevebring D, Sund M, Brage SE, Fuxe J, Rolny C, Li F, Ravetch JV,
Karlsson MC (2016) Reprogramming tumor-associated macrophages by antibody targeting
inhibits cancer progression and metastasis. Cell Rep 15:2000–2011
Li F, Ravetch JV (2013) Antitumor activities of agonistic anti-TNFR antibodies require differential
FccRIIB coengagement in vivo. Proc Natl Acad Sci U S A 110:19501–19506
Li F, Smith P, Ravetch JV (2014) Inhibitory Fcc receptor is required for the maintenance of
tolerance through distinct mechanisms. J Immunol 192:3021–3028
Li T, DiLillo DJ, Bournazos S, Giddens JP, Ravetch JV, Wang LX (2017) Modulating IgG effector
function by Fc glycan engineering. Proc Natl Acad Sci U S A 114:3485–3490
Lu CL, Murakowski DK, Bournazos S, Schoofs T, Sarkar D, Halper-Stromberg A, Horwitz JA,
Nogueira L, Golijanin J, Gazumyan A, Ravetch JV, Caskey M, Chakraborty AK,
Nussenzweig MC (2016) Enhanced clearance of HIV-1-infected cells by broadly neutralizing
antibodies against HIV-1 in vivo. Science 352:1001–1004
Lux A, Yu X, Scanlan CN, Nimmerjahn F (2013) Impact of immune complex size and
glycosylation on IgG binding to human FccRs. J Immunol 190:4315–4323
Nakagawa H, Hato M, Takegawa Y, Deguchi K, Ito H, Takahata M, Iwasaki N, Minami A,
Nishimura S (2007) Detection of altered N-glycan profiles in whole serum from rheumatoid
arthritis patients. J Chromatogr B Analyt Technol Biomed Life Sci 853:133–137
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin g subclass activity through
selective Fc receptor binding. Science 310:1510–1512
Pincetic A, Bournazos S, DiLillo DJ, Maamary J, Wang TT, Dahan R, Fiebiger BM, Ravetch JV
(2014) Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol
15:707–716
Qiu WQ, de Bruin D, Brownstein BH, Pearse R, Ravetch JV (1990) Organization of the human
and mouse low-affinity Fc gamma R genes: duplication and recombination. Science 248:
732–735
Scherer HU, van der Woude D, Ioan-Facsinay A, el Bannoudi H, Trouw LA, Wang J, Häupl T,
Burmester GR, Deelder AM, Huizinga TW, Wuhrer M, Toes RE (2010) Glycan profiling of
anti-citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis
Rheum 62:1620–1629
Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV (2012) Mouse model recapitulating human
Fcc receptor structural and functional diversity. Proc Natl Acad Sci U S A 109:6181–6186
Sondermann P, Pincetic A, Maamary J, Lammens K, Ravetch JV (2013) General mechanism for
modulating immunoglobulin effector function. Proc Natl Acad Sci U S A 110:9868–9872
Theodoratou E, Thaçi K, Agakov F, Timofeeva MN, Štambuk J, Pučić-Baković M, Vučković F,
Orchard P, Agakova A, Din FV, Brown E, Rudd PM, Farrington SM, Dunlop MG,
Campbell H, Lauc G (2016) Glycosylation of plasma IgG in colorectal cancer prognosis. Sci
Rep 6:28098
Unkeless JC, Shen Z, Lin CW, DeBeus E (1995) Function of human Fc c RIIA and Fc c RIIIB.
Semin Immunol 7:37–44
Wang TT, Maamary J, Tan GS, Bournazos S, Davis CW, Krammer F, Schlesinger SJ, Palese P,
Ahmed R, Ravetch JV (2015) Anti-HA glycoforms drive B cell affinity selection and determine
influenza vaccine efficacy. Cell 162:160–169
Zhou MJ, Brown EJ (1994) CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII
cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and
tyrosine phosphorylation. J Cell Biol 125:1407–1416
Role of FccRs in Antibody-Based
Cancer Therapy

Robert F. Graziano and John J. Engelhardt

Contents

1 Introduction.......................................................................................................................... 14
1.1 Mouse FccRs .............................................................................................................. 15
1.2 Human FccRs ............................................................................................................. 15
2 Tumor-Targeting Antibodies ............................................................................................... 16
3 Antibodies Against Immune Targets .................................................................................. 23
3.1 Depleting Antibodies: Regulatory T-Cell Targets ..................................................... 23
3.2 Antibodies Targeting Myeloid Cell Populations ....................................................... 26
3.3 Antagonist Antibodies ................................................................................................ 27
3.4 Agonist Antibodies ..................................................................................................... 27
4 Conclusions.......................................................................................................................... 29
References .................................................................................................................................. 30

Abstract Monoclonal antibodies can mediate antitumor activity by multiple

mechanisms. They can bind directly to tumor receptors resulting in tumor cell
death, or can bind to soluble growth factors, angiogenic factors, or their cognate
receptors blocking signals required for tumor cell growth or survival. Monoclonal
antibodies, upon binding to tumor cell, can also engage the host’s immune system
to mediate immune-mediated destruction of the tumor. The Fc portion of the
antibody is essential in engaging the host immune system by fixing complement
resulting in complement-mediated cytotoxicity (CDC) of the tumor, or by engaging
Fc receptors for IgG (FccR) expressed by leukocytes leading to antibody-dependent
cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP)
of tumor cells. Antibodies whose Fc portion preferentially engage activating FccRs
have shown greater inhibition of tumor growth and metastasis. Monoclonal anti-
bodies can also stimulate the immune system by binding to targets expressed on
immune cells. These antibodies may stimulate antitumor immunity by antagonizing
a negative regulatory signal, agonizing a costimulatory signal, or depleting immune

R. F. Graziano
J. J. Engelhardt (&)

Oncology Discovery, Bristol-Myers Squibb, Princeton, NJ, Redwood City, CA, USA
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2019) 423: 13–34

https://doi.org/10.1007/82_2019_150
© Springer Nature Switzerland AG 2019
Published online: 22 February 2019
14 R. F. Graziano and J. J. Engelhardt

cells that are inhibitory. The importance of Fc:FccR interactions in antitumor

therapy for each of these mechanisms have been demonstrated in both mouse
models and clinical trials and will be the focus of this chapter.

1 Introduction

The development of hybridoma technology described by Kohler and Milstein over

40 years ago triggered the concept of using monoclonal antibodies (mAbs) as ther-
apeutic drugs (Köhler and Milstein 1975). However, largely due to immunogenicity
and the short serum half-life of mouse antibodies in human patients, this potential was
not immediately realized. Advances in molecular biology, antibody engineering, and
transgenic mouse technology led to the development of chimeric (Boulianne et al.
1984; Morrison et al. 1984), humanized (Jones et al. 1986), and fully human(Taylor
et al. 1992; Jakobovits 1998; Tuaillon et al. 1993) mAbs, which significantly reduced
problems encountered with mouse mAbs. Rituximab, an anti-CD20 monoclonal
antibody developed for the treatment of B cell non-Hodgkin’s lymphoma (B-NHL)
and chronic lymphocytic leukemia (CLL), was the first FDA-approved monoclonal
antibody for cancer therapy (Maloney et al. 1997). Rituximab was FDA-approved in
1997 and since then more than 25 additional mAbs have been approved for treating
cancer and many more are in clinical development. With further technological
advancements in antibody engineering, tumor cell biology and cellular immunology
came more innovative approaches to inhibiting cancer growth with one or more mAbs
(reviewed further in (Weiner et al. 2009)).
Monoclonal antibodies can mediate their antitumor activity by several different,
but not mutually exclusive, mechanisms. Monoclonal antibodies to VEGF or to
VEGFR2 block the supply of blood to tumors by inhibiting angiogenesis (Ferrara
et al. 2004; Lu et al. 2003). One class of mAbs that binds directly to tumor-associated
antigens (TAA) can inhibit tumor growth by blocking a growth signal or by directly
inducing tumor cell death. In addition, as the antigen-binding fragment (Fab) of the
mAb binds to the TAA, the Fc portion of the mAb may engage the host immune
system by fixing complement, resulting in complement-dependent cytotoxicity
(CDC) of the tumor. The mAb can also bind to Fc receptors for IgG (FccR)
expressed on immune effector cells, which can result in antibody-dependent cellular
cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of the
tumor cell. ADCP mediated by dendritic cells or macrophages may also result in
augmented antigen presentation to tumor-specific T cells, engaging cells of the
adaptive immune system to eliminate tumors (DiLillo and Ravetch 2015).
Another class of therapeutic mAb binds to immune cells rather than the tumor
cell, leading to enhanced immune activation. This type of antibody can mediate its
effect by antagonizing a negative regulatory signal or by agonizing a costimulatory
signal leading to prolonged and/or enhanced antitumor immunity. Recent evidence
also suggests that mAb that bind to and deplete regulatory T cells (Treg) via ADCC
or ADCP can also augment antitumor activity (Selby et al. 2013; Simpson et al.
Role of FccRs in Antibody-Based Cancer Therapy 15

2013). This review will focus on the importance of Fc:FccR interactions in anti-
tumor immunity.

1.1 Mouse FccRs

Four FccRs have been molecularly characterized in the mouse system: FccRI,
FccRIIb, FccRIII, and FccRIV (Table 1). FccRI, FccRIII, and FccRIV are activating
receptors that are non-covalently associated with the immunoreceptor tyrosine-based
activation motif (ITAM)-containing common c-signaling chain. The activating
receptors mediate ADCC and ADCP of antibody-opsonized target cells. Conversely,
FccRIIb is an inhibitory FccR and contains an immunoreceptor tyrosine-based
inhibitory motif (ITIM) in its intracellular domain. Engagement of this receptor can
inhibit ADCC and ADCP. These four receptors have overlapping expression patterns
as well as differing affinities for the Fc portion of the four mouse subclasses of IgG:
IgG1, IgG2a, IgG2b, and IgG3. The Fc portion of antibodies of the IgG2a subclass,
and to a lesser extent the IgG2b subclass, bind preferentially to the activating
receptors FccRI and FccRIV, whereas antibodies of the IgG1 subclass have low
affinity for these receptors. However, IgG1 mAb has higher affinity than IgG2a for
the inhibitory FccRIIb. IgG1, IgG2a, and IgG2b bind FccRIII with similar affinities.
The avidity of IgG3 for each of the FccR is very low to non-detectable. As described
in the following section, the preferential binding of tumor-specific IgG2a subclass
mAb to activating FccR results in increased antitumor efficacy in vivo (Nimmerjahn
and Ravetch 2005; Ravetch and Lanier 2000), reviewed in (Clynes 2006)).

1.2 Human FccRs

The human FccR family has similarities to the mouse family but also differs in
several significant ways. The human FccR family does not contain a true homolog to
the mouse FccRIV and the mouse FccR family does not contain a true homolog of
the human FccRIIa. FccRIIa is an activating receptor that does not associate with the
ITAM-containing common c signaling chain, but contains an ITAM domain in its
intracellular domain. In addition, two versions of human FccRIII exist: FccRIIIa,
which contains a transmembrane region and associates with the ITAM-containing
common c signaling chain, and FccRIIIb, which is a glycophosphatidylinositol
(GPI)-linked FccR and does not directly mediate an activating or inhibitory signal
when engaged. Allelic variants of FccRIIa and FccRIIIa exist that have differing
avidities for Fc. The H131 allelic version of FccRIIa has overall higher affinity for Fc
than the R131 allelic version and is the only FccR with measurable affinity for the
IgG2 subclass. The V158 allelic version of FccRIIIa has overall higher avidity for Fc
than the F158 allelic version (Bruhns et al. 2009; reviewed in Bruhns 2012)
(Table 2).
16 R. F. Graziano and J. J. Engelhardt

Table 1 Mouse Fc receptors

FccR Affinity for Isotype Cellular distribution Activating or inhibitory
huIgG preference
FccRI High for IgG2a IgG2a o 2b Monocytes, Activating, associates
(KD * 10 nM)  1, 3 macrophages, DCs with ITAM-containing
signaling chain
FccRIIb Low IgG1 > 2b > 2a B cells, neutrophils, Inhibitory, contains
monocytes, ITIM domain in ICD
macrophages
FccRIII Low IgG1 = 2a = 2b NK cells, Activating, associates
neutrophils, with ITAM-containing
monocytes, signaling chain
macrophages, DCs
FccRIV Medium IgG2a >= 2b Neutrophils, Activating, associates
monocytes, with ITAM-containing
macrophages, DCs signaling chain
Source Nimmerjahn and Ravetch, Science 2005

Unlike the varying selectivity of mouse IgG1 subclasses for binding to activating
or inhibitory FccRs, IgG1 in the human system IgG1 is the isotype that binds best
to all types of FccRs. Fc variants have been engineered by altering Fc glycosylation
or by making amino acid substitutions that increase binding for specific FccRs.
Antibodies contain an N-linked glycosylation site at position 297. Antibodies
engineered to lack fucose moieties in their glycan chain have higher affinity for both
allelic versions of FccRIIIA and mediate more potent ADCC and ADCP (Shields
et al. 2002). Several amino acid-engineered variants of human IgG1 result in
preferential enhanced affinity for activating FccR and/or inhibitory FccR, each of
which may be utilized to enhance the activity of antitumor mAb, depending on the
mechanism of action of the mAb (reviewed in (Barnhart and Quigley 2017)). These
variants will be discussed throughout this review. Other alterations have been
created to eliminate binding to complement and/or FccR, resulting in an “inert” Fc
(Tam et al. 2017; Clynes et al. 2000). A mAb with an inert Fc may be utilized when
engagement of Fc effector mechanisms is undesirable, for example, to antagonize
negative regulatory signals on effector T cells without depleting them.

2 Tumor-Targeting Antibodies

The importance of Fc:FccR interactions in direct tumor targeting was established in

early preclinical studies. Clynes et al. demonstrated that full in vivo activity of
rituximab and trastuzumab required activating Fc:FccR engagement (Clynes et al.
2000). Mice that were deficient in the c signaling chain were unable to inhibit tumor
growth in vivo compared to wild-type mice. Furthermore, engineered Fc versions of
Role of FccRs in Antibody-Based Cancer Therapy 17

Table 2 Human Fc Receptors

FccR Allelic Affinity for Isotype preference Cellular Activating or
variants huIgG distribution inhibitory
FccRI None High IgG1 = 3 > 4  2 Monocytes, Activating,
described (KD * 10 nM) macrophages, associates with
activated ITAM-containing
neutrophils, DCs signaling chain
FccRIIa H131 Low to IgG1 > 2 = 3 > 4 Neutrophils, Activating,
medium monocytes, contains ITAM
R131 Low IgG1 > 3 > 4 > 2 macrophages, domain in ICD
eosinophils,
DCs, platelets
FccRIIb I232 Low IgG1 = 3 = 4 > 2 B cells, Inhibitory,
T232 Low IgG1 = 3 = 4 > 2 monocytes, contains ITIM
macrophages, domain in ICD
DCs, mast cells
FccRIIIa V158 Medium IgG1 = 3  4 > 2 NK cells, Activating,
F158 Low IgG1 = 3  4 > 2 monocytes, associates with
macrophages,, ITAM-containing
mast cells, signaling chain
eosinophils, DCs
FccRIIIb NA1 Low to IgG1 = 3  4 = 2 Neutrophils Neutral,
medium GPI-linked
NA2 Low to IgG1 = 3  4 = 2
medium

these antibodies that were unable to engage FccRs were also less active in elimi-
nating tumors. These data implied that, of the various antitumor activities attributed
to these mAbs (Table 3), FccR-mediated ADCC/ADCP was the most relevant for
efficacy in mouse tumor models and perhaps in treated patients. The authors also
demonstrated that mice deficient in the inhibitory FccR, FccRIIB, were more
capable of eliminating melanoma metastases than wild-type animals when treated
with a mAb to the melanoma TAA, gp75. This finding suggested that the optimal
design of antitumor mAb would preferentially engage activating FccR and not
inhibitory FccR. It was subsequently shown that TAA-specific mAbs, with mouse
Fcs that preferentially bind activating FccRs relative to inhibitory FccRs, were
significantly more potent in their ability to inhibit tumor growth and metastasis
in vivo. This suggested that isotypes that engage FccR with a high activating to
inhibitory ratio (A/I) accounted for this difference in activity (Ravetch and Lanier
2000; Nimmerjahn and Ravetch 2005).
Results from clinical studies also point to the importance of ADCC/ADCP in the
efficacy of antitumor mAb. In patients with lymphoma, expression of high-affinity
alleles of FccRIIIa (FccRIIIa-158 V) and FccRIIa (FccRIIa-131H) is associated
with improved response to rituximab therapy, likely due to enhanced ADCC and/or
ADCP (Cartron et al. 2002; Weng and Levy 2003). In fact, second-generation
Table 3 FDA-approved monoclonal antibodies for cancer therapy 1997–2018
18

Generic name Trade Target Fc format Cancer Target class Purported MoA(s) References
name
Rituximab Rituxan CD20 Chimeric B-NHL, CLL Tumor Direct tumor growth Reviewed in
mouse/human inhibition, CDC, Pierpont et al.
IgG1 ADCC, ADCP (2018)
Ofatamumab Arzerra CD20 Human IgG1 CLL Tumor Direct tumor growth Osterborg et al.
inhibition, CDC, (2016)
ADCC, ADCP
Obinutuzumab Gazyva CD20 Humanized B-NHL, CLL Tumor Direct tumor growth Goede et al.
IgG1 inhibition, CDC, (2014) and Salles
(nonfucosylated eADCC, eADCP et al. (2012)
Fc)
Ibritumomab Zevalin CD20 Mouse IgG1 B-NHL Tumor Delivery of a toxic Grillo-Lopez
90
Y-labeled radioisotope to tumor (2002)
Alemtuzumab Campath* CD52 Humanized B-CLLa Tumor Direct tumor growth Lundin et al.
IgG1 inhibition, CDC, (1998)
ADCC, ADCP?
Brentuximab vedotin Adcetris CD30 Chimeric HL, ALCL Tumor Delivery of a toxic (Younes et al.
mouse/human payload to tumor 2010)
IgG1
Gemtuzumab Myelotarg CD33 Humanized AML Tumor Delivery of a toxic Reviewed in
ozogamicin IgG4 ADC payload to tumor Gbadamosi et al.
(2018)
Inotuzumab Besponsa CD22 Humanized ALL Tumor Delivery of a toxic Reviewed in Tvito
ozogamicin IgG4 ADC payload to tumor and Rowe (2017)
Trastuzumab Herceptin HER2 Humanized Breast, stomach Tumor Direct tumor growth Albanell and
IgG1 inhibition, ADCC, Baselga (1999)
ADCP
(continued)
R. F. Graziano and J. J. Engelhardt
Table 3 (continued)
Generic name Trade Target Fc format Cancer Target class Purported MoA(s) References
name
Trastuzumab-dkst Ogivri HER2 Humanized Breast, stomach Tumor Direct tumor growth Serna-Gallegos
IgG1 inhibition, ADCC, et al. (2018)
ADCP
ado-Trastuzumab- Kadcyla HER2 Humanized Breast Tumor Delivery of a toxic Amiri-Kordestani
emtansine IgG1 ADC payload to tumor et al. (2014a)
Pertuzumab Perjeta HER2 Humanized Breast Tumor Direct tumor growth Amiri-Kordestani
IgG1 inhibition, ADCC, et al. (2014b)
ADCP
Cetuximab Erbitux EGFR Chimeric CRC, H&N Tumor Direct tumor growth Reviewed in
mouse/human inhibition, ADCC, Goldberg (2005)
IgG1 ADCP
Panitumumab Vectibix EGFR Human IgG2 CRC Tumor Direct tumor growth Giusti et al.
Role of FccRs in Antibody-Based Cancer Therapy

inhibition, ADCC, (2007)

ADCP
Necitumumab Portrazza EGFR Human IgG1 Lung Tumor Direct tumor growth Dienstmann and
inhibition, ADCC, Tabernero (2010)
ADCP
Dinutuximab Unituxin GD2 Chimeric Neuroblastoma Tumor ADCC, ADCP Dhillon (2015)
mouse/human
IgG1
Olaratumab Lartruvo PDGFRA Human IgG1 Sarcoma Tumor Direct tumor growth Shirley (2017)
inhibition, ADCC,
ADCP
Bevacizumab Avastin VEGF Humanized CRC, lung, brain, Growth Inhibition of Kerr (2004)
IgG1 kidney, cervical, factor angiogenesis
ovarian
(continued)
19
Table 3 (continued)
20

Generic name Trade Target Fc format Cancer Target class Purported MoA(s) References
name
Bevacizumab-awwb Mvasi VEGF Humanized CRC, lung, brain, Growth Inhibition of Serna-Gallegos
IgG1 kidney, cervical factor angiogenesis et al. (2018)
Ramucirumab Cyramza VEGFR2 Human IgG1 Stomach, CRC Growth Inhibition of Poole and Vaidya
factor angiogenesis (2014)
receptor,
endothelial
cell
Elotuzumab Empliciti SLAMF7 Humanized Multiple myeloma Tumor, ADCC, ADCP, Markham (2016)
IgG1 immune cell activation of NK cells
Daratumumab Darzalex CD38 Human IgG1 Multiple myeloma Tumor, Direct tumor growth McKeage (2016)
immune cell inhibition, ADCC,
ADCP, depletion of
Tregs
Ipilimumab Yervoy CTLA-4 Human IgG1 Melanoma Immune cell Checkpoint blockade, Cameron et al.
depletion of Tregs (2011)
Nivolumab Opdivo PD-1 Human IgG4 Melanoma, CRC, Immune cell Checkpoint blockade Kazandjian et al.
kidney, lung, H&N, (2016)
bladder, liver, HL
Pembrolizumab Keytruda PD-1 Humanized Melanoma, CRC, Immune cell Checkpoint blockade Poole (2014)
IgG4 stomach, lung,
H&N, bladder, liver,
HL
Atezolizumab Tecentriq PD-L1 Human IgG1 Bladder, lung Tumor, Checkpoint blockade Blair (2018)
(engineered immune cell
“inert” Fc)
(continued)
R. F. Graziano and J. J. Engelhardt
Table 3 (continued)
Generic name Trade Target Fc format Cancer Target class Purported MoA(s) References
name
Avelumab Bavencio PD-L1 Human IgG1 Merkel cell Tumor, Checkpoint blockade Kim (2017)
carcinoma immune cell
Durvalumab Imfinzi PD-L1 Human IgG1 Bladder Tumor, Checkpoint blockade Syed (2017)
(engineered immune cell
“inert” Fc)
Mogamulizumab-kpkc Poteligeo CCR4 IgG1-NF CTCL Tumor Direct tumor growth Kim et al. (2018)
inhibition, ADCC?
a
Campath was withdrawn from market as an agent for treating B-CLL in 2012 and subsequently approved for multiple sclerosis in 2014
Role of FccRs in Antibody-Based Cancer Therapy
21
22 R. F. Graziano and J. J. Engelhardt

anti-CD20 mAbs have been created that increase affinity for activating FccR
(Table 3). Ocaratuzumab is an IgG1 anti-human CD20 antibody in which two
amino-acid substitutions were made in the human IgG1 Fc in order to increase
affinity to both 158 V and 158F allotypes of FccRIIIA (Forero-Torres et al. 2012;
Klein et al. 2013). The enhanced FccRIIIA binding and increased affinity to human
CD20 have resulted in increased in vitro ADCC activity regardless of donor
FccRIIIA allotype. In addition, promising clinical responses have been seen in
patients with follicular lymphoma, including patients who received prior rituximab
and those harboring the 158F allotype of FccRIIIA (Forero-Torres et al. 2012;
Cheney et al. 2014; Tobinai et al. 2011).
Nonfucosylated antibodies can be generated via mutations at N-linked glyco-
sylation sites in the Fc or a cell line lacking fucosyltransferases and fucose trans-
porters (Yamane-Ohnuki and Satoh 2009). Increased function can be achieved with
a nonfucosylated antibody, as demonstrated by clinical responses to rituximab
(anti-human CD20 with wild-type IgG1) and obinutuzumab (anti-human CD20
with a nonfucosylated IgG1). Compared with rituximab plus chemotherapy, non-
fucosylated obinutuzumab plus chemotherapy has demonstrated greater response
rate and survival in patients with chronic lymphocytic leukemia (Goede et al. 2014).
As observed with rituximab, patients who are homozygous for the 158 V allotype
of FccRIIIA tend to have stronger responses to obinutuzumab (Salles et al. 2012).
Recently two monoclonal antibodies received FDA approval for the treatment of
multiple myeloma (MM). Daratumumab is a human IgG1 antibody that binds to
CD38, which is expressed at high levels on the surface of MM tumors.
Daratumumab manifests its activity through multiple mechanisms including CDC,
ADCC, and ADCP of MM tumor cells (van der Veer et al. 2011; Overdijk et al.
2015). In addition to being expressed on MM tumor cells, CD38 is expressed on
normal T cells, monocytes, B cells, NK cells, and, importantly, at high levels on
Tregs, Bregs, and myeloid-derived suppressor cells (MDSC). An additional mech-
anism of action of daratumumab is to deplete Treg, Breg, and MDSC, cells that
inhibit the antitumor immunity (Krejcik et al. 2016). This mechanism of action will
be discussed in detail later in this review. A second monoclonal antibody that
received FDA approval for the treatment of MM is elotuzumab (Lonial et al. 2015).
Elotuzumab is a humanized IgG1 antibody that binds to SLAMF7, another receptor
that is expressed at high levels on MM tumor cells. Elotuzumab was shown to
mediate ADCC and ADCP of SLAMF7-expressing MM tumor cell lines in vitro
(Hsi et al. 2008; Tai et al. 2008). SLAMF7 is also expressed as an activating receptor
on normal NK cells, and binding of elotuzumab to NK cells can activate these
effector cells to control tumor growth (Bouchon et al. 2001; Collins et al. 2013).
Elotuzumab treatment inhibits tumor growth of human SLAMF7 positive MM
tumor xenografts in immunocompromised mice (Hsi et al. 2008; Tai et al. 2008).
This efficacy is dependent on FccRIII engagement on NK cells—depletion of NK
cells or blocking FccRIII abrogates its efficacy (Hsi et al. 2008). In clinical studies,
similar to what was observed with rituximab, benefits in progression-free survival
were reported in patients with relapsed/refractory multiple myeloma (RRMM) who
Role of FccRs in Antibody-Based Cancer Therapy 23

were homozygous for the FccRIIIa-158 V allele and treated with elotuzumab in
combination with bortezomib and dexamethasone (Jakubowiak et al. 2016).
FccRs are expressed on cells of the innate immune system and, as discussed
above, innate effector cells such as NK cells and myeloid cells are often required for
the therapeutic efficacy of tumor-targeted mAbs in mouse tumor models. However,
some studies of immunocompetent mice with syngeneic tumor allografts showed
that depletion of cells of the adaptive immune response, in particular CD8+ T cells,
reduced the therapeutic effects of tumor-targeted mAbs (Kohrt et al. 2012; Park
et al. 2010; Stagg et al. 2011). Furthermore, patients with lymphoma have devel-
oped lymphoma-specific CD4+ and CD8+ T cell responses after rituximab treat-
ment, suggesting that tumor-targeted mAbs may initiate an antitumor adaptive
immune response (Abès et al. 2010 #244). Experiments combining mAb to TAA
with agents that antagonize immune checkpoints or agonize costimulatory receptors
on the surface of T cells have been performed in mouse tumor models. For
example, Stagg et al. (2011) demonstrated that the efficacy of an anti-HER2 anti-
body was dependent on NK cells and CD8+ T cells and on release of type I and type
II interferons. Furthermore, the combination of the anti-HER2 antibody with
anti-PD-1 or anti-CD137 mAb enhanced antitumor efficacy toward HER2 + tumors
versus single agents. Bezman et al. (Bezman et al. 2017) used mouse tumor cell
lines that were engineered to express human SLAMF7 to look at the ability of the
mouse IgG2a version of elotuzumab (elotuzumab-g2a) to reduce or eliminate tumor
growth in immunocompetent mouse models. They showed that antitumor efficacy
required both FccRIII-expressing NK cells and CD8+ T cells and was significantly
enhanced by co-administration of anti-PD-1 antibody. Elotuzumab-g2a and
anti-PD-1 combination treatment promoted tumor-infiltrating NK and CD8+ T cell
activation, as well as increased intratumoral cytokine and chemokine release.

3 Antibodies Against Immune Targets

In addition to directly targeting antigens expressed by tumor cells, antibodies are

also used to influence the function of cells of the immune system. Antibodies
against immune targets can be separated into three main mechanisms: antagonists,
agonists, or depleting mAbs. In some cases, it is unclear whether more than one of
these strategies is associated with a particular mAb, and Fc:FccR interactions or
lack thereof can determine which effect is occurring.

3.1 Depleting Antibodies: Regulatory T-Cell Targets

Anti-CTLA-4 treatment was the first example of an immune cell-targeted mAb

having antitumor efficacy in mouse models (Leach et al. 1996). This finding was
later translated to clinical treatment of patients with ipilimumab, the first checkpoint
24 R. F. Graziano and J. J. Engelhardt

inhibitor approved for the treatment of human cancer. While the success of treat-
ment with anti-CTLA-4 was originally attributed to blockade of CTLA-4 binding to
its ligands CD80 and CD86, recent data have called that conclusion into question.
Studies by three different research groups implicated the depletion of
tumor-infiltrating Treg cells as being critical for the activity of anti-CTLA-4 in
mouse tumor models. In Selby et al., the anti-mouse CTLA-4 mAb 9D9 (mouse
IgG2b) was reformatted as multiple different isotypes (mIgG1 D265A, mIgG1,
mIgG2a) to determine the role of Fc:FcR interactions on antitumor efficacy (Selby
et al. 2013). Antitumor effects were found to correlate with the ability to engage
activating FccRs, i.e., the mIgG2a, which bound best to activating receptors, had
the highest activity, and was followed by the original mIgG2b. The mIgG1 D265A
isotype, which does not bind FccRs, had no antitumor activity, and neither did the
mIgG1, which preferentially binds to the inhibitory FccRIIb. When the tumors of
treated mice were examined by flow cytometry, it was found that Tregs were
specifically reduced by the IgG2a isotype and to a lesser extent by the IgG2b
isotype but not the mIgG1 or mIgG1 D265A. Depletion of peripheral Tregs in the
spleen, lymph node or blood was not observed for any of the mAbs. Taken together
this data revealed that binding to activating FccRs was critical to the activity of
anti-CTLA-4 treatment, and that blockade in the absence of Treg depletion had little
to no antitumor activity.
In complementary work, Simpson et al. examined the effects of another
anti-CTLA-4 mAb, 9H10, which is a hamster anti-mouse CTLA-4 mAb. Using an
antigen-specific T-cell transfer system, they were able to demonstrate that 9H10
depleted tumor antigen-specific Tregs and also reduced endogenous Tregs
(Simpson et al. 2013). Depletion was not changed when the experiments were
performed in C3 KO mice or when NK cells were depleted, suggesting that
complement or NK cells were not responsible for depletion. However, depletion
was reduced in either Fcc or FccRIV KO mice, but not in FccRIII KO mice.
FccRIV-expressing phagocytes were found to be more prevalent in the tumor
microenvironment and, taken together with the lack of a role for NK cells in the
process, FccRIV-expressing macrophages/monocytes are implicated as the
responsible effector cell population. Considering the results from these two papers,
two different anti-mouse CTLA-4 mAbs, 9D9 and 9H10, were shown to deplete
Tregs, and this effect was shown in three different mouse tumor models—MC38,
CT26, and B16. The strength of this finding across different mAbs and models
suggests this is indeed a critical mediator of antitumor efficacy in mice.
In another recent publication, Bulliard et al. also showed that efficacy and Treg
depletion depended on isotype for both anti-CTLA-4 and anti-GITR mAbs
(Bulliard et al. 2013). The finding that depletion of immune suppressive cells is also
the primary mechanism of action of another T cell-directed antibody therapy
confirms that this is a potent effector mechanism. Because GITR is a costimulatory
receptor, antitumor efficacy mediated by the GITR mAb, DTA-1, was originally
attributed to agonist activity against the receptor (Ko et al. 2005), but depletion of
Tregs was later identified by Coe et al. (Coe et al. 2010). Determining that efficacy
is dependent on ADCC of Tregs challenges this assumption and suggests that
Role of FccRs in Antibody-Based Cancer Therapy 25

activity of antibodies against other T-cell costimulatory receptors should be

re-evaluated.
Indeed, it has been found that mAbs against other receptors such as OX-40
(Bulliard et al. 2014), and ICOS are most efficacious when they mediate Treg
depletion. While it is clear that mAbs against multiple T-cell surface receptors can
mediate depletion of Tregs, it is less clear what effects these mAbs have on effector
T-cell populations that can also express the receptors. Bulliard et al. noted reduction
of effector CD4 s and CD8 s after both GITR and CTLA-4 mAb treatments, but
this effect was not observed by Selby and Simpson for CTLA-4 or by Mahne for
GITR (Mahne et al. 2017). However, the fact that no difference in abundance of
bulk effector T cells was observed does not necessarily rule out an effect on
effectors. Some effectors may be depleted by antibody treatment, but since Treg
depletion would likely be inducing effector expansion separately, that expansion
may mask some amount of effector depletion. Both GITR and CTLA-4 are
expressed at higher levels on Treg cells than effector T cells, so this difference may
result in preferential depletion of Tregs.
In addition to the costimulatory receptors described above, chemokine receptors
are also being investigated as targets that can induce depletion of Treg cells. Two in
particular have drawn increased attention. The chemokine receptor CCR4 is cur-
rently being targeted by the nonfucosylated mAb mogamulizumab. This mAb was
originally developed for the treatment of cutaneous T-cell lymphoma, which
expresses CCR4, but was also found to deplete Tregs from peripheral blood of
patients treated with the mAb (Ogura et al. 2014). This has prompted testing of the
mAb for treatment of solid tumors in addition to its original indication. However,
CCR4 expression is not only found on Treg cells but also effector CD4 T cells,
which could limit clinical activity due to depletion of these effectors in addition to
Tregs. Plitas et al. recently identified another chemokine receptor, CCR8 that is
more highly expressed by Treg cells at the tumor site compared to those in
peripheral blood or tissues (Plitas et al. 2016). The CCR8 + Tregs appear to be the
most activated Tregs, and CD4 and CD8 effector cells express little to no CCR8.
These attributes make CCR8 an intriguing target that may avoid significant
depletion of either peripheral Tregs or activated effector cells from the tumor site.
While the discovery of Treg depletion in mouse models suggests that this is a
powerful means to improve immune responses to tumors, it also brings up
important questions about how this process may occur in patients. Mouse syngeneic
models are useful for determining antitumor efficacy of mAb therapies and were
critical in demonstrating how both anti-CTLA-4 and anti-PD-1/L1 could enhance
immune responses to tumors. However, extrapolating from the interactions of
mouse Fc regions with mouse FccRs to those of human Fcs and human FccRs is
not straightforward.
A critical tool for studying human Fc-FcR interactions in the context of mouse
tumor model is the human FcR transgenic mouse model developed by Ravetch
et al. These mice have had the mouse FccRs knocked out, and the human FccRs
introduced transgenically (Smith et al. 2012). The mice thus express only human
FccRs and can be used to model the effect of human Fcs on the function of
26 R. F. Graziano and J. J. Engelhardt

therapeutic mAbs. These mice have been used to investigate the roles of human Fcs
on therapeutic targets for multiple immunotherapy targets as well as tumor-targeted
mAbs. Expression of the human FcRs seems to match the expected expression
pattern (Smith et al. 2012). Of course, while these mice are an invaluable tool, they
are still subject to the inherent weaknesses of mouse tumor models, where immune
infiltrates and specifically immune cells expressing FcRs are different from human
tumors.

3.2 Antibodies Targeting Myeloid Cell Populations

In addition to depletion of Treg cells, other suppressive immune cell types can be
removed by mAb treatment. One example being explored clinically is CSF1R,
which targets myeloid cells thought to restrain T cell responses to tumors. CSF1 is a
growth factor required for the survival of these cells, and the cell type can be
targeted with either small molecules that inhibit the kinase function of the receptor
or mAbs that prevent binding of CSF1R to its ligands CSF1 and IL-34. The mAb
cabiralizumab, which is in clinical testing in combination with nivolumab, is an
IgG4, and is not thought to require effector function for its activity. A mouse
surrogate (mIgG1) of this mAb has been shown to have antitumor activity in mouse
models and to specifically reduce M2 macrophages at the tumor site. While both the
surrogate and human mAbs employ Fcs that should not induce ADCC, a mAb that
does induce ADCC may be more effective at eliminating this cell type. Inhibiting
CSF1R signaling is also being investigated using the humanized Ab, emactuzumab,
which blocks the dimerization interface of CSF1R and is in ongoing Phase 1
clinical trials in combination with atezolizumab.
Myeloid-derived suppressor cells (MDSC) are another suppressive cell type that
could be targeted with depleting mAbs. Using a Ly6G mAb, Clavijo et al. showed
depletion of Ly6G+ , Ly6Cint myeloid cells that they classified as granulocytic
MDSCs (Clavijo et al. 2017). This depletion of MDSCs combined with CTLA-4
treatment resulted in complete rejection of the MOC1 tumor model, but had no
effect on tumor growth in the MOC2 model. The authors speculated that this
difference in responses was due to the lack of antigen and T-cell infiltrate in the
MOC2 model. While this data suggests targeting MDSCs could be a powerful
therapeutic strategy in combination with other immunotherapies, the lack of specific
markers of MDSCs in humans complicates the application of this strategy to cancer
patients. As tumor-infiltrating macrophages may also be critical effector cells for
cytotoxic and checkpoint control antibodies, a highly specific targeting of MDSCs
is of critical importance (Lehmann et al. 2017; Simpson et al. 2013).
A mAb can also be used to target multiple cell populations at once, based on
expression of its target. Daratumumab, a human IgG1 mAb specific for CD38, has
been approved for the treatment of multiple myeloma. CD38 is a promising target
for MM as it is highly expressed on these tumor cells. It is also highly expressed on
a variety of immune suppressive cell populations such as granulocytic MDSCs,
Role of FccRs in Antibody-Based Cancer Therapy 27

Bregs, and a highly suppressive fraction of human Tregs. Daratumumab has been
shown to mediate ADCC of these populations in vitro and in the case of Bregs and
CD38-expressing Tregs, patients treated with daratumumab have reduced numbers
of these cell types, consistent with ADCC (Krejcik et al. 2016).

3.3 Antagonist Antibodies

While a depleting mAb targeting CTLA-4 has been shown to confer superior
antitumor activity to a mAb that does not engage FcR, this is not true for all T-cell
inhibitory receptors. In contrast to CTLA-4 and most costimulatory receptors, PD-1
is expressed at higher levels on CD8 T cells in the tumor than in Tregs. Targeting
PD-1 with a depleting mAb isotype can preferentially deplete CD8 cells and result
in an abrogated antitumor response in comparison with a non-depleting isotype
(Dahan et al. 2015). Therefore, the expression pattern of the mAb target on Tregs
and T effector cells is likely to determine the strategy that should be employed to
target the receptor.

3.4 Agonist Antibodies

Antibodies can be used to initiate signaling through a receptor as well as to block

binding to the receptor’s ligand. In some cases, such as the CD28 superagonist, this
may occur simply through soluble binding of the mAb to the receptor, but signaling
may also be improved by Ab binding to FcR bearing cells (Bartholomaeus et al,
2014). More commonly there is a requirement for the mAb to bind to another
surface in order to achieve the higher valency required. In vitro, this is often
accomplished by binding the mAb to plastic in the form of a plate or bead. The
mAb then mediates the formation of clustered receptors necessary for signaling. In
vivo, it is thought that FcRs can mediate this higher valency and allow mAbs to
work as agonists even if they cannot trigger signaling without crosslinking.
Agonist mAbs have been most well studied in the context of the TNF receptor
superfamily. TNFRs require the formation of higher-order clusters to signal, and
hence, often require mAbs to be crosslinked by FcR to mediate signaling in vivo.
CD40 is expressed on antigen presenting cells and is critical in promoting immune
activation by inducing expression of costimulatory ligands on APCs. The activation
of CD40 can be studied by using a DEC205-based vaccination system. Using this
system Li and Ravetch (Li and Ravetch 2011) demonstrated that the inhibitory Fc
receptor, FccRIIb was required for CD40 agonist activity, and that mAbs that
preferentially bind to activating receptors such as mIgG2a or mAbs that did not
bind FcR such as the mIgG1 D265A mutant, did not confer agonist activity. Similar
results were found in human FcR transgenic mice, where a human IgG1 S267E
mutation significantly enhanced agonist activity compared to the human IgG1 mAb
28 R. F. Graziano and J. J. Engelhardt

or an FcR nonbinder (N297A) (Li and Ravetch 2013). Importantly, the requirement
for preferential FccRIIb binding was also confirmed in mouse tumor models, where
a mIgG1 mAb against CD40 was more efficacious than mIgG2a or mIgG1 D265A
mAbs. Separate work from White et al. using a different vaccination system also
confirmed the requirement for FccRIIb interactions while utilizing a different CD40
agonist mAb (White et al. 2011).
In addition to CD40, the importance of FccRIIb binding has also been
demonstrated for other agonistic mAbs such as Fas, DR4, and DR5. Anti-Fas
antibodies were shown to have significantly less hepatotoxicity in FccRIIb KO
mice than in wild-type mice, indicating that the inhibitory FcR is necessary for Fas
agonism (Xu et al. 2003). For DR4, the mIgG1 isotype had higher antitumor
efficacy in the Colo205 mouse tumor model than the mIgG2a version and similar
results were seen in multiple tumor models for a DR5 mAb (Chuntharapai et al.
2001). In the case of DR5, the S267E mutation was able to enhance activity
compared to the human IgG1 in the MC38 and 4T1 tumor models when these
expressed human FccRIIb in place of the mouse homolog (Li and Ravetch 2012).
Together these data confirm the role of CD32B in mediating agonist signaling for
targets expressed on APCs or tumors themselves.
Extrapolating this data to T cell costimulatory targets is more complicated. As
mentioned earlier, mAbs against GITR and OX-40 are more active when formatted
to engage with activating receptors. Similar data have been observed for mAbs
against ICOS (Engelhardt et al. unpublished observations). These observations are
driven primarily by the depletion of Tregs at the tumor site and likely are not
representative of which mAb formats induce the best agonist signal through the
receptor in question. Antibodies against another TNFRSF member, CD27, have
also been shown to have antitumor activity, in this case in human CD27 transgenic
mice (wild-type mouse FcR). This activity was dependent on FcR binding, as the
human IgG1 mAb had activity while the N297S version did not (He et al. 2013).
These results do not indicate whether activity is due to a depleting or agonistic
mechanism (Wasiuk et al. 2017). For most if not all mAbs directed against cos-
timulatory receptors expressed on Tregs and effector T cells, activity in mouse
tumor models will be highest with depleting isotypes rather than those that pref-
erentially engage the inhibitory receptor. This hierarchy may not translate to
humans though, as it is unclear that Treg depletion occurs as readily in patients as in
mice. If depletion is not a relevant mechanism in humans or in cases where an
agonist is potentially being combined with another agent that depletes Tregs, it may
be advantageous to have a mAb that is optimized for agonist function via
crosslinking by CD32B. It is possible that binding to activating Fc receptors may
lead to depletion in some circumstances and agonism in others further complicating
the interpretation of the data from mouse tumor models.
Most T-cell agonist programs that have moved to the clinic so far have utilized
human IgG1 mAbs or in the case of urelumab an IgG4 mAb. These programs have
thus shown limited antitumor efficacy as monotherapy, with no agonist mAb having
gained approval to date. It may be that a superior CD32B binding mutant would
have more activity than what has been observed so far.
Role of FccRs in Antibody-Based Cancer Therapy 29

4 Conclusions

The use of monoclonal mAbs in cancer treatment allows for the targeting of pro-
teins and cell populations with high specificity. An improved understanding of the
interactions between mAb Fc regions and FccRs has led to the design of thera-
peutics that lead to distinct effects after the binding of the mAb to its antigen.
Antibodies can be optimized for specific purposes as shown in Fig. 1. Initial
therapeutics targeted the cancer cells themselves and relied on FcR+ cells as
effectors to kill the cancer cells through ADCC or ADCP. Human IgG1 mAbs were
first used to mediate this killing, but advances in the field of mAb engineering have
led to the use of nonfucosylated or Fc mutated mAbs that enhance the interactions
leading to ADCC or ADCP. With the advent of immunotherapy, mAbs have also
been used to alter the immune response to tumors, that is, to target, and eradicate
inhibitory immune cells by ADCC or ADCP, thereby allowing other immune cells
to recognize and kill the cancer cells. It has long been known that mAbs can
stimulate signaling through a receptor and mimic ligand binding. This often
requires clustering of the receptors on the cell surface that can be accomplished
in vivo by the binding of the mAb Fc to FcRs. In multiple systems, this is most
efficiently accomplished by binding to the inhibitory receptor CD32B and this
binding can be enhanced and made more selective to CD32B through mutation to
the Fc region. Finally, mAbs can be used to block binding of a particular receptor or
ligand on an immune cell. In this case, an absence of binding to FcR is often
preferable and can be accomplished by choosing an isotype with low affinity for
FcRs such as IgG4 or including Fc mutations that further reduce FcR binding.

Fig. 1 Critical role of FccRs in promoting antibody-mediated tumor immunity

30 R. F. Graziano and J. J. Engelhardt

Antibodies are powerful tools for treating cancer patients, and understanding the
role of FccRs in the function of therapeutic mAbs will enable the design of more
powerful therapeutics.

Acknowledgements The authors thank Catherine A. Bolger for her review and editing, and
Alan J. Korman for his critical reading and insightful perspective on this chapter.

References

Abès R et al (2010) Long-lasting antitumor protection by anti-CD20 antibody through cellular

immune response. Blood 116(6):926–934
Albanell J, Baselga J (1999) Trastuzumab, a humanized anti-HER2 monoclonal antibody, for the
treatment of breast cancer Drugs Today (Barc) 35:931–946
Amiri-Kordestani L et al. (2014a) FDA approval: ado-trastuzumab emtansine for the treatment of
patients with HER2-positive metastatic breast cancer Clin Cancer Res 20:4436–4441 https://
doi.org/10.1158/1078-0432.ccr-14-0012
Amiri-Kordestani L et al. (2014b) First FDA approval of neoadjuvant therapy for breast cancer:
pertuzumab for the treatment of patients with HER2-positive breast cancer Clin Cancer Res
20:5359–5364 https://doi.org/10.1158/1078-0432.ccr-14-1268
Barnhart BC, Quigley M (2017) Role of Fc-FcgammaR interactions in the antitumor activity of
therapeutic antibodies. Immunol Cell Biol 95:340–346. https://doi.org/10.1038/icb.2016.121
Bartholomaeus P et al (2014) Cell contact-dependent priming and Fc interaction with
CD32 + immune cells contribute to the TGN1412-triggered cytokine response. J Immunol
192:2091–2098. https://doi.org/10.4049/jimmunol.1302461
Bezman NA et al (2017) PD-1 blockade enhances elotuzumab efficacy in mouse tumor models
blood advances 1:753–765
Blair HA (2018) Atezolizumab: A Review in Previously Treated Advanced Non-Small. Cell Lung
Cancer Target Oncol 13:399–407. https://doi.org/10.1007/s11523-018-0570-5
Bouchon A, Cella M, Grierson HL, Cohen JI, Colonna M (2001) Activation of NK cell-mediated
cytotoxicity by a SAP-independent receptor of the CD2 family. J Immunol 167:5517–5521
Boulianne GL, Hozumi N, Shulman MJ (1984) Production of functional chimaeric mouse/human
antibody. Nature 312:643–646
Bruhns P (2012) Properties of mouse and human IgG receptors and their contribution to disease
models. Blood 119:5640–5649. https://doi.org/10.1182/blood-2012-01-380121
Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, Daeron M (2009)
Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human
IgG subclasses. Blood 113:3716–3725. https://doi.org/10.1182/blood-2008-09-179754
Bulliard Y et al (2013) Activating Fc gamma receptors contribute to the antitumor activities of
immunoregulatory receptor-targeting antibodies. J Exp Med 210:1685–1693. https://doi.org/
10.1084/jem.20130573
Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL (2014) OX40 engagement
depletes intratumoral Tregs via activating FcgammaRs, leading to antitumor efficacy Immunol
Cell Biol 92:475–480. https://doi.org/10.1038/icb.2014.26
Cameron F, Whiteside G, Perry C (2011) Ipilimumab: first global approval. Drugs 71:1093–1104.
https://doi.org/10.2165/11594010-000000000-00000
Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H (2002)
Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG
Fc receptor FcgammaRIIIa gene. Blood 99:754–758
Role of FccRs in Antibody-Based Cancer Therapy 31

Cheney CM et al (2014) Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced

antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia. MAbs 6:749–
755. https://doi.org/10.4161/mabs.28282
Chuntharapai A et al (2001) Isotype-dependent inhibition of tumor growth in vivo by monoclonal
antibodies to death receptor 4. J Immunol 166:4891–4898
Clavijo PE et al (2017) Resistance to CTLA-4 checkpoint inhibition reversed through selective
elimination of granulocytic myeloid cells. Oncotarget 8:55804–55820. https://doi.org/10.
18632/oncotarget.18437
Clynes R (2006) Antitumor antibodies in the treatment of cancer: Fc receptors link opsonic
antibody with cellular immunity. Hematol Oncol Clin North Am 20:585–612. https://doi.org/
10.1016/j.hoc.2006.02.010
Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo
cytotoxicity against tumor targets. Nat Med 6:443–446. https://doi.org/10.1038/74704
Coe D, Begom S, Addey C, White M, Dyson J, Chai JG (2010) Depletion of regulatory T cells by
anti-GITR mAb as a novel mechanism for cancer immunotherapy. Cancer Immunol
Immunother 59:1367–1377. https://doi.org/10.1007/s00262-010-0866-5
Collins SM et al (2013) Elotuzumab directly enhances NK cell cytotoxicity against myeloma via
CS1 ligation: evidence for augmented NK cell function complementing ADCC. Cancer
Immunol Immunother 62:1841–1849. https://doi.org/10.1007/s00262-013-1493-8
Dahan R, Sega E, Engelhardt J, Selby M, Korman AJ, Ravetch JV (2015) FcgammaRs modulate
the anti-tumor activity of antibodies targeting the PD-1/PD-L1 axis. Cancer Cell 28:285–295.
https://doi.org/10.1016/j.ccell.2015.08.004
Dhillon S (2015) Dinutuximab: first global approval. Drugs 75:923–927. https://doi.org/10.1007/
s40265-015-0399-5
Dienstmann R, Tabernero J (2010) Necitumumab, a fully human IgG1 mAb directed against the
EGFR for the potential treatment of cancer Curr Opin Investig. Drugs 11:1434–1441
DiLillo DJ, Ravetch JV (2015) Fc-receptor interactions regulate both cytotoxic and immunomod-
ulatory therapeutic antibody effector functions cancer. Immunol Res 3:704–713. https://doi.
org/10.1158/2326-6066.CIR-15-0120
Ferrara N, LeCouter J, Lin R, Peale F (2004) EG-VEGF and Bv8: a novel family of
tissue-restricted angiogenic factors. Biochim Biophys Acta 1654:69–78. https://doi.org/10.
1016/j.bbcan.2003.07.001
Forero-Torres A et al (2012) Results of a phase 1 study of AME-133v (LY2469298), an
Fc-engineered humanized monoclonal anti-CD20 antibody, in FcgammaRIIIa-genotyped
patients with previously treated follicular lymphoma. Clin Cancer Res 18:1395–1403. https://
doi.org/10.1158/1078-0432.ccr-11-0850
Gbadamosi M, Meshinchi S, Lamba JK (2018) Gemtuzumab ozogamicin for treatment of newly
diagnosed CD33-positive acute myeloid leukemia. Future Oncol. https://doi.org/10.2217/fon-
2018-0325
Giusti RM, Shastri KA, Cohen MH, Keegan P, Pazdur R (2007) FDA drug approval summary:
panitumumab (Vectibix). Oncologist 12:577–583. https://doi.org/10.1634/theoncologist.12-5-
577
Goede V et al (2014) Obinutuzumab plus chlorambucil in patients with CLL and coexisting
conditions. N Engl J Med 370:1101–1110. https://doi.org/10.1056/NEJMoa1313984
Goldberg RM (2005) Cetuximab. Nat Rev Drug Discov Suppl:S10–11
Grillo-Lopez AJ (2002) Zevalin: the first radioimmunotherapy approved for the treatment of
lymphoma.Expert Rev Anticancer Ther 2:485–493. https://doi.org/10.1586/14737140.2.5.485
He LZ et al (2013) Agonist anti-human CD27 monoclonal antibody induces T cell activation and
tumor immunity in human CD27-transgenic mice. J Immunol 191:4174–4183. https://doi.org/
10.4049/jimmunol.1300409
Hsi ED et al (2008) CS1, a potential new therapeutic antibody target for the treatment of multiple
myeloma. Clin Cancer Res 14:2775–2784. https://doi.org/10.1158/1078-0432.ccr-07-4246
32 R. F. Graziano and J. J. Engelhardt

Jakobovits A (1998) The long-awaited magic bullets: therapeutic human monoclonal antibodies
from transgenic mice. Expert Opin Investig Drugs 7:607–614. https://doi.org/10.1517/
13543784.7.4.607
Jakubowiak AJ et al (2016) Cost-effectiveness of adding carfilzomib to lenalidomide and
dexamethasone in relapsed multiple myeloma from a US perspective J. Med Econ 19:1061–
1074. https://doi.org/10.1080/13696998.2016.1194278
Jones PT, Dear PH, Foote J, Neuberger MS, Winter G (1986) Replacing the
complementarity-determining regions in a human antibody with those from a mouse. Nature
321:522–525. https://doi.org/10.1038/321522a0
Kazandjian D et al (2016) FDA approval summary: Nivolumab for the treatment of metastatic
non-small cell lung cancer with progression on or after platinum-based chemotherapy.
Oncologist 21:634–642. https://doi.org/10.1634/theoncologist.2015-0507
Kerr DJ (2004) Targeting angiogenesis in cancer: clinical development of bevacizumab. Nat Clin
Pract Oncol 1:39–43. https://doi.org/10.1038/ncponc0026
Kim ES (2017) Avelumab: first global approval. Drugs 77:929–937. https://doi.org/10.1007/
s40265-017-0749-6
Kim YH et al (2018) Mogamulizumab versus vorinostat in previously treated cutaneous T-cell
lymphoma (MAVORIC): an international, open-label, randomised, controlled phase 3 trial.
Lancet Oncol. https://doi.org/10.1016/s1470-2045(18)30379-6
Klein C et al (2013) Epitope interactions of monoclonal antibodies targeting CD20 and their
relationship to functional properties. MAbs 5:22–33. https://doi.org/10.4161/mabs.22771
Ko K et al (2005) Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on
tumor-infiltrating Foxp3 + CD25 + CD4 + regulatory. T cells J Exp Med 202:885–891.
https://doi.org/10.1084/jem.20050940
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined
specificity. Nature 256:495
Kohrt HE et al (2012) Combination strategies to enhance antitumor ADCC. Immunotherapy
4:511–527. https://doi.org/10.2217/imt.12.38
Krejcik J et al (2016) Daratumumab depletes CD38 + immune regulatory cells, promotes T-cell
expansion, and skews T-cell repertoire in multiple myeloma. Blood 128:384–394
Leach DR, Krummel MF, Allison JP (1996) Enhancement of antitumor immunity by CTLA-4
blockade. Science 271:1734–1736
Lehmann B et al (2017) Tumor location determines tissue-specific recruitment of tumor-associated
macrophages and antibody-dependent immunotherapy response. Sci Immunol 2.pii: eaah6413.
https://doi.org/10.1126/sciimmunol.aah6413
Li F, Ravetch JV (2011) Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor
activities of agonistic CD40 antibodies. Science 333:1030–1034. https://doi.org/10.1126/
science.1206954
Li F, Ravetch JV (2012) Apoptotic and antitumor activity of death receptor antibodies require
inhibitory Fcgamma receptor engagement. Proc Natl Acad Sci U S A 109:10966–10971.
https://doi.org/10.1073/pnas.1208698109
Li F, Ravetch JV (2013) Antitumor activities of agonistic anti-TNFR antibodies require differential
FcgammaRIIB coengagement in vivo. Proc Natl Acad Sci U S A 110:19501–19506. https://
doi.org/10.1073/pnas.1319502110
Lonial S et al (2015) Elotuzumab Therapy for Relapsed or Refractory Multiple Myeloma. N Engl J
Med 373:621–631. https://doi.org/10.1056/NEJMoa1505654
Lu D et al (2003) Tailoring in vitro selection for a picomolar affinity human antibody directed
against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity. J Biol
Chem 278:43496–43507. https://doi.org/10.1074/jbc.m307742200
Lundin J et al (1998) CAMPATH-1H monoclonal antibody in therapy for previously treated
low-grade non-Hodgkin’s lymphomas: a phase II multicenter study. European Study Group of
CAMPATH-1H Treatment in Low-Grade Non-Hodgkin’s Lymphoma. J Clin Oncol 16:3257–
3263. https://doi.org/10.1200/jco.1998.16.10.3257
Role of FccRs in Antibody-Based Cancer Therapy 33

Mahne AE et al (2017) Dual roles for regulatory T-cell depletion and costimulatory signaling in
agonistic GITR targeting for tumor immunotherapy. Cancer Res 77:1108–1118. https://doi.org/
10.1158/0008-5472.CAN-16-0797
Maloney DG et al (1997) IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in
patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90:2188–2195
Markham A (2016) Elotuzumab: first global approval drugs 76:397–403. https://doi.org/10.1007/
s40265-016-0540-0
McKeage K (2016) Daratumumab: first global approval. Drugs 76:275–281. https://doi.org/10.
1007/s40265-015-0536-1
Morrison SL, Johnson MJ, Herzenberg LA, Oi VT (1984) Chimeric human antibody molecules:
mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S
A 81:6851–6855
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin g subclass activity through
selective Fc receptor binding. Science 310:1510–1512
Ogura M et al (2014) Multicenter phase II study of mogamulizumab (KW-0761), a defucosylated
anti-cc chemokine receptor 4 antibody, in patients with relapsed peripheral T-cell lymphoma
and cutaneous T-cell lymphoma. J Clin Oncol 32:1157–1163. https://doi.org/10.1200/jco.2013.
52.0924
Osterborg A et al (2016) Phase III, randomized study of ofatumumab versus physicians’ choice of
therapy and standard versus extended-length ofatumumab in patients with bulky
fludarabine-refractory chronic lymphocytic leukemia Leuk. Lymphoma 57:2037–2046.
https://doi.org/10.3109/10428194.2015.1122783
Overdijk MB et al (2015) Antibody-mediated phagocytosis contributes to the anti-tumor activity of
the therapeutic antibody daratumumab in lymphoma and multiple myeloma. In: MAbs, vol 2.
Taylor & Francis, pp 311–320
Park KU et al (2010) Gene expression analysis of ex vivo expanded and freshly isolated NK cells
from cancer patients. J Immunother 33:945–955. https://doi.org/10.1097/CJI.
0b013e3181f71b81
Pierpont TM, Limper CB, Richards KL (2018) Past, present, and future of rituximab—the world’s
first oncology monoclonal antibody therapy. Front Oncol 8:163. https://doi.org/10.3389/fonc.
2018.00163
Plitas G et al (2016) Regulatory T cells exhibit distinct features in human breast cancer. Immunity
45:1122–1134. https://doi.org/10.1016/j.immuni.2016.10.032
Poole RM (2014) Pembrolizumab: first global approval. Drugs 74:1973–1981. https://doi.org/10.
1007/s40265-014-0314-5
Poole RM, Vaidya A (2014) Ramucirumab: first global approval. Drugs 74:1047–1058. https://
doi.org/10.1007/s40265-014-0244-2
Ravetch JV, Lanier LL (2000) Immune inhibitory receptors Science 290:84–89
Salles G et al. (2012) Phase 1 study results of the type II glycoengineered humanized anti-CD20
monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients. Blood 119:5126–
5132. https://doi.org/10.1182/blood-2012-01-404368
Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, Korman AJ (2013)
Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of
intratumoral regulatory T cells. Cancer Immunol Res 1:32–42. https://doi.org/10.1158/2326-
6066.cir-13-0013
Serna-Gallegos TR, La-Fargue CJ, Tewari KS (2018) The ecstacy of gold: patent expirations for
Trastuzumab, Bevacizumab, Rituximab, and Cetuximab Recent Pat. Biotechnol 12:101–112.
https://doi.org/10.2174/1872208311666171122152131
Shields RL et al (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding
to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–
26740. https://doi.org/10.1074/jbc.m202069200
Shirley M (2017) Olaratumab: first global approval. Drugs 77:107–112. https://doi.org/10.1007/
s40265-016-0680-2
34 R. F. Graziano and J. J. Engelhardt

Simpson TR et al (2013) Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines

the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 210:1695–1710. https://doi.
org/10.1084/jem.20130579
Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV (2012) Mouse model recapitulating human
Fcgamma receptor structural and functional diversity. Proc Natl Acad Sci U S A 109:6181–
6186. https://doi.org/10.1073/pnas.1203954109
Stagg J, Divisekera U, Duret H, Sparwasser T, Teng MW, Darcy PK, Smyth MJ (2011)
CD73-deficient mice have increased antitumor immunity and are resistant to experimental
metastasis. Cancer Res 71:2892–2900. https://doi.org/10.1158/0008-5472.can-10-4246
Syed YY (2017) Durvalumab: first global approval. Drugs 77:1369–1376. https://doi.org/10.1007/
s40265-017-0782-5
Tai YT et al (2008) Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell
adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu
Blood 112:1329–1337. https://doi.org/10.1182/blood-2007-08-107292
Tam SH et al (2017) Functional, biophysical, and structural characterization of human IgG1 and
IgG4 variants with ablated immune functionality. Antibodies 6:1–34
Taylor LD et al (1992) A transgenic mouse that expresses a diversity of human sequence heavy
and light chain immunoglobulins. Nucleic Acids Res 20:6287–6295
Tobinai K et al (2011) Phase I study of LY2469298, an Fc-engineered humanized anti-CD20
antibody, in patients with relapsed or refractory follicular lymphoma. Cancer Sci 102:432–438.
https://doi.org/10.1111/j.1349-7006.2010.01809.x
Tuaillon N, Taylor LD, Lonberg N, Tucker PW, Capra JD (1993) Human immunoglobulin
heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma
transcripts. Proc Natl Acad Sci U S A 90:3720–3724
Tvito A, Rowe JM (2017) Inotuzumab ozogamicin for the treatment of acute lymphoblastic
leukemia. Expert Opin Biol Ther 17:1557–1564. https://doi.org/10.1080/14712598.2017.
1387244
van der Veer MS et al (2011) The therapeutic human CD38 antibody daratumumab improves the
anti-myeloma effect of newly emerging multi-drug therapies. Blood Cancer J 1:e41
Wasiuk A et al (2017) CD27-mediated regulatory T cell depletion and effector T cell costimulation
both contribute to antitumor efficacy. J Immunol 199:4110–4123. https://doi.org/10.4049/
jimmunol.1700606
Weiner LM, Dhodapkar MV, Ferrone S (2009) Monoclonal antibodies for cancer immunotherapy.
Lancet 373:1033–1040. https://doi.org/10.1016/S0140-6736(09)60251-8
Weng WK, Levy R (2003) Two immunoglobulin G fragment C receptor polymorphisms
independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol
21:3940–3947. https://doi.org/10.1200/JCO.2003.05.013
White AL et al (2011) Interaction with FcgammaRIIB is critical for the agonistic activity of
anti-CD40 monoclonal antibody. J Immunol 187:1754–1763. https://doi.org/10.4049/
jimmunol.1101135
Xu Y et al (2003) Fc gamma Rs modulate cytotoxicity of anti-Fas antibodies: implications for
agonistic antibody-based therapeutics. J Immunol 171:562–568
Yamane-Ohnuki N, Satoh M (2009) Production of therapeutic antibodies with controlled
fucosylation. MAbs 1:230–236
Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, Forero-Torres A
(2010) Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med
363:1812–1821. https://doi.org/10.1056/nejmoa1002965
Anti-inflammatory Activity of IgG-Fc

Christopher Beneduce, Elma Kurtagic and Carlos J. Bosques

Contents

1 Introduction.......................................................................................................................... 36
2 Clinical Relevance of IgG Glycosylation in Humans ........................................................ 38
3 Role of Sialylation in the Anti-inflammatory Activity of Intravenous
Immunoglobulin................................................................................................................... 41
4 Impact of Sialylation on IgG Structure and Anti-inflammatory Activity .......................... 43
5 Impact of IgG Sialylation on the Adaptive Immune System............................................. 46
6 Modulation of Autoantibody Pathogenicity by Sialylation ................................................ 48
7 Anti-inflammatory Activity of Multivalent IgG-Fc ............................................................ 51
8 Conclusion ........................................................................................................................... 53
References .................................................................................................................................. 54

Abstract Over 80 different autoimmune disorders have been identified. A common

denominator across most of these disorders is the presence of pathogenic autoan-
tibodies. The pathogenic and inflammatory nature of antibodies is well accepted,
and over the last three decades, evidence in humans and rodent models has revealed
that antibodies can induce anti-inflammatory activities. The discovery of the rela-
tionship between immunoglobulin G (IgG) glycovariants and disease activity in
autoimmune patients has provided insight into the structural and functional char-
acteristics of IgG associated with its pro- and anti-inflammatory activity. In this
chapter, we discuss evidence of the anti-inflammatory nature of IgG and the
mechanisms by which this activity is exerted. Current clinical evidence of this
anti-inflammatory activity is also discussed.

C. Beneduce
E. Kurtagic
C. J. Bosques (&)

Research Department, Momenta Pharmaceuticals, Cambridge, USA
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2019) 423: 35–62

https://doi.org/10.1007/82_2019_148
© Springer Nature Switzerland AG 2019
Published online: 22 February 2019
36 C. Beneduce et al.

1 Introduction

The role of immunoglobulin G (IgG) in the pathogenesis of many autoimmune

disorders such as systemic lupus erythematosus (SLE), myasthenia gravis, immune
thrombocytopenia, and many other diseases is well defined. Autoantibodies can
form immune complexes with their cognate antigen and trigger inflammatory
processes through their interaction with cellular Fc gamma receptors (FccRs) and
complement. It is now understood that IgG can also have anti-inflammatory
activities. The pro- and anti-inflammatory activity of IgG can be modulated by
different properties of the Ig such as the IgG subclass, glycosylation, and valency.
The composition of the Fc domain, although it is considered the invariant region of
the IgG molecule, is highly heterogeneous. In addition to the four distinct sub-
classes of human IgG (IgG1, IgG2, IgG3, and IgG4), the Fc domain exhibits
additional diversity in individual allotypes and glycosylation states. Furthermore,
IgG can form complexes with target ligands, antigens, cells, or particles, leading to
different degrees of Fc valency. The combination of subclass, allotype, glycosy-
lation, and valency leads to a substantial amount of heterogeneity in Fc structure
(Arnold et al. 2007; Vidarsson et al. 2014), which can influence the interaction of
IgG with its receptors and ultimately influence its function. Effector functions of
human IgG are carried out by complement and by the FccRs. Humans express
several activating FccRs, including FccRI, FccIIa, FccIIc, FccIIIa, and the lone
inhibitory FccRIIb. Activating FccRs signal via a highly conserved immunore-
ceptor tyrosine-based activation motif. FccRIIa and FccRIIc intrinsically express
this motif in their c chain, whereas FccRI and FccRIIIa rely on the common FcR c
chain (c2) to trigger activating signals. In contrast, FccRIIb contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its a chain. Activating
FccRs are commonly found on myeloid cells, dendritic cells (DCs), natural killer
(NK) cells, and neutrophils. The inhibitory FccRIIb is typically expressed together
with activating FccRs. B cells are a notable exception as they express FccRIIb only
and do not express activating FccRs.
FccRs exhibit different affinities for IgG subclasses. FccRI can bind all human
IgG subclasses with relatively high affinity, with the exception of IgG2. FccRI has
the unique ability to interact with monomeric IgG. FccRIIa, the most widely
expressed receptor, can bind all IgG subclasses, and it is the only FccR to bind to
IgG2a. IgG3 binding affinity is highest for FccRIIa, followed by IgG1, with weak
but measurable binding affinity to IgG2 and IgG4. Variants of FccRIIa have been
described to modulate affinity for IgG2a, with the 131R variant having higher
affinity than the 131H variant. IgG binding affinity to FccRIIb is weak for all
subclasses, and affinity for IgG2 is the lowest. FccRIIIa, which binds all subclasses,
has two well-described variants: FV158 and V158. V158 has a greatly increased
affinity for all IgG subclasses and a particularly high affinity for IgG3. FccRIIIb,
which is mostly expressed on neutrophils, binds IgG1 and IgG3, with little binding
to IgG2 and IgG4. FccRIIIb also has two allotypic variants, denoted NA1 and NA2.
Anti-inflammatory Activity of IgG-Fc 37

The NA1 allotype exhibits higher binding affinity to IgG1 and IgG3 subclasses and
an increased ability to phagocytose IgG1- and IgG3-opsonized particles (Bruhns
et al. 2009; Vidarsson et al. 2014).
Overall, human and mouse FccRs, with the exception of FccRI, have low
affinity for monomeric IgG. They preferentially bind IgG in the form of immune
complexes. Immune complex multivalent binding results in multimeric clustering
of FccRs and signaling cascades that involves spleen tyrosine kinase (SYK),
phospholipase C (PLC), and calcium release, ultimately resulting in activation of
the ERK/AKT pathways. Depending on the cell type, signaling via activating
FccRs results in cellular proliferation, generation of reactive oxygen species (ROS),
antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular
cytotoxicity (ADCC), and cytokine production. Conversely, immune complex
interaction with FccRIIb results in quelling of the proinflammatory activities of the
activating FccRs. This process is mediated by the FccRIIb ITIM domain and the
SH2-containing inositol phosphatase (SHIP). It is well accepted that FccRIIb
counterbalances signals triggered by the activating FccRs in cells of the innate
immune system and modulates signaling of the B-cell receptor (BCR) on B cells. In
2000, Clynes et al. demonstrated that FccRIIb could modulate the in vivo ADCC
activity of the anti-CD20 antibody rituximab (Clynes et al. 2000). Co-ligation of the
inhibitory FccRIIb with activating FccRs was shown to inhibit the activation and
maturation of dendritic cells and inhibit FccRIIa-mediated cytokine release
(Boruchov et al. 2005). Co-ligation of FccRIIb and the B-cell receptor on B cells is
known to inhibit BCR-mediated signaling in a SHIP-mediated manner resulting in
reduced B-cell activation and proliferation (Chu et al. 2008; Heyman 2003; Kiener
et al. 1997; Ono et al. 1997).
IgG glycosylation is crucial in modulating the in vivo activity of IgG. All IgG
molecules contain a single conserved N-linked glycosylation site (Asn-297) in each
of the constant heavy two (CH2) domains, critical for maintaining their
proinflammatory and anti-inflammatory effector functions (Arnold et al. 2007;
Schwab and Nimmerjahn 2013). The sugar moiety attached to Asp-297 predomi-
nantly consists of an octasaccharide biantennary structure, composed of four N-
acetylglucosamines (GlcNac), three mannoses, and one fucose, which may contain
terminal galactose or sialic acid residues. These glycans help maintain the quater-
nary structure and the stability of the Fc (Mimura et al. 2000, 2001) and are vitally
involved in FccR binding through maintenance of an open conformation of the two
heavy chains (Jefferis and Lund 2002; Sondermann et al. 2001). Deglycosylated
IgG has been shown to lose nearly all significant FccR and C1q binding affinity
(Nose and Wigzell 1983). Evidence that deglycosylated IgG antibodies are unable
to mediate in vivo inflammatory responses accounts for the requirement of the
glycans for FccR binding (Nimmerjahn and Ravetch 2006). Fc glycans are critical
for maintaining both the proinflammatory and the anti-inflammatory effector
functions of IgG (Arnold et al. 2007). Branching fucose residues at the core of the
biantennary glycan have had a significant effect on the IgG-Fc binding affinity for
FccRIIIa, with afucosylated IgG having a much higher affinity for the receptor
38 C. Beneduce et al.

(Ferrara et al. 2011; Houde et al. 2010; Nimmerjahn and Ravetch 2005; Shields
et al. 2002; Shinkawa et al. 2003).
The terminal sugar residues in the IgG-Fc can consist of N-acetylglucosamine,
galactose, or sialic acid. Sialylated IgG glycoforms have decreased binding affinity
to several FcRs (Kaneko et al. 2006b) and can adversely impact IgG-Fc
proinflammatory function (Scallon et al. 2007). In addition, changes in Fc glyco-
sylation have been shown to modulate complement-dependent cytotoxicity where
increasing amounts of terminal galactose increase the binding affinity of the com-
plement C1q to the IgG-Fc (Raju 2008).

2 Clinical Relevance of IgG Glycosylation in Humans

A common feature of most autoimmune diseases is the formation of immune

complexes between pathogenic IgG autoantibodies and cognate antigens. These
immune complexes activate inflammatory effector cells expressing FccRs and the
complement cascade, contributing to tissue damage. Apart from antibody isotype
and subclass, the function of autoantibodies is in part modulated by their Fc
N-linked glycans. An example of the role of autoantibody glycosylation in disease
pathogenicity was provided by Lood et al. The authors demonstrated that enzymatic
deglycosylation of SLE immune complexes in vitro abolished their proinflamma-
tory properties (Lood et al. 2012). Immune complex deglycosylation eliminated
several important proinflammatory properties of immune complexes that are
important in SLE pathogenesis: FccR-mediated phagocytosis, interferon alfa
(IFN-a) production, polymorphonuclear leukocytes chemotaxis, and classical
pathway of complement activation. Furthermore, deglycosylation of immune
complexes from SLE patients also had a direct effect on the molecular structure of
immune complexes, causing decreased immune complex size, emphasizing the
crucial role of Fc glycans for immune complex-mediated pathological signals.
In contrast to studies that link hypoglycosylation of immune complexes and the
resulting loss of proinflammatory activity, results of other studies have shown that
glycoforms that specifically lack terminal sialic acid or galactose residues (G0
glycoforms) might have increased proinflammatory activity. Original observations
from the 1980s showed that IgG glycosylation differs in patients with rheumatoid
arthritis, with decreased Fc galactosylation and sialylation compared with normal
individuals (Parekh et al. 1985). Since then, a difference in the glycosylation profile
of total IgG has been reported in patients with various other autoimmune diseases,
including SLE, inflammatory bowel disease, myasthenia gravis, ankylosing
spondylitis, primary Sjögren’s syndrome, psoriatic arthritis, and multiple sclerosis
(Dube et al. 1990; Selman et al. 2011; Trbojevic Akmacic et al. 2015; Vuckovic
et al. 2015; Watson et al. 1999; Wuhrer et al. 2015a). In addition to their association
with autoimmune diseases, agalactosylated polyclonal antibodies are associated
Anti-inflammatory Activity of IgG-Fc 39

with increased inflammation in infectious diseases such as HIV (Ackerman et al.

2013). Besides the evidence of disparate IgG glycans in different autoimmune
disease, therapeutic treatments and changes in the physiologic state can also result
in differential IgG glycosylation. Namely, treatment of patients with rheumatoid
arthritis with methotrexate can lead to increased levels of IgG galactosylation and
hence a decrease in the abundance of agalactosylated IgG-G0 glycoforms (Pasek
et al. 2006)). Furthermore, IgG-G0 glycoforms were decreased in women with
rheumatoid arthritis during pregnancy, correlating with reduced incidence of flares
(Rook et al. 1991; van de Geijn et al. 2009). Additional evidence suggests that
serum levels of IgG-G0 glycans increase shortly before the onset of disease in
patients with rheumatoid arthritis (Rombouts et al. 2015). Presumably, a window of
time for initiation of preventative treatment could be estimated based on the
abundance of G0 glycoforms. More recently, it was shown that treatment with
estrogen in postmenopausal women with rheumatoid arthritis significantly
increased IgG-Fc sialylation (Engdahl et al. 2018).
One of the best examples of specific monosaccharide residue impacting the
proinflammatory activity of IgG is the core N-linked glycan fucose. Glycoforms
lacking core fucose residues have been shown to have altered affinities for indi-
vidual FccRs (Shields et al. 2002). Defucosylation of IgG resulted in 10- to 50-fold
enhanced affinity for mouse FccRIV and human FccRIII (Ferrara et al. 2011;
Nimmerjahn and Ravetch 2005; Shields et al. 2002; Shinkawa et al. 2003).
Consequently, defucosylation in the Fc domain of antibodies leads to increased
antibody-dependent cell-mediated cytotoxic activity (Ferrara et al. 2011;
Nechansky et al. 2007). Furthermore, results of a series of studies showed enhanced
cytotoxic and phagocytic activity in mouse tumor and autoimmune models using
these IgG glycovariants, indicating the importance of glycoengineering of anti-
bodies for improved clinical effectiveness. In fact, therapeutic antibodies that lack
fucose residues have shown enhanced tumor killing in cancer patients and have
been approved as therapy for different B-cell cancers. Although there is abundant
evidence of strongly increased IgG-G0 glycoforms in patients during active
autoimmune disease, fucosylated IgG seems to remain stable during inflammation
and vaccination in mice and humans (Kao et al. 2017; Kemna et al. 2017;
Rombouts et al. 2015; Scherer et al. 2010; Selman et al. 2011; Sjowall et al. 2015).
However, there are recent reports of disparate fucosylation levels of alloantibodies
in hemolytic disease of the fetus or newborn (HDFN) (Kapur et al. 2014a) and fetal
and neonatal alloimmune thrombocytopenia (FNAIT) (Kapur et al. 2014b;
Sonneveld et al. 2016; Wuhrer et al. 2009), diseases that arise because of maternal
alloimmunization against paternally inherited red blood cell antigen Rh-D or pla-
telets, respectively. These alloantibodies are transported across the placenta and
result in severe fetal anemia or thrombocytopenia. Systematical analysis of
IgG-derived glycopeptides from 70 anti-D alloantibodies from pregnant women
revealed a decrease in Fc-fucosylation (Kapur et al. 2014b), which correlated sig-
nificantly with FccRIIIa-mediated ADCC. Furthermore, low Fc-fucosylation
40 C. Beneduce et al.

correlated with severe fetal anemia (Sonneveld et al. 2017). Thus, even though IgG
fucosylation levels are generally stable during various autoimmune diseases, there
is a strong correlation between antibody fucosylation and disease severity during
pregnancy, and those antibodies play an active role in disease pathogenicity.
The anti-inflammatory activity of sialylated IgG is evident in patients as several
studies have reported changes in IgG sialylation levels during inflammation and
presence of autoimmune diseases. For instance, patients with rheumatoid arthritis,
granulomatosis with polyangiitis (GPA), antiphospholipid syndrome (APS), vas-
culitis, and SLE have low serum levels of IgG or low autoantibody sialylation (Espy
et al. 2011; Fickentscher et al. 2015; Kemna et al. 2017; Vuckovic et al. 2015; Wuhrer
et al. 2015b). One particular study reported significantly higher sialylation of IgG
recognizing anti-2-glycoprotein 1 autoantibodies (2GP1-IgG) isolated from the sera
of healthy children and asymptomatic adults compared with that of patients with
clinically apparent anti-phospholipid syndrome (APS) (Fickentscher et al. 2015).
Another group demonstrated that histone IgG autoantibodies purified from SLE
patients displayed significantly lower sialylation than total IgG of the same patients,
suggesting that the SLE utoantibodies contain proinflammatory activities
(Magorivska et al. 2016). In patients with GPA, characterized by the presence of
antineutrophil cytoplasmic antibodies (ANCA) against proteinase-3 (PR3), patho-
genic proteinase 3 autoantibodies are less sialylated in patients with active Wegener’s
vasculitis (GPA) than in those with inactive disease (Espy et al. 2011). The authors
further demonstrated that purified anti-PR3 antibodies from patients with active
disease were proinflammatory, and enzymatic desialylation of anti-PR3 antibodies
from patients with inactive disease restored pathogenic activity (Espy et al. 2011).
This study provided direct evidence of the importance of autoantibody sialylation as it
pertains to the modulation of antibody activity in an autoimmune disease.
Furthermore, it was shown that total IgG-Fc of patients with severe ANCA-associated
vasculitis (AAV) exhibits lower levels of galactosylation, sialylation, and bisecting N-
acetylglucosamine (GlcNAc) compared with healthy controls (Wuhrer et al. 2015b).
This finding was more pronounced for PR3-ANCA antibodies than with total IgG.
Potential use of circulating levels of sialylated IgG as a disease biomarker is
noteworthy. Serum levels of sialylated IgG have decreased shortly before disease
relapses, similar to galactosylated IgG, showing promise as a predictive biomarker
(Kemna et al. 2017; Rombouts et al. 2015). Patients with GPA exhibited low
galactosylation or low sialylation of total IgG1 and were highly prone to relapse
after ANCA increase. When relapsing patients were examined, total IgG1 sialyla-
tion decreased from the time of the PR3-ANCA increase to the relapse, while the
glycosylation profile remained similar in nonrelapsing patients (Kemna et al. 2017).
An intriguing observation is that low sialylation was observed in total IgG1 and not
in antigen-specific PR3, and low sialylation of total IgG1 predicted disease reac-
tivation in patients with GPA who experienced ANCA increase during follow-up.
Along the same lines, studies have shown a reduction in sialylation of IgG in
chronic inflammatory demyelinating polyneuropathy (CIDP), and this reduction
Anti-inflammatory Activity of IgG-Fc 41

correlated with clinical severity of the disease. This observation implies potential
use of sialylated IgG as a biomarker to monitor disease severity in CIDP (Wong
et al. 2016). Restoring serum antibody sialylation correlated with treatment
response in patients with Guillain-Barre syndrome (Fokkink et al. 2014) and
Kawasaki disease (Ogata et al. 2013), where lower sialylation of endogenous IgG
predicted resistance to therapy (Ogata et al. 2013). Taken together all the evidence
summarized above, monitoring of sialylated IgG antibodies during different phases
of an antibody-mediated disease may have diagnostic and prognostic potential and
could help optimize the treatment of patients with autoimmune diseases.

3 Role of Sialylation in the Anti-inflammatory Activity

of Intravenous Immunoglobulin

Despite the decade-long clinical effectiveness of intravenous immunoglobulin

(IVIg) in treating inflammatory and autoimmune diseases, the mechanism of action
of IVIg has remained enigmatic. IVIg is effective in a wide range of diseases in
which many mechanisms of the underlying disease are not well understood, further
complicating the understanding of all the details of IVIg anti-inflammatory mech-
anisms. Moreover, the fact that IgG can form many different binding interactions
through antigen binding and Fc domains adds to the complexity. Many hypotheses
have been proposed to explain the reasons for the seemingly paradoxical activity of
high-dose IVIg. One theory attributes IVIg activity to the polyclonal binding
specificities that are encoded in the variable region of the administered antibodies,
which in turn may counteract the activity of autoantibodies and inflammatory
mediators, as shown in toxic epidermal necrolysis (Viard et al. 1998). However,
intact IVIg and its Fc fragments have almost identical activity in many animal
models (Bruhns et al. 2003; Kaneko et al. 2006b; Samuelsson et al. 2001) and in the
clinical treatment of idiopathic thrombocytopenic purpura (ITP) (Debre et al. 1993).
Therefore, the emergence of evidence attributing the anti-inflammatory activity of
IVIg to its Fc portion has promoted further investigation and focus on Fc-mediated
mechanisms. For example, saturation of the neonatal FcRn—leading to increased
catabolism of pathogenic autoantibodies—has been proposed as one of the primary
mechanisms of IVIg (Akilesh 2004; Hansen and Balthasar 2002; Li et al. 2005).
However, the evidence to support this proposal is limited because of the difficulty in
experimentally modeling FcRn dependence since removing FcRn in mice results in
rapid clearance of exogenous autoantibodies and in strongly reduced serum IgG
levels (Vaccaro et al. 2005).
Another proposed mechanism is the saturation of activating FccRs and prevention
of pathogenic autoantibodies from triggering FccRs activation, thereby limiting their
pathogenic potential. For example, monoclonal antibodies that block activating
FccRs have mimicked the anti-inflammatory properties of IVIg (Siragam et al. 2006).
42 C. Beneduce et al.

Furthermore, the dimeric and aggregate fraction in IVIg may facilitate multivalent
avid interactions with low-affinity FccRs and inhibit their activation by pathogenic
immune complexes. Indeed, it has been experimentally shown that the protective
effects of IVIg can be mimicked by the injection of opsonized red blood cells or by
soluble immune complexes (Siragam et al. 2005). More recently, recombinant mul-
tivalent Fc molecules with avid binding to low-affinity FccRs have also demonstrated
similar anti-inflammatory properties to IVIg (Zuercher et al. 2016).
Another proposed mechanism of action for IVIg is the induction of
anti-inflammatory activities by altering the relative surface expression of activating
and inhibiting FccRs in favor of the inhibitory FccRIIb, thereby increasing the
signal threshold necessary to activate immune cells (Anthony et al. 2011; Schwab
and Nimmerjahn 2013). As it was long postulated, immune complex-induced
inflammatory responses are regulated by the relative expression of activating versus
inhibitory FccRs on effector cells, dictating the threshold of immune
complex-induced inflammation (Ravetch and Clynes 1998). In fact, it was
demonstrated in animal models of ITP, autoimmune hemolytic anemia, rheumatoid
arthritis, and nephrotoxic nephritis that the ability of IVIg to protect mice from
pathogenic IgG was dependent on FccRIIb (Akilesh 2004; Crow et al. 2003; Huang
et al. 2010; Kaneko et al. 2006a, b; Samuelsson et al. 2001). Similarly, results of
several studies showed that FccRIIb is upregulated on innate immune effector cells
and B cells after IVIg treatment in mice (Bruhns et al. 2003; Kaneko et al. 2006b;
Samuelsson et al. 2001). Furthermore, this was confirmed in patients with CIDP, in
whom FccRIIb expression levels on B cells were impaired. Interestingly, FccRIIb
protein expression was upregulated on monocytes and B cells after clinically
effective IVIg therapy (Tackenberg et al. 2009).
Given all the evidence of anti-inflammatory activities of high-dose IVIg treat-
ment, it has become apparent that the preparations of IVIg from normal human
donors indeed contain a small fraction of active therapeutic that is not necessarily an
oligomer. The first study to strongly correlate Fc glycosylation and biological
activity of IVIg was reported by Kaneko 2006 and was later substantiated by results
of several studies in a variety of animal models of autoimmune disease (Anthony
et al. 2008a; Kaneko et al. 2006b; Schwab et al. 2012; Tackenberg et al. 2009).
Biochemical characterizations of IVIg preparations that display anti-inflammatory
activities in animal models pointed to the absolute requirement for the Fc fragment
and its N-linked biantennary glycan attached at the Asn-297 (Kaneko et al. 2006b).
The authors demonstrated that highly sialylated forms of monoclonal mouse IgG
have reduced affinity for FccR and cytotoxic function. Furthermore, the
anti-inflammatory activity of IVIg was shown to be mediated mainly by a small
fraction of antibodies containing terminal sialic acid on their oligosaccharide
structures. Interestingly, a stronger protective effect of the IVIg fraction enriched for
sialic acid was also shown. Enzymatic removal of the sialic acid residues abrogated
the anti-inflammatory activity of IVIg in a mouse model of rheumatoid arthritis.
These studies also showed that sialylation of the IVIg Fc fragment was sufficient for
anti-inflammatory activities (Kaneko et al. 2006b).
Anti-inflammatory Activity of IgG-Fc 43

4 Impact of Sialylation on IgG Structure

and Anti-inflammatory Activity

Work by Ravetch and others has helped shed light on how different IgG glycoforms
can impact the anti-inflammatory activity of IgG (Anthony et al. 2008a; Crow et al.
2003; Kaneko et al. 2006b; Samuelsson et al. 2001; Schwab et al. 2014). Structural
studies revealed that the IgG-Fc may adopt “open” or “closed” confirmations
depending on the glycosylation state. For example, the Fc glycans at position 297
have been shown to contribute to an “open” conformation of the IgG-Fc in which
the CH2 domains are separated by larger distances than in deglycosylated Fc
structures (Krapp et al. 2003). Conversely, the deglycosylated IgG displays a
conformation in which the CH2 region adopts a “closed” state (Feige et al. 2009).
More recent crystallographic work highlighted a higher conformational hetero-
geneity of the CH2 domain in sialylated IgG-Fc protein fragments. The specific
interactions between the N297 glycans and the amino acid backbone of the CH2
domain are interrupted upon terminal sialylation, which can help explain these
conformational changes (Ahmed et al. 2014). It was demonstrated how a specific
point mutation (F241A) in the Fc domain could recapitulate the flexibility seen with
sialylated glycoforms (Fiebiger et al. 2015). These structural characterizations may
help explain how earlier studies showed that highly sialylated IgG significantly
reduced affinity for the canonical FccRs (Kaneko et al. 2006b). These changes in Fc
structure could also help explain how highly sialylated IgG shows reduced ability to
induce effector functions such as ADCC (Scallon et al. 2007). Some findings
suggest otherwise, where highly sialylated species were unchanged or only slightly
reduced in their ability to bind activating FccRs (Li et al. 2017; Subedi and Barb
2016). One study suggested that Fc sialylation can also modulate an antibody Fab
binding to its cognate antigen via altered flexibility of the hinge region. This
reduced flexibility may restrict the antibody to monovalent Fab binding to its target,
thus reducing its apparent affinity (Scallon et al. 2007).
In addition to modulating classic FccR binding, Fc sialylation can increase IgG
affinity for type II FcRs including DC-SIGN and CD23 (Anthony et al. 2008a;
Sondermann et al. 2013). DC-SIGN (Dendritic Cell-Specific ICAM3-Grabbing
Non-Integrin), a C-type Lectin, is found most commonly on the surface of dendritic
cells and macrophages. It has been shown to recognize bacterial and viral
pathogen-associated molecular patterns and is involved in HIV pathogenesis
(Gringhuis et al. 2014; Mogensen et al. 2010). The murine analog of DC-SIGN,
SIGN-R1, was shown to be necessary for the anti-inflammatory activity of IVIg in
the KBX/N mouse model of arthritis (Anthony et al. 2008a). The activity of IVIg
could be restored in SIGN-R1−/− mice when reconstituted with human DC-SIGN.
Although SIGN-R1 is not expressed on dendritic cells in mice, it is highly
expressed on a population of CSF1-dependent macrophages that reside in the
marginal zone of the spleen (Anthony et al. 2008a; Schwab and Nimmerjahn 2013).
In 2003, Bruhns et al. had shown that CSF1-dependent macrophages were
responsible for the in vivo activity of IVIg. Studies using op/op mice deficient in
44 C. Beneduce et al.

CSF1 implied that IVIg induced FccRIIb upregulation through an indirect mech-
anism. It was suggested that IVIg may interact with CSF1-dependent regulatory
cells, which in turn leads to the upregulated expression of FccRIIb on separate
CSF1-independent effector cells (Bruhns et al. 2003). Work by Anthony and col-
leagues in 2011 presented a unified hypothesis to help reconcile IVIg requirements
for Fc sialylation, FccRIIb, and SIGN-R1 for its anti-inflammatory activity in
various models of autoimmunity. Using the KBX/N model, the researchers showed
how IVIg, via interaction with DC-SIGN-positive splenic myeloid cells, caused the
release of interleukin (IL) 33, which in turn induced the production of IL-4. IL-4
was then shown to be involved in upregulation of FccRIIb on effector macrophages,
thereby increasing their threshold of immune complex-mediated activation. Mice
deficient in the IL-4 receptor were refractory to IVIg therapy, and systemic
administration of IL-33 or IL-4 suppressed serum-induced arthritis. The source of
IL-4 in these mice was determined to be basophils, which produce Il-4 in response
to IL-33 stimulation (Anthony et al. 2011). Figure 1 summarizes the mechanism
proposed by Ravetch and coworkers to explain the requirement of Fc sialylation for
the anti-inflammatory activity of IVIg. This study laid the groundwork for under-
standing how intrinsic sialylated IgG could help maintain immune homeostasis.
Both IVIg and sialylated IVIg, but not unsialylated IVIg, upregulated IL-33 mRNA
in spleens of treated animals, and sialylated IVIg was more potent in a model of
antiganglioside antibody-mediated inhibition of axon regeneration, supporting the
previously proposed mechanisms (Zhang et al. 2016).
The question of whether this particular anti-inflammatory mechanism of IVIg
plays a role in human patients is unclear given that marginal zone macrophages are
absent in the human spleen (Steiniger et al. 1997). Furthermore, some ITP patients
who underwent splenectomies still respond to IVIg therapy (Cines and Bussel
2005), and studies in ITP mouse models demonstrated that the inhibitory effects of
sialylated IVIg were not impaired after splenectomy (Leontyev et al. 2012; Schwab
et al. 2012), suggesting a potential different pathway in humans or in specific
disease settings. Schwab et al. showed that, in addition to IVIg activity not being
impaired in mice that underwent splenectomy, neither the activating FccRs FccRI
and FccRIII nor the cytokines requiring the common cytokine c chain or the IL-33
receptor were involved in this anti-inflammatory pathway suppressing murine
ITP. However, IVIg activity was still fully dependent on the terminal sialic acid and
SIGN-R1, most likely on a cell type outside the spleen (Schwab et al. 2012).
Perhaps IVIg mechanisms might be different depending on whether IVIg is
administered before induction of autoimmune disease or inflammation (such is the
case in the majority of the reported studies) versus IVIg administration during acute
autoimmune disease. Recent studies addressed this question and showed that, under
therapeutic conditions in mouse models of ITP, inflammatory arthritis, and the
autoimmune skin blistering disease epidermolysis bullosa acquisita, IVIg activity
was fully dependent on IgG sialylation and FccRIIb. However, SIGN-R1 was only
partially required, suggesting that select pathways may be involved in mediating
IVIg activity when treating established autoimmune disease (Schwab et al. 2014).
Anti-inflammatory Activity of IgG-Fc 45

DCs or
SIGN-R1+ / DC-SIGN+ Sialylated IgG Asialylated IgG Autoantibody
Regulatory immune complex
macrophages

Activating Fc R Inhibitory DC-SIGN CD23 or other

Fc RIIb type II FcR

IL-33

Basophil

Unidentified
IL-4 immune cell types

Upregulation of Effector
inhibitory Fc RIIb M

NO INFLAMMATION

Fig. 1 Schematic representation of mechanism proposed by Ravetch and coworkers to explain the
requirement of Fc sialylation for IVIg anti-inflammatory activity (Anthony et al. 2008b; Bruhns
et al. 2003; Sondermann et al. 2013; Maamary et al. 2017) Ig, immunoglobulin; IL, interleukin

However, the exact pathways during patient treatment with IVIg were not addressed
in those studies.
Some recent studies have helped elucidate whether this T-helper type 2 (TH2)
cascade occurs in human patients treated with IVIg. IVIg studies in humans have
shown that high-dose IVIg does induce IL-33 and IL-4 in patients with various
inflammatory myopathies (Schwab et al. 2014; Tjon et al. 2014). Similar results
with IL-33 were seen in IVIg-treated patients with Guillain-Barre syndrome, but
this was not a predictor of therapeutic success (Maddur et al. 2017). The study by
Tjon et al. aimed to address the variations between mice and humans and
demonstrated that the IL-33 TH2 pathway by which IVIg inhibits myeloid cells in
mice may also be used in humans but with slight variations (Tjon et al. 2014).
Namely, instead of FccRIIb upregulation, FccRIIa was downregulated on circu-
lating myeloid dendritic cells (mDCs) from IVIg-treated patients while FccRIIb
46 C. Beneduce et al.

expression remained unchanged. In vitro, IL-4 induced this change in FcR

expression on purified mDCs. Although not in line with observations in mice of
FccRIIb upregulation, the change in the ratio of activating and inhibitory Fc
receptors may produce the same result of higher threshold for immune
complex-mediated activation of these cells. The source of IL-33 in humans remains
undiscovered. One study did note an increased expression of IL-33 mRNA in
human lymph node cells and monocyte-derived macrophages in the presence of
IVIg and LPS, but this effect was independent of DC-SIGN (Tjon et al. 2014). More
controlled studies with IVIg in the clinic are necessary to further elucidate this TH2
cascade in human patients.
Further evidence of the crucial role of Fc sialylation in IVIg efficacy was
demonstrated more recently, where tetra-Fc-sialylated IVIg enhanced
anti-inflammatory activity up to 10-fold higher than IVIg across different animal
models (Washburn et al. 2015). Specifically, the tetra-Fc-sialylated IVIg was more
potent than the parent IVIg product when dosed prophylactically in a model of
collagen antibody-induced arthritis (CAIA) and a murine skin blistering model.
Therapeutic anti-inflammatory benefit with the tetra-Fc-sialylated IVIg treatment at
the time of active inflammation was also shown in murine K/BxN-induced arthritis
and ITP models. One study also extended the importance of IVIg sialylation in bone
loss in patients with rheumatoid arthritis. Desialylated immune complexes
enhanced osteoclastogenesis in vivo and in vitro. Mice treated with the sialic acid
precursor N-acetylmannosamine, which increases IgG sialylation, were less sus-
ceptible to inflammatory bone loss, suggesting a protective role of sialylated IgG in
autoimmune-mediated bone loss (Harre et al. 2015).

5 Impact of IgG Sialylation on the Adaptive Immune

System

The observation that T cells, purified from IVIg-treated nonimmunodeficient chil-

dren, significantly suppressed pokeweed mitogen-induced B-cell and T-cell pro-
liferation provided the first indication that IVIg could modulate the adaptive
immune system (Durandy et al. 1981). In vitro experiments from the same study
showed that this modulation of T-cell suppressor activity was Fc-mediated. As a
follow-up, other publications noted the increase in the suppressive activities of T
cells after IVIg treatment in ITP and AIDs (Delfraissy et al. 1985; Gupta et al.
1986). Since then, IVIg has been shown to increase the number of circulating
T-regulatory cells (Tregs) in Guillain-Barre syndrome, SLE, eosinophilic granu-
lomatosis with polyangiitis (Churg–Strass syndrome), Kawasaki disease, and a
peripheral neuropathy known as mononeuritis multiplex, among others (Barreto
et al. 2009; Burns et al. 2013; Chi et al. 2007; Tsurikisawa et al. 2012, 2014).
Various mechanisms have been proposed to explain how IVIg can modulate sup-
pressor T-cell function and frequency. One possible Fc-specific mechanism was
Anti-inflammatory Activity of IgG-Fc 47

described in patients with Kawasaki disease where IVIg treatment induces the
expansion of IL-10-producing Tregs with TCR specificity for peptides derived from
IgG-Fc (Franco et al. 2014). It was later shown by the same group that these Tregs
could be activated in a major histocompatibility complex (MHC)-restricted manner
by autologous IgG-positive B cells in the absence of exogenous Fc. Interestingly,
these Fc-specific Tregs were present in IVIg-treated patients with Kawasaki disease
patients and in healthy adult donors (Burns and Franco 2015).
Successful induction of Treg responses requires signals in the form of soluble
cytokines and costimulatory molecules, typically provided by professional
antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells.
IVIg has been shown to modulate dendritic cell function in various ways.
Decreased expression of costimulatory markers such as CD80/86 and MHC class II
on IVIg-exposed dendritic cells results in decreased capacity for these cells to drive
lymphocyte proliferation (Bayry et al. 2003; Kaufman et al. 2011; Qian et al. 2011).
In addition, IVIg has been shown to induce dendritic cell production of inhibitory
and anti-inflammatory cytokines such as IL-10 (Bayry et al. 2003; Massoud et al.
2012; Ohkuma et al. 2007; Ramakrishna et al. 2011). Work from De Groot et al.
and others has suggested a role for dendritic cells in the presentation of IgG-derived
T-cell epitopes known as “Tregitopes,” which contain peptides from both the Fab
and the Fc fragments of IgG (De Groot et al. 2008; Ephrem et al. 2008). In the
DeGroot model, dendritic cells uptake IVIg and process it for loading of peptides
onto MHC class II, allowing for activation and expansion of Tregs.
Specifically, it is debatable which receptors on APC IVIg might interact with.
The obvious candidates are the type I and type II FcRs. FccRIIa and FccRIIb are
found on subsets of dendritic cells and macrophages. Activating FccRs on dendritic
cells are necessary for successful treatment of ITP (Siragam et al. 2006). In this
model, the researchers hypothesized that IVIg formed immune complexes in vivo,
which acted on dendritic cells, thus priming their regulatory activity. The investi-
gators developed an adoptive transfer model in which isolated dendritic cells were
primed with IVIg in vitro and subsequently transferred to mice with active ITP. The
IVIg-primed dendritic cells were able to ameliorate ITP in these mice even when
the dendritic cells lacked FccRIIb. Interestingly, the IVIg-primed dendritic cells
were unable to ameliorate ITP when the host mouse lacked FccRIIb expression.
This suggests that the IVIg-dendritic cell interaction is independent of FccRIIb, but
the receptor was still necessary for later phases of IVIg action (Siragam et al. 2006).
Substantiating this finding, IVIg and immune complexes containing sialylated IgGs
were able to inhibit the LPS-induced maturation of bone marrow-derived dendritic
cells independently of FccRIIb (Oefner et al. 2012). One study, using a similar
model of adoptive transfer, showed that IVIg-primed bone marrow-derived den-
dritic cells (BMDCs) could induce Treg activity and be used to successfully treat
mice with ovalbumin-induced airway hyperresponsiveness (AHR), but IVIg
effectiveness was not disturbed when the BMDCs lacked activating FcRs (Massoud
et al. 2014). In this same study, the researchers found that IVIg ability to ameliorate
AHR was almost entirely dependent on the dendritic cell immunoreceptor (DCIR).
DCIR is an ITIM-bearing C-type lectin receptor found mostly on monocyte and
48 C. Beneduce et al.

dendritic cell populations (Kanazawa 2007). Massoud et al. showed that Treg
induction in AHR by IVIg was dependent on Cd11c-positive dendritic cells, but
they were unable to detect expression of the type II Fc receptor SIGN-R1 on these
cells. Instead, they observed DCIR-dependent binding of sialylated IVIg glyco-
forms and DCIR-dependent induction of Tregs.
The type II FccR SIGN-R1 is expressed mainly on macrophages in mice, whereas
DC-SIGN is expressed on myeloid and plasmacytoid dendritic cells and on some
macrophages in humans. Fieberger et al. reported that IVIg required DC-SIGN/
SIGN-R1 in amelioration of both T-cell-mediated and autoantibody-mediated dis-
eases (Fiebiger et al. 2015). They demonstrated that a single-point mutation at F241A
in recombinant Fc, even when lacking terminal sialic acid, conferred the same pro-
tections as sialylated Fc in the KBX/N model of arthritis. IVIg and the F241A Fc but
not unsialylated IVIg were shown to expand FoxP3+ Tregs in KBX/N-naive and
KBX/N-challenged mice. Both IVIg and F241A Fc were also able to expand Treg and
suppress T-cell-mediated autoimmunity in the experimental autoimmune encephalitis
(EAE) model in a SIGN-R1/DC-SIGN-dependent manner. Finally, inhibition of
IL-33 signaling abrogated IVIg and F241A Fc ability to induce Treg and suppress
inflammation, lending more support to this group’s original proposal of TH2 cytokine
cascade as the underlying cause of sialylated IVIg anti-inflammatory activity
(Fiebiger et al. 2015). Do DCIR and DC-SIGN/SIGN-R1 operate independently of
one another to induce Treg expansion and activity in response to sialylated IVIg
treatment? More studies are necessary to address how these receptors and the cells
upon which they are expressed mediate IVIg ability to induce Treg.
Modulation of T-cell responses in models of delayed-type hypersensitivity
(DTH) by sialylated IgGs was reported by Oefner et al. in 2012. DTH reactions
require antigen uptake, processing, and presentation to T-helper cells, which in turn
produce proinflammatory cytokines, resulting in tissue inflammation. In the Oefner
et al. model of ovalbumin-induced DTH injection of sialic-rich ovalbumin-specific
IgG, but not antibodies of different antigen specificity, they were able to suppress
DTH responses. Sialylated immune complexes were shown to inhibit dendritic cell
maturation in an FccRIIb-independent manner, which could explain this inhibition
of DTH. Although both the antigen specificity and the FccRIIb independence of the
sialylated IgG ability to inhibit DTH in this model are in contrast to reports of
IVIg-mediated suppression of antibody responses, modulation of T-cell responses is
still a common denominator (Oefner et al. 2012).

6 Modulation of Autoantibody Pathogenicity

by Sialylation

Although most understanding of the anti-inflammatory activity of IgG comes from

experience with IVIg, more recent studies have helped translate the IVIg findings to
antigen-specific IgG autoantibodies. One particular study elegantly demonstrated
Anti-inflammatory Activity of IgG-Fc 49

how antigen-specific antibodies, when sialylated, could be used not only to reverse
autoantibody-mediated inflammation, but also to prevent development of disease.
Ohmi et al. showed that genetic knockdown of mSt6gal1, a glycotransferase
responsible for transferring sialic acid to galactose, specifically in activated B cells
resulted in higher incidence and earlier development of collagen-induced arthritis
(CIA), along with more severe joint swelling in the affected mice (Ohmi et al.
2016). Enforcing sialylation of the ACC4 monoclonal antibody used to stimulate
collagen antibody-induced arthritis in DBA-1 mice abolished the development of
disease in all mice. Using the CIA model, the researchers demonstrated that, after
immunization with bovine type II collagen, treatment of mice with sialylated
ACPAs resulted in delayed incidence of disease and less severe joint swelling. In
addition, treatment of CIA with the sialylated ACPAs resulted in in vivo production
of sialylated anti-type II collagen antibodies (Ohmi et al. 2016).
The anti-inflammatory activity of specific sialylated antibodies was also shown in
models of IgG- and IgE-mediated anaphylaxis. Although allergen-induced anaphy-
laxis is mostly induced by IgE, it can also be mediated by IgG produced in response to
high levels of allergen. In the presence of excess allergen, these IgGs have the potential
to activate complement and FccRs on various effector cells, leading to anaphylaxis.
However, allergen-specific IgG antibodies, which are often induced by
allergen-specific immunotherapies (AITs), have also been shown to inhibit
IgE-mediated anaphylaxis (Burton et al. 2014; Strait et al. 2006). This is accomplished
through a combination of allergen masking and FccRIIb engagement. Just as FccRIIb
can mediate inhibition of the activating FccRs, cross-linking of FccRIIb with the IgE
receptor FcɛRI on basophils and mast cells can prevent activation of these cells and,
thus, limit release of various proinflammatory mediators implicated in
allergen-induced anaphylaxis (Strait et al. 2006). In line with these observations,
TNP-specific IgG1 antibodies were unable to inhibit anti-TNP IgE-mediated ana-
phylaxis in response to challenge with TNP-Ova in FccRIIb−/− −mice (Epp et al.
2018). In a similar model, it was shown that desialylated and degalactosylated IgG1
and IgG2 anti-TNP antibodies induce more severe anaphylaxis in response to chal-
lenge with TNP-Ova. In contrast, sialylated anti-TNP IgG1 effectively inhibited
TNP-Ova-induced anaphylaxis. This effect was dependent on FccRIIb and SIGN-R1.
In humans, AITs typically elicit allergen-specific IgG4 antibodies. These studies used
mouse IgG1 antibodies, which resemble human IgG4 in their complement and clas-
sical FccR binding (Epp et al. 2018). This study highlights the possibility that AIT
protocols that induce sialylated IgGs might limit IgG-mediated anaphylaxis in patients
exposed to high doses of allergen. To this end, the researchers showed that immu-
nization of mice with Ova in the adjuvant monophosphoryl lipid A, which was
recently approved for AIT, resulted in production of sialylated and galactosylated
IgG1 and IgG2, which were limited in their ability to induce IgG-mediated anaphy-
laxis (Epp et al. 2018).
Immune complexes formed by autoantibodies and their cognate antigens have
also been shown, in certain scenarios, to mediate anti-inflammatory processes. As
discussed in previous sections, it is clear that IgG glycosylation changes during
50 C. Beneduce et al.

disease progression and sialylation is often correlated with low disease activity
(Espy et al. 2011; Fickentscher et al. 2015; Kemna et al. 2017; Parekh et al. 1985;
Pasek et al. 2006). In vitro analysis of these sialylated autoantibodies and immune
complexes has helped shed light on some of the mechanisms by which inflam-
mation is controlled by antibody glycosylation. In the case of SLE, antihistone
antibodies isolated from patients were shown to be mostly asialylated and were able
to mediate efficient phagocytosis when used to opsonize necrotic cells, a hallmark
of SLE pathology (Magorivska et al. 2016). Sialylated autoantibodies isolated from
these patients were less effective at mediated neutrophil phagocytosis and switched
inflammatory cytokine production form IL-6/IL-8 to IL-1b/TNF-a (Magorivska
et al. 2016). Earlier studies had shown similar results in anti-PR3 antibodies iso-
lated from patients with active GPA (Espy et al. 2011). The authors demonstrated
that autoantibodies isolated from patients with active disease, exhibiting low sialic
acid content, induced a greater oxidative burst in neutrophils than those from
patients with quiescent disease. Enzymatic desialylation of anti-PR3 antibodies
from those patients with inactive GPA resulted in restoration of pathogenic activity
(Espy et al. 2011). In rheumatoid arthritis, anticitrullinated protein antibodies
(ACPAs) are known to induce innate cell-mediated inflammation in and around
joints, resulting in bone destruction (Kurowska et al. 2017). These ACPAs can
contain less Fc sialylation than other circulating IgGs (Ohmi et al. 2016; Rombouts
et al. 2015; Scherer et al. 2010). Studies in mice have shown that these ACPAs
promote osteoclastogenesis, resulting in bone resorption. In following up on these
findings, it was found that desialylated immune complexes increased osteoclasto-
genesis in an FccRII- and FccRIII-mediated manner. Furthermore, treatment of
CIA in mice with ManNAc, a precursor to sialic acid, resulted in increased sialy-
lation of circulating IgG1, delayed induction of arthritis, and reduced severity of the
disease (Harre et al. 2015).
Taken together, these studies show that, when sialylated, antigen-specific
autoantibodies possess anti-inflammatory properties. The results of these studies
have large implications for the development of antigen-specific immunotherapies
for autoimmune disorders whose autoantigens are well defined. In line with this
notion, a recent study took a different approach and attempted to sialylate antibodies
in vivo as a means of making them anti-inflammatory. The researchers investigated
how administration of B4GALT1 and ST6GAL1, glycotransferases that transfer
galactose and sialic acid, respectively, modulated autoantibody function in murine
models of arthritis. Coadministration of these enzymes before the transfer of K/BxN
sera resulted in marked reduction in inflammation similar to that seen with IVIg
treatment (Pagan et al. 2018). This treatment was also effective in a model of
Goodpasture syndrome, in which coadministration of the enzymes markedly
inhibited kidney disease. Keeping with the aforementioned TH2 anti-inflammatory
cascade initiated by sialylated IgG, in vivo sialylation and the resulting
anti-inflammatory activity achieved with these enzymes had a strict requirement of
FccRIIb and SIGN-R1 (Pagan et al. 2018).
Anti-inflammatory Activity of IgG-Fc 51

7 Anti-inflammatory Activity of Multivalent IgG-Fc

Immune complexes formed by the interaction between autoantibodies and

self-antigens are drivers of pathogenicity in many inflammatory and autoimmune
diseases. The inflammatory mechanisms are driven by activation of complement
pathways or through FccR-mediated cellular activation. Briefly, all FccRs except
FccRI bind Fc domains with low affinity. For effective cell activation, FccRs
require immune complexes for their aggregation, resulting in ultimate activation of
downstream signaling. Inhibition of these pathways through competition with
IgG-Fc molecules with high valency has been explored as a potential therapeutic
alternative. In fact, commercial IVIg preparations may contain a small fraction of
higher-order IgG structures in the form of dimers and aggregates, which presum-
ably are important for the anti-inflammatory activities of IVIg (Teeling et al. 2001).
All these observations have stimulated the development of recombinant oligo-
meric Fc proteins as potential replacement for IVIg (Zuercher et al. 2016). Figure 2
provides an overview of multimeric Fc drug candidates currently in preclinical or
clinical studies. Stradomer (GL-2045) generated by Gliknik/Pfizer (Gliknik.
Gliknik pipeline products. http://www.gliknik.com/pipeline/) is a drug candidate
consisting of a mixture of heterogenous IgG-Fc oligomeric structures. In particular,
stradomers are fully recombinant fusion proteins composed of murine IgG2a-Fc
hinge and either the human IgG2H or isoleucine zipper multimerization domains,
existing as homodimers and as a high percentage of covalently linked highly

Drug Candidate Description Institution Structure

Stradomer Heterogeneous IgG-Fc
Gliknik/Pfizer
GL2045 multimer with IgG2 hinge

. . . n Fc units

Fc3Y Momenta
or M230/CSL730 3 IgG1-Fc domains
Pharmaceuticals/CSL

HexaGard: Liverpool School of

Hexameric IgG-Fc Tropical Medicine
with IgM tailpiece L309C
with L309/H310L

CSL777: Hexameric CSL

Hexamers IgG-Fc with IgM
tailpiece with L309C

γ1 or γ4: IgG1 or IgG4 IgG1 or IgG4Fc

Hexameric Fc

UCB

Fig. 2 Summary of multimeric Fc drug candidates being explored as anti-inflammatory agents.

Ig, immunoglobulin
52 C. Beneduce et al.

ordered multimers (Jain et al. 2012). Stradomers prevented the onset of ITP and
ameliorated symptoms in other autoimmune disease models, including
collagen-induced arthritis (Jain et al. 2012), experimental autoimmune neuritis
(Niknami et al. 2013), and autoimmune myasthenia gravis (Thiruppathi et al. 2014).
This drug candidate is in preclinical development, and, in 2015, it received orphan
drug status in the USA for the treatment of chronic inflammatory demyelinating
polyradiculoneuropathy (Adis Insight. Research program: autoimmune disorder
therapeutics—Gliknik GL-2045. http://adisinsight.springer.com/drugs/800035077).
The knowledge of how IgM polymerizes via Fc domains was leveraged to
develop a novel approach to generating recombinant polymeric Fc-fusion proteins.
Specifically, more controlled oligomers—hexameric Fc proteins—were generated
by Richard Pleass as potential alternatives to IVIg (Czajkowsky et al. 2015;
Mekhaiel et al. 2011). These hexameric proteins were shown to bind low-affinity
inhibitory receptors FcRL5, FccRIIb, and DC-SIGN with high avidity and speci-
ficity, although they can also activate complement pathways (Czajkowsky et al.
2015). These proteins have yet to advance to the clinic, but they have great potential
as more effective and less expensive to produce than IVIg. Another hexameric
Fc-fusion protein was created by UCB, which was shown to alter receptor density
and trigger internalization and degradation of FccRs in vitro (Qureshi et al. 2017).
As a result, the molecule caused Fc binding disruption and phagocytosis in vitro
and the inhibition of platelet phagocytosis in a mouse ITP model. However, the
molecule also exhibited a short half-life in mice and cynomolgus monkeys. This
work pointed to yet another mechanism through which oligomeric Fc compounds
can exert anti-inflammatory activity. This molecule has not been advanced to
clinical use. Most recently, CSL generated an rFc hexamer, termed Fc-mTP-L309C,
by fusing IgM µ-tailpiece to the C terminus human IgG1-Fc (Spirig et al. 2018).
The hexamer bound FccRs with high avidity and could inhibit a range of in vitro
FccR-mediated effector functions such as ADCC, phagocytosis, and respiratory
burst. In addition, the molecule potently inhibited the activation of the classic
complement pathway. In vivo, Fc-mTP-L309C suppressed inflammatory arthritis in
mice at a 10-fold lower dose than IVIg, and, in a mouse model of immune
thrombocytopenia, it successfully restored platelet counts. The authors hypothesize
that the mechanism of action exerted by this hexamer is through blockade of FccRs
and through its unique inhibition of complement activation.
The concept of selectively antagonizing the activating FccR system without
activating proinflammatory pathways was introduced by Momenta Pharmaceuticals
(Ortiz et al. 2016). Through novel insight into the modulation of the FccR path-
ways, a trivalent-IgG-Fc recombinant product candidate—Fc3Y, or M230—was
designed. M230 was designed to preferentially assemble into a homogeneous tri-
meric IgG-Fc using knobs into hole and electrostatic steering molecular
heterodimerization technology. Trivalent M230 exerts avid binding to FccRs,
without inadvertently activating immune cell effector functions, resulting in a potent
molecule that inhibits immune complex-driven processes in many cells that express
FccRs. A main driver for the design of M230 was the identification of a threshold
for activation of the FccR signaling pathways with Fc-multimers containing five or
Anti-inflammatory Activity of IgG-Fc 53

Activating
FcγR

Antibody

Antigen

IgG-Fc
Fc valency 1 2 3 5 >5 >5

- + + +

Threshold for activating FcγR signaling

Fig. 3 Schematic representation of the IgG1-Fc valency threshold required for FccR signaling
identified by Bosques and coworkers

more Fc units but not with multivalent Fc containing three or less Fc units (Fig. 3).
Furthermore, M230 has been shown to have 40-fold greater potency than IVIg in
animal models of autoimmune diseases such as arthritis, immune thrombocytope-
nia, and epidermolysis bullosa acquisita skin blistering. This drug candidate is in
clinical development in collaboration with CSL, and it may represent an effective
therapeutic candidate for FccR-mediated autoimmune diseases.

8 Conclusion

Studies from the past 15 years have shed new light on the mechanisms by which
IgG can perform pro- and anti-inflammatory activities. Through detailed biophys-
ical characterization, use of in vitro and in vivo models, and biomarker studies in
clinical investigation, the role of glycosylation in the anti-inflammatory and
immunoregulatory properties of IgG has been well established. At the molecular
level, studies have shown the far-reaching effects of terminal Fc sialylation and
have demonstrated the ability of this sugar moiety to modulate Fc structure and
interactions with FcRs. In addition to the use of IVIg in the clinic, the relationship
between Fc glycosylation states and disease activity in various autoimmune dis-
orders has provided valuable human evidence of the immunoregulatory effects of
IgG. Detailed in vitro and in vivo studies have elucidated the key receptors, cells,
and signaling cascades involved in these anti-inflammatory processes. This
knowledge has enabled the evaluation of new therapeutics, such as multivalent Fc
drug candidates and hypersialylated IVIg, which are being developed to harness the
anti-inflammatory effects of IgG.
54 C. Beneduce et al.

References

Ackerman ME, Crispin M, Yu X, Baruah K, Boesch AW, Harvey DJ, Dugast AS, Heizen EL,
Ercan A, Choi I, Streeck H, Nigrovic PA, Bailey-Kelogg C, Scanlan C, Alter G (2013) Natural
variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity. J Clin Invest
123(5):2183–2192
Ahmed AA, Giddens J, Pincetic A, Lomino JV, Ravetch JV, Wang LX, Bjorkman PJ (2014)
Structural characterization of anti-inflammatory immunoglobulin G Fc proteins. J Molec Biol
426(18):3166–3179. https://doi.org/10.1016/j.jmb.2014.07.006
Akilesh S (2004) The MHC class I-like Fc receptor promotes humorally mediated autoimmune
disease. J Clin Invest 113(9):1328–1333. https://doi.org/10.1172/jci200418838
Anthony RM, Nimmerjahn F, Ashline D, Reinhold V, Paulson J, Ravetch JV (2008a) A
recombinant IgG Fc that recapitulates the anti-inflammatory activity of IVIg. Science 320
(5874):373–376. https://doi.org/10.1126/science.1154315
Anthony RM, Kobayashi T, Wermeling F, Ravetch JV (2011) Intravenous gammaglobulin
suppresses inflammation through a novel T(H)2 pathway. Nature 475(7354):110–113. https://
doi.org/10.1038/nature10134
Anthony R, Wermeling F, Karlsson M, Ravetch JV (2008b) Identification of a receptor required
for the anti-inflammatory activity of IVIG. Proc Natl Acad Sci U S A 105(50):19571–19578.
https://doi.org/10.1073/pnas.0810163105
Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA (2007) The impact of glycosylation on
the biological function and structure of human immunoglobulins. Ann Rev Immunol 25:21–50.
https://doi.org/10.1146/annurev.immunol.25.022106.141702
Barreto M, Ferreira RC, Lourenco L, Moraes-Fontes MF, Santos E, Alves M, Carvalho C,
Martins B, Andreia R, Viana JF, Vasconcelos C, Mota-Vieira L, Ferreira C, Demengeot J,
Vicente AM (2009) Low frequency of CD4+ CD25+ Treg in SLE patients: a heritable trait
associated with CTLA4 and TGFb gene variants. BMC Immunol 10:5. https://doi.org/10.1186/
1471-2172-10-5
Bayry J, Lacroix-Desmazes S, Carbonneil C, Misra N, Donkova V, Pashov A, Chevailler A,
Mouthon L, Weill B, Bruneval P, Kazatchkine MD, Kaveri SV (2003) Inhibition of maturation
and function of dendritic cells by intravenous immunoglobulin. Blood 101(2):758–765. https://
doi.org/10.1182/blood-2002-05-1447
Boruchov AM, Heller G, Veri MC, Bonvini E, Ravetch JV, Young JW (2005) Activating and
inhibitory IgG Fc receptors on human DCs mediate opposing functions. J Clin Invest 115
(10):2914–2923. https://doi.org/10.1172/JCI24772
Bruhns P, Samuelsson A, Pollard J, Ravetch JV (2003) Colony-stimulating factor-1-dependent
macrophages are responsible for IVIG Protection in antibody-induced autoimmune disease.
Immunity 18(4):573–581. https://doi.org/10.1016/S1074-7613(03)00080-3
Bruhns P, Iannascoli B, England P, Mancardi D, Fernandez N, Jorieux S, Daeron M (2009)
Specificity and affinity of human Fcc receptors and their polymorphic variants for human IgG
subclasses. Blood 113(16):3716–3725. https://doi.org/10.1182/blood2008-09-179754
Burns JC, Franco A (2015) The immunomodulatory effects of intravenous immunoglobulin
therapy in Kawasaki disease. Expert Rev Clin Immunol 11(7):819–825. https://doi.org/10.
1586/1744666X.2015.1044980
Burns JC, Song Y, Bujold M, Shimizu C, Kanegaye JT, Tremoulet AH, Franco A (2013)
Immune-monitoring in Kawasaki disease patients treated with infliximab and intravenous
immunoglobulin. Clin Exp Immunol 174(3):337–344. https://doi.org/10.1111/cei.12182
Burton OT, Logsdon SL, Zhou JS, Medina-Tamayo J, Abdel-Gadir A, Noval Rivas M,
Koleoglou KJ, Chatila TA, Schneider LC, Rachid R, Umetsu DT, Oettgen HC (2014) Oral
immunotherapy induces IgG antibodies that act through FccRIIb to suppress IgE-mediated
hypersensitivity. J Allergy Clin Immunol 134(6):1310–1317 e1316. https://doi.org/10.1016/j.
jaci.2014.05.042
Anti-inflammatory Activity of IgG-Fc 55

Chi LJ, Wang HB, Zhang Y, Wang WZ (2007) Abnormality of circulating CD4(+) CD25(+)
regulatory T cell in patients with Guillain-Barre syndrome. J Neuroimmunol 192(1–2):206–
214. https://doi.org/10.1016/j.jneuroim.2007.09.034
Chu SY, Vostiar I, Karki S, Moore GL, Lazar GA, Pong E, Joyce PF, Szymkowski DE,
Desjarlais JR (2008) Inhibition of B cell receptor-mediated activation of primary human B cells
by coengagement of CD19 and FccRIIb with Fc-engineered antibodies. Mol Immunol 45
(15):3926–3933. https://doi.org/10.1016/j.molimm.2008.06.027
Cines DB, Bussel JB (2005) How I treat idiopathic thrombocytopenic purpura (ITP). Blood 106
(7):2244–2251. https://doi.org/10.1182/blood-2004-12-4598
Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo
cytoxicity against tumor targets. Nat Med 6(4):443–446
Crow AR, Song S, Freedman J, Helgason CD, Humphries RK, Siminovitch KA, Lazarus AH
(2003) IVIg-mediated amelioration of murine ITP via FccRIIB is independent of SHIP1,
SHP-1, and Btk activity. Blood 102(2):558–560. https://doi.org/10.1182/blood-2003-01-0023
Czajkowsky DM, Andersen JT, Fuchs A, Wilson TJ, Mekhaiel D, Colonna M, He J, Shao Z,
Mitchell DA, Wu G, Dell A, Haslam S, Lloyd KA, Moore SC, Sandlie I, Blundell PA,
Pleass RJ (2015) Developing the IVIG biomimetic, hexa-Fc, for drug and vaccine applications.
Sci Rep 5:9526. https://doi.org/10.1038/srep09526
De Groot AS, Moise L, McMurry JA, Wambre E, Van Overtvelt L, Moingeon P, Scott DW,
Martin W (2008) Activation of natural regulatory T cells by IgG Fc-derived peptide
“Tregitopes”. Blood 112(8):3303–3311. https://doi.org/10.1182/blood-2008-02-138073
Debre M, Griscelli C, Bonnet MC, Carosella E, Philippe N, Reinert P, Vilmer E, Kaplan C,
Fridman WH, Teillaud JL (1993) Infusion of Fcc fragments for treatment of children with
acute immune thrombocytopenic purpura. Lancet 342(8877):945–949. https://doi.org/10.1016/
0140-6736(93)92000-J
Delfraissy JF, Tchernia G, Laurian Y, Wallon C, Galanaud P, Dormont J (1985) Suppressor cell
function after intravenous gammaglobulin treatment in adult chronic idiopathic thrombocy-
topenic purpura. Br J Haematol 60(2):315–322. https://doi.org/10.1111/j.1365-2141.1985.
tb07417.x
Dube R, Rook GA, Steele J, Brealey R, Dwek RA, Rademacher T, Lennard-Jones J (1990)
Agalactosyl IgG in inflammatory bowel disease: correlation with C-reactive protein. Gut 31
(4):431–434. https://doi.org/10.1136/gut.31.4.431
Durandy A, Fischer A, Griscelli C (1981) Dysfunctions of pokeweed mitogen-stimulated T and B
lymphocyte responses induced by gammaglobulin therapy. J Clin Invest 67(3):867–877.
https://doi.org/10.1172/JCI110104
Engdahl C, Bondt A, Harre U, Raufer J, Pfeifle R, Camponeschi A, Wuhrer M, Seeling M,
Martensson IL, Nimmerjahn F, Kronke G, Scherer HU, Forsblad-d’Elia H, Schett G (2018)
Estrogen induces St6gal1 expression and increases IgG sialylation in mice and patients with
rheumatoid arthritis: a potential explanation for the increased risk of rheumatoid arthritis in
postmenopausal women. Arthritis Res Ther 20(1):84. https://doi.org/10.1186/s13075-018-
1586-z
Ephrem A, Chamat S, Miquel C, Fisson S, Mouthon L, Caligiuri G, Delignat S, Elluru S, Bayry J,
Lacroix-Desmazes S, Cohen JL, Salomon BL, Kazatchkine MD, Kaveri SV, Misra N (2008)
Expansion of CD4+ CD25+ regulatory T cells by intravenous immunoglobulin: a critical factor
in controlling experimental autoimmune encephalomyelitis. Blood 111(2):715–722. https://doi.
org/10.1182/blood-2007-03-079947
Epp A, Hobusch J, Bartsch YC, Petry J, Lilienthal GM, Koeleman CAM, Eschweiler S, Mobs C,
Hall A, Morris SC, Braumann D, Engellenner C, Bitterling J, Rahmoller J, Leliavski A,
Thurmann R, Collin M, Moremen KW, Strait RT, Blanchard V, Petersen A, Gemoll T,
Habermann JK, Petersen F, Nandy A, Kahlert H, Hertl M, Wuhrer M, Pfutzner W, Jappe U,
Finkelman FD, Ehlers M (2018) Sialylation of IgG antibodies inhibits IgG-mediated allergic
reactions. J Allergy Clin Immunol 141(1):399–402 e398. https://doi.org/10.1016/j.jaci.2017.
06.021
56 C. Beneduce et al.

Espy C, Morelle W, Kavian N, Grange P, Goulvestre C, Viallon V, Chereau C, Pagnoux C,

Michalski JC, Guillevin L, Weill B, Batteux F, Guilpain P (2011) Sialylation levels of
anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis
(Wegener’s). Arthritis Rheum 63(7):2105–2115. https://doi.org/10.1002/art.30362
Feige MJ, Nath S, Catharino SR, Weinfurtner D, Steinbacher S, Buchner J (2009) Structure of the
murine unglycosylated IgG1 Fc fragment. J Molec Biol 391(3):599–608. https://doi.org/10.
1016/j.jmb.2009.06.048
Ferrara C, Grau S, Jager C, Sondermann P, Brunker P, Waldhauer I, Hennig M, Ruf A, Rufer AC,
Stihle M, Umana P, Benz J (2011) Unique carbohydrate–carbohydrate interactions are required
for high affinity binding between FccRIII and antibodies lacking core fucose. Proc Natl Acad
Sci U S A 108(31):12669–12674. https://doi.org/10.1073/pnas.1108455108
Fickentscher C, Magorivska I, Janko C, Biermann M, Bilyy R, Nalli C, Tincani A, Medeghini V,
Meini A, Nimmerjahn F, Schett G, Munoz LE, Andreoli L, Herrmann M (2015) The
Pathogenicity of anti-b2GP1-IgG Autoantibodies depends on Fc glycosylation. J Immunol Res
2015:638129. https://doi.org/10.1155/2015/638129
Fiebiger BM, Maamary J, Pincetic A, Ravetch JV (2015) Protection in antibody- and T
cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs. Proc
Natl Acad Sci U S A 112(18):E2385–E2394. https://doi.org/10.1073/pnas.1505292112
Fokkink WJ, Selman MH, Dortland JR, Durmus B, Kuitwaard K, Huizinga R, van Rijs W,
Tio-Gillen AP, van Doorn PA, Deelder AM, Wuhrer M, Jacobs BC (2014) IgG Fc N-
glycosylation in Guillain-Barre syndrome treated with immunoglobulins. J Proteome Res 13
(3):1722–1730. https://doi.org/10.1021/pr401213z
Franco A, Touma R, Song Y, Shimizu C, Tremoulet AH, Kanegaye JT, Burns JC (2014)
Specificity of regulatory T cells that modulate vascular inflammation. Autoimmunity 47(2):95–
104. https://doi.org/10.3109/08916934.2013.860524
Gringhuis SI, Kaptein TM, Wevers BA, van der Vlist M, Klaver EJ, van Die I, Vriend LE, de
Jong MA, Geijtenbeek TB (2014) Fucose-based PAMPs prime dendritic cells for follicular T
helper cell polarization via DC-SIGN-dependent IL-27 production. Nat Commun 5:5074.
https://doi.org/10.1038/ncomms6074
Gupta A, Novik BE, Rubinstein A (1986) Restoration of suppressor T-cell functions in children
With AIDS following intravenous gamma globulin treatment. Am J Dis Child 140(2):143–146.
https://doi.org/10.1001/archpedi.1986.02140160061033
Hansen RJ, Balthasar JP (2002) Effects of intravenous immunoglobulin on platelet count and
antiplatelet antibodydisposition in a rat model of immune thrombocytopenia. Blood 100
(6):2087–2093
Harre U, Lang SC, Pfeifle R, Rombouts Y, Fruhbeisser S, Amara K, Bang H, Lux A,
Koeleman CA, Baum W, Dietel K, Grohn F, Malmstrom V, Klareskog L, Kronke G,
Kocijan R, Nimmerjahn F, Toes RE, Herrmann M, Scherer HU, Schett G (2015) Glycosylation
of immunoglobulin G determines osteoclast differentiation and bone loss. Nat Commun
6:6651. https://doi.org/10.1038/ncomms7651
Heyman B (2003) Feedback regulation by IgG antibodies. Immunol Lett 88(2):157–161. https://
doi.org/10.1016/s0165-2478(03)00078-6
Houde D, Peng Y, Berkowitz S, Engen JR (2010) Post-translational modifications differentially
affect IgG1 conformation and receptor binding. Mol Cell Proteomics 9(8):1716–1728. https://
doi.org/10.1074/mcp.M900540-MCP200
Huang HS, Sun DS, Lien TS, Chang HH (2010) Dendritic cells modulate platelet activity in
IVIg-mediated amelioration of ITP in mice. Blood 116(23):5002–5009. https://doi.org/10.
1182/blood-2010-03-275123
Jain A, Olsen HS, Vyzasatya R, Burch E, Sakoda Y, Merigeon EY, Cai L, Lu C, Tan M,
Tamada K, Schulze D, Block DS, Strome SE (2012) Fully recombinant IgG2a Fc multimers
(stradomers) effectively treat collagen-induced arthritis and prevent idiopathic thrombocy-
topenic purpura in mice. Arthritis Res Ther 14(4):R192
Jefferis R, Lund J (2002) Interaction sites on human IgG-Fc for FccR: current models. Immunol
Lett 82(1–2):57–65
Anti-inflammatory Activity of IgG-Fc 57

Kanazawa N (2007) Dendritic cell immunoreceptors: C-type lectin receptors for

pattern-recognition and signaling on antigen-presenting cells. J Dermatol Sci 45(2):77–86.
https://doi.org/10.1016/j.jdermsci.2006.09.001
Kaneko Y, Nimmerjahn F, Madaio MP, Ravetch JV (2006a) Pathology and protection in
nephrotoxic nephritis is determined by selective engagement of specific Fc receptors. J Exp
Med 203(3):789–797. https://doi.org/10.1084/jem.20051900
Kaneko Y, Nimmerjahn F, Ravetch JV (2006b) Anti-inflammatory activity of immunoglobulin G
resulting from Fc sialylation. Science 313(5787):670–673. https://doi.org/10.1126/science.
1129594
Kao D, Lux A, Schaffert A, Lang R, Altmann F, Nimmerjahn F (2017) IgG subclass and
vaccination stimulus determine changes in antigen specific antibody glycosylation in mice.
Eur J Immunol 47(12):2070–2079. https://doi.org/10.1002/eji.201747208
Kapur R, Della Valle L, Sonneveld M, Hipgrave Ederveen A, Visser R, Ligthart P, de Haas M,
Wuhrer M, van der Schoot CE, Vidarsson G (2014a) Low anti-RhD IgG-Fc-fucosylation in
pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn.
Br J Haematol 166(6):936–945. https://doi.org/10.1111/bjh.12965
Kapur R, Kustiawan I, Vestrheim A, Koeleman CA, Visser R, Einarsdottir HK, Porcelijn L,
Jackson D, Kumpel B, Deelder AM, Blank D, Skogen B, Killie MK, Michaelsen TE, de
Haas M, Rispens T, van der Schoot CE, Wuhrer M, Vidarsson G (2014b) A prominent lack of
IgG1-Fc fucosylation of platelet alloantibodies in pregnancy. Blood 123(4):471–480. https://
doi.org/10.1182/blood-2013-09-527978
Kaufman GN, Massoud AH, Audusseau S, Banville-Langelier AA, Wang Y, Guay J, Garellek JA,
Mourad W, Piccirillo CA, McCusker C, Mazer BD (2011) Intravenous immunoglobulin
attenuates airway hyperresponsiveness in a murine model of allergic asthma. Clin Exp Allergry
41(5):718–728. https://doi.org/10.1111/j.1365-2222.2010.03663.x
Kemna MJ, Plomp R, van Paassen P, Koeleman CAM, Jansen BC, Damoiseaux J, Cohen
Tervaert JW, Wuhrer M (2017) Galactosylation and sialylation levels of IgG predict relapse in
patients with PR3-ANCA associated vasculitis. EBioMedicine 17:108–118. https://doi.org/10.
1016/j.ebiom.2017.01.033
Kiener PA, Lioubin MN, Rohrschneider LR, Ledbetter JA, Nadler SG, Diegel ML (1997)
Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of
intracellular signaling in B cells. J Biol Chem 272(6):3838–3844
Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P (2003) Structural analysis of human
IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol
Biol 325(5):979–989. https://doi.org/10.1016/s0022-2836(02)01250-0
Kurowska W, Kuca-Warnawin EH, Radzikowska A, Maslinski W (2017) The role of
anti-citrullinated protein antibodies (ACPA) in the pathogenesis of rheumatoid arthritis. Cent
Eur J Immunol 42(4):390–398. https://doi.org/10.5114/ceji.2017.72807
Leontyev D, Katsman Y, Ma XZ, Miescher S, Kasermann F, Branch DR (2012)
Sialylation-independent mechanism involved in the amelioration of murine immune throm-
bocytopenia using intravenous gammaglobulin. Transfusion 52(8):1799–1805. https://doi.org/
10.1111/j.1537-2995.2011.03517.x
Li N, Zhao M, Hilario-Vargas J, Prisayanh P, Warren S, Diaz LA, Roopenian DC, Liu Z (2005)
Complete FcRn dependence for intravenous Ig therapy in autoimmune skin blistering diseases.
J Clin Invest 115(12):3440–3450. https://doi.org/10.1172/JCI24394
Li T, DiLillo DJ, Bournazos S, Giddens JP, Ravetch JV, Wang LX (2017) Modulating IgG effector
function by Fc glycan engineering. Proc Natl Acad Sci U S A 114(13):3485–3490. https://doi.
org/10.1073/pnas.1702173114
Lood C, Allhorn M, Lood R, Gullstrand B, Olin AI, Ronnblom L, Truedsson L, Collin M,
Bengtsson AA (2012) IgG glycan hydrolysis by endoglycosidase S diminishes the
proinflammatory properties of immune complexes from patients with systemic lupus
erythematosus: a possible new treatment? Arthritis Rheum 64(8):2698–2706. https://doi.org/
10.1002/art.34454
58 C. Beneduce et al.

Maamary J, Wang TT, Tan GS, Palese P, Ravetch JV (2017) Increasing the breadth and potency of
response to the seasonal influenza virus vaccine by immune complex immunization. Proc Natl
Acad Sci U S A 114(38):10172–10177
Maddur MS, Stephen-Victor E, Das M, Prakhar P, Sharma VK, Singh V, Rabin M, Trinath J,
Balaji KN, Bolgert F, Vallat JM, Magy L, Kaveri SV, Bayry J (2017) Regulatory T cell
frequency, but not plasma IL-33 levels, represents potential immunological biomarker to
predict clinical response to intravenous immunoglobulin therapy. J Neuroinflammation 14
(1):58. https://doi.org/10.1186/s12974-017-0818-5
Magorivska I, Munoz LE, Janko C, Dumych T, Rech J, Schett G, Nimmerjahn F, Bilyy R,
Herrmann M (2016) Sialylation of anti-histone immunoglobulin G autoantibodies determines
their capabilities to participate in the clearance of late apoptotic cells. Clin Exp Immunol 184
(1):110–117. https://doi.org/10.1111/cei.12744
Massoud AH, Guay J, Shalaby KH, Bjur E, Ablona A, Chan D, Nouhi Y, McCusker CT,
Mourad MW, Piccirillo CA, Mazer BD (2012) Intravenous immunoglobulin attenuates airway
inflammation through induction of forkhead box protein 3-positive regulatory T cells. J Allergy
Clin Immunol 129(6):1656–1665 e1653. https://doi.org/10.1016/j.jaci.2012.02.050
Massoud AH, Yona M, Xue D, Chouiali F, Alturaihi H, Ablona A, Mourad W, Piccirillo CA,
Mazer BD (2014) Dendritic cell immunoreceptor: a novel receptor for intravenous
immunoglobulin mediates induction of regulatory T cells. J Allergy Clin Immunol 133
(3):853–863 e855. https://doi.org/10.1016/j.jaci.2013.09.029
Mekhaiel DN, Czajkowsky DM, Andersen JT, Shi J, El-Faham M, Doenhoff M, McIntosh RS,
Sandlie I, He J, Hu J, Shao Z, Pleass RJ (2011) Polymeric human Fc-fusion proteins with
modified effector functions. Sci Rep 1:124. https://doi.org/10.1038/srep00124
Mimura Y, Church S, Ghirlando R, Ashton PR, Dong S, Goodall M, Lund J, Jefferis R (2000) The
influence of glycosylation on the thermal stability and effector function expression of human
IgG1-Fc: properties of a series of truncated glycoforms. Mol Immunol 37(12):697–706
Mimura Y, Sondermann P, Ghirlando R, Lund J, Young SP, Goodall M, Jefferis R (2001) Role of
oligosaccharide residues of IgG1-Fc in FccRIIb binding. J Biol Chem 276(49):45539–45547.
https://doi.org/10.1074/jbc.M107478200
Mogensen TH, Melchjorsen J, Larsen CS, Paludan SR (2010) Innate immune recognition and
activation during HIV infection. Retrovirology 7:54. https://doi.org/10.1186/1742-4690-7-54
Nechansky A, Schuster M, Jost W, Siegl P, Wiederkum S, Gorr G, Kircheis R (2007)
Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular
cytotoxicity by glyco-engineering of therapeutic antibodies. Mol Immunol 44(7):1815–1817.
https://doi.org/10.1016/j.molimm.2006.08.013
Niknami M, Wang MX, Nguyen T, Pollard JD (2013) Beneficial effect of a multimerized
immunoglobulin Fc in an animal model of inflammatory neuropathy (experimental autoim-
mune neuritis). J Peripher Nerv Syst 18(2):141–152
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin G subclass activity through
selective Fc receptor binding. Science 310(5759):1510–1512
Nimmerjahn F, Ravetch JV (2006) Fcc receptors: old friends and new family members. Immunity
24(1):19–28. https://doi.org/10.1016/j.immuni.2005.11.010
Nose M, Wigzell H (1983) Biological significance of carbohydrate chains on monoclonal
antibodies. Proc Natl Acad Sci U S A 80(21):6632–6636
Oefner CM, Winkler A, Hess C, Lorenz AK, Holecska V, Huxdorf M, Schommartz T, Petzold D,
Bitterling J, Schoen AL, Stoehr AD, Vu Van D, Darcan-Nikolaisen Y, Blanchard V,
Schmudde I, Laumonnier Y, Strover HA, Hegazy AN, Eiglmeier S, Schoen CT, Mertes MM,
Loddenkemper C, Lohning M, Konig P, Petersen A, Luger EO, Collin M, Kohl J, Hutloff A,
Hamelmann E, Berger M, Wardemann H, Ehlers M (2012) Tolerance induction with T
cell-dependent protein antigens induces regulatory sialylated IgGs. J Allergy Clin Immunol
129(6):1647–1655 e1613. https://doi.org/10.1016/j.jaci.2012.02.037
Ogata S, Shimizu C, Franco A, Touma R, Kanegaye JT, Choudhury BP, Naidu NN, Kanda Y,
Hoang LT, Hibberd ML, Tremoulet AH, Varki A, Burns JC (2013) Treatment response in
Kawasaki disease is associated with sialylation levels of endogenous but not therapeutic
Anti-inflammatory Activity of IgG-Fc 59

intravenous immunoglobulin G. PLoS ONE 8(12):e81448. https://doi.org/10.1371/journal.

pone.0081448
Ohkuma K, Sasaki T, Kamei S, Okuda S, Nakano H, Hamamoto T, Fujihara K, Nakashima I,
Misu T, Itoyama Y (2007) Modulation of dendritic cell development by immunoglobulin G in
control subjects and multiple sclerosis patients. Clin Exp Immunol 150(3):397–406. https://doi.
org/10.1111/j.1365-2249.2007.03496.x
Ohmi Y, Ise W, Harazono A, Takakura D, Fukuyama H, Baba Y, Narazaki M, Shoda H,
Takahashi N, Ohkawa Y, Ji S, Sugiyama F, Fujio K, Kumanogoh A, Yamamoto K,
Kawasaki N, Kurosaki T, Takahashi Y, Furukawa K (2016) Sialylation converts arthritogenic
IgG into inhibitors of collagen-induced arthritis. Nat Commun 7:11205. https://doi.org/10.
1038/ncomms11205
Ono M, Okada H, Bolland S, Yanagi S, Tomohiro K, Ravetch JV (1997) Deletion of SHIP or
SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90(2):293–301
Ortiz DF, Lansing JC, Rutitzky L, E. K, Prod’homme T, Choudhury A, Washburn N,
Bhatnagar N, Beneduce C, K. H, Prenovitz R, Child M, Killough J, Tyler S, Brown J,
Nguyen S, Schwab I, Hains M, Meccariello R, Markowitz L, Wang J, Zouaoui R, Simpson A,
Schultes B, Capila I, Ling L, Nimmerjahn F, Manning AM, Bosques CJ (2016) Elucidating the
interplay between IgG-Fc valency and FccR activation for the design of immune complex
inhibitors. Sci Transl Med 8(365):ra158
Pagan JD, Kitaoka M, Anthony RM (2018) Engineered sialylation of pathogenic antibodies
in vivo attenuates autoimmune disease. Cell 172(3):564–577 e513. https://doi.org/10.1016/j.
cell.2017.11.041
Parekh RB, Dwek RA, Sutton BJ, Fernandes DL, Leung A, Stanworth D, Rademacher T,
Mizouchi T, Takahashi N, Matsuta K (1985) Association of rheumatoid arthritis and primary
osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316
Pasek M, Duk M, Podbielska M, Sokolik R, Szechinski J, Lisowska E, Krotkiewski H (2006)
Galactosylation of IgG from rheumatoid arthritis (RA) patients—changes during therapy.
Glycoconjugate J 23(7–8):463–471. https://doi.org/10.1007/s10719-006-5409-0
Qian J, Zhu J, Wang M, Wu S, Chen T (2011) Suppressive effects of intravenous immunoglobulin
(IVIG) on human umbilical cord blood immune cells. Pediatr Allergy Immunol 22(2):211–220.
https://doi.org/10.1111/j.1399-3038.2010.01049.x
Qureshi OS, Rowley TF, Junker F, Peters SJ, Crilly S, Compson J, Eddleston A, Bjorkelund H,
Greenslade K, Parkinson M, Davies NL, Griffin R, Pither TL, Cain K, Christodoulou L,
Staelens L, Ward E, Tibbitts J, Kiessling A, Smith B, Brennan FR, Malmqvist M, Fallah-Arani
F, Humphreys DP (2017) Multivalent Fcc-receptor engagement by a hexameric Fc-fusion
protein triggers Fcc-receptor internalisation and modulation of Fcc-receptor functions. Sci Rep
7(1):17049. https://doi.org/10.1038/s41598-017-17255-8
Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr
Opin Immunol 20(4):471–478. https://doi.org/10.1016/j.coi.2008.06.007
Ramakrishna C, Newo AN, Shen YW, Cantin E (2011) Passively administered pooled human
immunoglobulins exert IL-10 dependent anti-inflammatory effects that protect against fatal
HSV encephalitis. PLoS Pathog 7(6):e1002071. https://doi.org/10.1371/journal.ppat.1002071
Ravetch JV, Clynes RA (1998) Divergent roles for Fc receptors and complement in vivo. Ann Rev
Immunol 16:421–432
Rombouts Y, Ewing E, van de Stadt LA, Selman MH, Trouw LA, Deelder AM, Huizinga TW,
Wuhrer M, van Schaardenburg D, Toes RE, Scherer HU (2015) Anti-citrullinated protein
antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of
rheumatoid arthritis. Ann Rheum Dis 74(1):234–241. https://doi.org/10.1136/annrheumdis-
2013-203565
Rook GA, Steele J, Brealey R, Whyte A, Isenberg D, Sumar N, Nelson JL, Bodman KB, Young A,
Roitt IM (1991) Changes in IgG glycoform levels are associated with remission of arthritis
during pregnancy. J Autoimmun 4(5):779–794
Samuelsson A, Towers TL, Ravetch JV (2001) Anti-inflammatory activity of IVIG mediated
through the inhibitory Fc receptor. Science 291(5503):484–486
60 C. Beneduce et al.

Scallon BJ, Tam SH, McCarthy SG, Cai AN, Raju TS (2007) Higher levels of sialylated Fc
glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 44
(7):1524–1534. https://doi.org/10.1016/j.molimm.2006.09.005
Scherer HU, van der Woude D, Ioan-Facsinay A, el Bannoudi H, Trouw LA, Wang J, Haupl T,
Burmester GR, Deelder AM, Huizinga TW, Wuhrer M, Toes RE (2010) Glycan profiling of
anti-citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis
Rheum 62(6):1620–1629. https://doi.org/10.1002/art.27414
Schwab I, Nimmerjahn F (2013) Intravenous immunoglobulin therapy: how does IgG modulate
the immune system? Nat Rev Immunol 13(3):176–189. https://doi.org/10.1038/nri3401
Schwab I, Biburger M, Kronke G, Schett G, Nimmerjahn F (2012) IVIg-mediated amelioration of
ITP in mice is dependent on sialic acid and SIGNR1. Eur J Immunol 42(4):826–830. https://
doi.org/10.1002/eji.201142260
Schwab I, Mihai S, Seeling M, Kasperkiewicz M, Ludwig RJ, Nimmerjahn F (2014) Broad
requirement for terminal sialic acid residues and FccRIIB for the preventive and therapeutic
activity of intravenous immunoglobulins in vivo. Eur J Immunol 44(5):1444–1453. https://doi.
org/10.1002/eji.201344230
Selman MH, Niks EH, Titulaer MJ, Verschuuren JJ, Wuhrer M, Deelder AM (2011) IgG fc N-
glycosylation changes in Lambert-Eaton myasthenic syndrome and myasthenia gravis.
J Proteome Res 10(1):143–152
Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002)
Lack of fucose on human IgG1N-linked oligosaccharide improves binding to human FccRIII
and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740. https://doi.org/
10.1074/jbc.M202069200
Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K,
Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not
the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type
oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.
J Biol Chem 278(5):3466–3473. https://doi.org/10.1074/jbc.M210665200
Siragam V, Brinc D, Crow AR, Song S, Freedman J, Lazarus AH (2005) Can antibodies with
specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the
treatment of autoimmune disease. J Clin Invest 115(1):155–160. https://doi.org/10.1172/
jci200522753ds1
Siragam V, Crow AR, Brinc D, Song S, Freedman J, Lazarus AH (2006) Intravenous
immunoglobulin ameliorates ITP via activating Fcc receptors on dendritic cells. Nat Med 12
(6):688–692. https://doi.org/10.1038/nm1416
Sjowall C, Zapf J, von Lohneysen S, Magorivska I, Biermann M, Janko C, Winkler S, Bilyy R,
Schett G, Herrmann M, Munoz LE (2015) Altered glycosylation of complexed native IgG
molecules is associated with disease activity of systemic lupus erythematosus. Lupus 24
(6):569–581
Sondermann P, Kaiser J, Jacob U (2001) Molecular basis for immune complex recognition: a
comparison of Fc-receptor structures. J Mol Biol 309(3):737–749
Sondermann P, Pincetic A, Maamary J, Lammens K, Ravetch JV (2013) General mechanism for
modulating immunoglobulin effector function. Proc Natl Acad Sci U S A 110(24):9868–9872
Sonneveld ME, Natunen S, Sainio S, Koeleman CA, Holst S, Dekkers G, Koelewijn J, Partanen J,
van der Schoot CE, Wuhrer M, Vidarsson G (2016) Glycosylation pattern of anti-platelet IgG
is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia. Br J
Haematol 174(2):310–320. https://doi.org/10.1111/bjh.14053
Sonneveld ME, Koelewijn J, de Haas M, Admiraal J, Plomp R, Koeleman CA, Hipgrave
Ederveen AL, Ligthart P, Wuhrer M, van der Schoot CE, Vidarsson G (2017) Antigen
specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby
severity of haemolytic disease of the fetus and newborn. Br J Haematol 176(4):651–660.
https://doi.org/10.1111/bjh.14438
Spirig R, Campbell IK, Koernig S, Chen CG, Lewis BJB, Butcher R, Muir I, Taylor S, Chia J,
Leong D, Simmonds J, Scotney P, Schmidt P, Fabri L, Hofmann A, Jordi M, Spycher MO,
Anti-inflammatory Activity of IgG-Fc 61

Cattepoel S, Brasseit J, Panousis C, Rowe T, Branch DR, Baz Morelli A, Kasermann F,

Zuercher AW (2018) rIgG1 Fc Hexamer inhibits antibody-mediated autoimmune disease via
effects on complement and FccRs. J Immunol 200(8):2542–2553. https://doi.org/10.4049/
jimmunol.1701171
Steiniger B, Barth P, Herbst B, Hartnell A, Crocker PR (1997) The species-specific structure of
microanatomical compartments in the human spleen: strongly sialoadhesin-positive macro-
phages occur in the perifollicular zone, but not in the marginal zone. Immunology 92(2):307–
316
Strait RT, Morris SC, Finkelman FD (2006) IgG-blocking antibodies inhibit IgE-mediated
anaphylaxis in vivo through both antigen interception and FccRIIb cross-linking. J Clin Invest
116(3):833–841. https://doi.org/10.1172/JCI25575
Subedi GP, Barb AW (2016) The immunoglobulin G1 N-glycan composition affects binding to
each low affinity Fcc receptor. mAbs 8(8):1512–1524. https://doi.org/10.1080/19420862.2016.
1218586
Tackenberg B, Jelcic I, Baerenwaldt A, Oertel WH, Sommer N, Nimmerjahn F, Lunemann JD
(2009) Impaired inhibitory Fcc receptor IIB expression on B cells in chronic inflammatory
demyelinating polyneuropathy. Proc Natl Acad Sci U S A 106(12):4788–4792. https://doi.org/
10.1073/pnas.0807319106
Teeling JL, Jansen-Hendriks T, Juijpers TW, de Haas M, van de Winkel JG, Hack CE,
Bleeker WK (2001) Therapeutic efficacy of intravenous immunoglobulin preparations depends
on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood
98(4):1095–1099
Thiruppathi M, Sheng JR, Li L, Prabhakar BS, Meriggioli MN (2014) Recombinant IgG2a Fc
(M045) multimers effectively suppress experimental autoimmune myasthenia gravis.
J Autoimmun 52:64–73. https://doi.org/10.1016/j.jaut.2013.12.014
Tjon AS, van Gent R, Jaadar H, Martin van Hagen P, Mancham S, van der Laan LJ, te
Boekhorst PA, Metselaar HJ, Kwekkeboom J (2014) Intravenous immunoglobulin treatment in
humans suppresses dendritic cell function via stimulation of IL-4 and IL-13 production.
J Immunol 192(12):5625–5634. https://doi.org/10.4049/jimmunol.1301260
Trbojevic Akmacic I, Ventham NT, Theodoratou E, Vuckovic F, Kennedy NA, Kristic J,
Nimmo ER, Kalla R, Drummond H, Stambuk J, Dunlop MG, Novokmet M, Aulchenko Y,
Gornik O, Campbell H, Pucic Bakovic M, Satsangi J, Lauc G, Consortium I-B (2015)
Inflammatory bowel disease associates with proinflammatory potential of the immunoglobulin
G glycome. Inflamm Bowel Dis 21(6):1237–1247. https://doi.org/10.1097/MIB.
0000000000000372
Tsurikisawa N, Saito H, Oshikata C, Tsuburai T, Akiyama K (2012) High-dose intravenous
immunoglobulin treatment increases regulatory T cells in patients with eosinophilic
granulomatosis with polyangiitis. J Rheumatol 39(5):1019–1025. https://doi.org/10.3899/
jrheum.110981
Tsurikisawa N, Saito H, Oshikata C, Tsuburai T, Akiyama K (2014) High-dose intravenous
immunoglobulin therapy for eosinophilic granulomatosis with polyangiitis. Clin Transl Allergy
4(38)
Vaccaro C, Zhou J, Ober RJ, Ward ES (2005) Engineering the Fc region of immunoglobulin G to
modulate in vivo antibody levels. Nat Biotechnol 23(10):1283–1288. https://doi.org/10.1038/
nbt1143
van de Geijn FE, Wuhrer M, Selman MH, Willemsen SP, de Man YA, Deelder AM, Hazes JM,
Dolhain RJ (2009) Immunoglobulin G galactosylation and sialylation are associated with
pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from
a large prospective cohort study. Arthritis Res Ther 11(6):R193. https://doi.org/10.1186/ar2892
Viard I, Wehrli P, Bullani R, Schneider P, Holler N, Salomon D, Hunziker T, Saurat JH,
Tschopp J, French LE (1998) Inhibition of toxic epidermal necrolysis by blockade of CD95
with human intravenous immunoglobulin. Science 282(5388):490–493
Vidarsson G, Dekkers G, Rispens T (2014) IgG subclasses and allotypes: from structure to effector
functions. Front Immunol 5:520. https://doi.org/10.3389/fimmu.2014.00520
62 C. Beneduce et al.

Vuckovic F, Kristic J, Gudelj I, Teruel M, Keser T, Pezer M, Pucic-Bakovic M, Stambuk J,

Trbojevic Akmacic I, Barrios C, Pavic T, Menni C, Wang Y, Zhou Y, Cui L, Song H, Zeng Q,
Guo X, Pns-Estel BA, Mckeigue P, Leslie Patrick A, Gornik O, Spector TD, Harjacek M,
Alarcon-Riquelme M, Molokhia M, Wang W, Luac G (2015) Association of systemic lupus
erythematosus with decreased immunosuppressive potential of the IgG glycome. Arthritis
Rheumatol 67(11):2798–3789
Washburn N, Schwab I, Ortiz D, Bhatnagar N, Lansing JC, Medeiros A, Tyler S, Mekala D,
Cochran E, Sarvaiya H, Garofalo K, Meccariello R, Meador JW 3rd, Rutitzky L, Schultes BC,
Ling L, Avery W, Nimmerjahn F, Manning AM, Kaundinya GV, Bosques CJ (2015)
Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced
anti-inflammatory activity. Proc Natl Acad Sci U S A 112(11):E1297–E1306. https://doi.org/
10.1073/pnas.1422481112
Watson M, Rudd PM, Bland M, Dwek RA, Axford JS (1999) Sugar printing rheumatic diseases: a
potential method for disease differentiation using immunoglobulin G oligosaccharides.
Arthritis Rheum 42(8):1682–1690
Wong AH, Fukami Y, Sudo M, Kokubun N, Hamada S, Yuki N (2016) Sialylated IgG-Fc: a novel
biomarker of chronic inflammatory demyelinating polyneuropathy. J Neurol Neurosurg
Psychiatry 87(3):257–259. https://doi.org/10.1136/jnnp2014-309964
Wuhrer M, Porcelijn L, Kapur R, Koeleman CA, Deelder AM, De Haas M, Vidarsson G (2009)
Regulated glycosylation patterns of IgG during alloimmune responses against human platelet
antigens. J Proteome Res 8(2):450–456
Wuhrer M, Selman MH, McDonnell LA, Kumpfel T, Derfuss T, Khademi M, Olsson T,
Hohlfeld R, Meinl E, Krumbholz M (2015a) Pro-inflammatory pattern of IgG1 Fc
glycosylation in multiple sclerosis cerebrospinal fluid. J Neuroinflammation 12:235. https://
doi.org/10.1186/s12974-015-0450-1
Wuhrer M, Stavenhagen K, Koeleman CA, Selman MH, Harper L, Jacobs BC, Savage CO,
Jefferis R, Deelder AM, Morgan M (2015b) Skewed Fc glycosylation profiles of
anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis
patients show low levels of bisection, galactosylation, and sialylation. J Proteome Res 14
(4):1657–1665. https://doi.org/10.1021/pr500780a
Zhang G, Massaad CA, Gao T, Pillai L, Bogdanova N, Ghauri S, Sheikh KA (2016) Sialylated
intravenous immunoglobulin suppress anti-ganglioside antibody mediated nerve injury. Exp
Neurol 282:49–55. https://doi.org/10.1016/j.expneurol.2016.05.020
Zuercher AW, Spirig R, Baz Morelli A, Kasermann F (2016) IVIG in autoimmune disease—
potential next generation biologics. Autoimmun Rev 15(8):781–785. https://doi.org/10.1016/j.
autrev.2016.03.018
IgG Fc Glycosylation in Human
Immunity

Taia T. Wang

Contents

1 Introduction.......................................................................................................................... 64
2 Structure and Assembly of Fc Glycans .............................................................................. 65
3 IgG Fc Glycans in Inflammation ........................................................................................ 67
4 IgG Fc Glycans and Adaptive Immunity............................................................................ 69
5 Heterogeneity and Regulation of Fc Glycoforms............................................................... 70
6 Concluding Remarks ........................................................................................................... 72
References .................................................................................................................................. 72

Abstract Glycosylation of IgG Fc domains is a central mechanism in the diver-

sification of antibody function. Modifications to the core Fc glycan impact antibody
function by shifting the balance of Type I and Type II Fc gamma receptors (FccR)
that will be engaged by immune complexes. This, in turn, modulates the effector
cells and functions that can be recruited during immune activation. Critically,
humans have evolved to regulate Fc glycan modifications for immune homeostasis.
Dysregulation in Fc glycan modifications can lead to loss of immune tolerance,
symptomatic autoimmunity, and susceptibility to infectious diseases. Here, we
discuss IgG Fc glycosylation and its role in human health and disease.

T. T. Wang (&)
Department of Medicine, Division of Infectious Diseases, Department of Microbiology
and Immunology, Program in Immunology, Stanford University School of Medicine,
Stanford University, Stanford, CA 94305, USA
e-mail: [email protected]
T. T. Wang
Chan Zuckerberg Biohub, San Francisco, CA, USA

Current Topics in Microbiology and Immunology (2019) 423: 63–75

https://doi.org/10.1007/82_2019_152
© Springer Nature Switzerland AG 2019
Published online: 26 February 2019
64 T. T. Wang

1 Introduction

Recent studies have defined the considerable heterogeneity that exists among
people in specific IgG Fc glycan modifications that impact antibody activity in vivo
(Selman et al. 2012a, b; Wang et al. 2015, 2017; Mahan et al. 2016; Mahan et al.
2015). Intriguingly, individuals produce distinct basal repertoires of Fc glycoforms
which are quite stable over periods of weeks to months (Wang et al. 2015). Because
Fc glycan modifications directly affect the ability of IgGs to recruit various effector
cell populations, this heterogeneity is likely a significant driver of immune diversity
across the population.
The ability of IgG antibodies to mediate effector functions arises from their
capacity to bridge antigen binding through the Fab domain with the recruitment of
effector cells through interactions between the Fc domain and FccRs. Because the
majority of FccRs have low affinity for monomeric IgGs, Fc-FccR interactions
occur when multivalent IgG-antigen immune complexes are formed, thus enabling
avidity-based interactions and conferring specificity to the effector cell response.
The structure of the Fc domains contained in a given immune complex deter-
mines which effector cells and FccRs can be engaged by the complex. Fc structure,
in turn, is determined by two variables: the IgG subclass and the composition of a
complex biantennary glycan that is present on all IgG heavy chains within the CH2
domain. Four IgG subclasses are found in humans (IgG1-4), with IgG1 and IgG3
having highest affinity for activating Type I FccRs (FccRI, FccRIIa, FccRIIIa). In
contrast, IgG2 has highest affinity of all subclasses for the inhibitory FccR, FccRIIb
(Fig. 1).
The activity of different IgG subclasses is further tuned by modifications to the
Fc glycan which can impart diverse and potent effector functions to IgG1s and
likely to the other subclasses, though how Fc glycosylation impacts the activity of
IgG2, IgG3 and IgG4 has yet to be described. Overall, the composition of IgG

Fig. 1 Heterogeneity in the human IgG Fc domain repertoire. IgG repertoires vary across the
population by ratios of activating to inhibitory IgG subclasses ((IgG1+IgG3)/IgG2) and in the
abundance of Fc glycoforms that impact Fc domain structure and antibody function. Fucosylated,
sialylated Fc glycoforms impart reduced Type I FccR binding activity and enable binding to the
Type II FccRs. Afucosylated, sialylated or asialylated Fc glycans mediate pro-inflammatory
effector functions by virtue of increased affinity for the activating Type I FccR, FccRIIIa
IgG Fc Glycosylation in Human Immunity 65

subclasses and Fc glycans within immune complexes determines whether they will
trigger pro- or anti-inflammatory effector cell activity and regulates the quality of
the adaptive immune response against the antigen(s) in complex (Wang et al. 2015;
Regnault et al. 1999; Getahun et al. 2004; de Jong et al. 2006; Ding et al. 2016;
Hjelm et al. 2008).

2 Structure and Assembly of Fc Glycans

Mature Fc glycoforms are N-linked, complex biantennary structures, present at

asparagine 297 of all heavy chains (Kao et al. 2015). The core Fc glycan is composed
of seven saccharides and is required for maximal binding to FccRs: 4 N-acet-
ylglucosamine (GlcNAc) and 3 mannose (Man) residues (Lux et al. 2013). This core
glycan can be modified by additional sugars, including a core fucose (Fuc), bisecting
GlcNAc, galactose (Gal) at one or both arms and, in the presence of galactose,
N-acetylneuraminic acid (NeuAc) or sialic acid (Fig. 2). Two modifications to the
IgG1 Fc, fucosylation and sialylation, have well defined functions in vivo and will be
discussed in more detail below. How bisecting GlcNAc impacts IgG function is not
yet well understood. Some studies indicate a role for bisection in modulation of
FccRIIIa-mediated activities, however data on this are not consistent and any phe-
notype related to FcgRIIIa binding is clearly less pronounced than what can be
achieved through afucosylation of Fc glycans (Hodoniczky et al. 2005; Shinkawa
et al. 2003). Galactosylation of the Fc is significant as a precursor to sialylation but
does not significantly limit the abundance of Fc sialylation as galactosylation occurs
with several fold greater frequency than sialylation (Wang et al. 2015; Wuhrer et al.

Fig. 2 Structure of IgG Fc glycans. Fc glycoforms are present at Asn 297 within CH2 domains
and are complex biantennary glycans. The core Fc glycan (boxed in red) is composed of seven
saccharides: 4 N-acetylglucosamine (GlcNAc) and 3 mannose (Man) residues. This core glycan
can be modified by a core fucose (Fuc), bisecting GlcNAc, galactose (Gal) at one or both arms and,
in the presence of galactose, N-acetyl-neuraminic acid or sialic acid (NeuAc)
66 T. T. Wang

Table 1 Set of 11 Core glycan modification IgG1 Fc (% total)

modifications to the core Fc
glycan which, together, G0 0.18
comprise *90% of human G1 2.04
IgG1 Fc glycoforms. Relative G2 3.16
quantification of the 11 G0F 1.72
modifications is shown.
G1F 27.11
G: Galactose; 0,1,2 indicate
the number of Gal residues; G2F 38.28
F: Fucose; N: bisecting G0FN 0.72
GlcNAc; S: sialic acid. G1FN 7.00
Data are from a commercial
G2FN 0.81
IVIg preparation
G1FS 1.03
G2FS 17.96
100.00

2015). A direct role for galactosylated Fc glycans in modulation of immune function

has not been defined. Overall, 11 distinct complex biantennary Fc glycoforms
comprise *90% of the human IgG1 repertoire (Table 1). In addition to complex
biantennary glycans, up to *2% of Fc glycans on serum IgG1 may be a combination
of hybrid and high mannose forms (Flynn et al. 2010 and Wang, unpublished
observation).
Assembly of Fc glycans begins with oligomannose forms that are pre-assembled
on a lipid, polyprenol dolichol pyrophosphate. The precursor Fc glycan is assem-
bled first by transfer of sugar residues on the cytoplasmic face of the endoplasmic
reticulum (ER), followed by transport of the partial precursor across the ER
membrane to the luminal side where the full oligomannose N-glycan precursor is
assembled. The triantennary precursor is comprised of two GlcNAc, nine Man and
three glucose (Glc) residues. Within the ER, this Glc3Man9GlcNac2 oligosac-
charide is transferred to Asn 297 of the IgG heavy chain. The Fc glycan then
undergoes remodeling by membrane-anchored glycosidases and glycosyltrans-
ferases within the ER and the golgi apparatus, ultimately forming the mature,
complex, biantennary glycan structure (Fig. 2). Addition of bisecting GlcNAc is
accomplished by the activity of b-1,4-N-Acetylglucosaminyltransferase III
(GNT-III) while addition of a core fucose is performed by a-1,6-fucosyltransferase
(FUT8). It has been observed that FUT8 activity is inhibited by the presence of
bisecting GlcNAc. Thus, fucosylation is likely the initial modification in glycans
that are both fucosylated and bisected. Increasing the abundance of afucosylated Fc
glycoforms in in vitro expression systems has been achieved by increasing
expression of GNT-III and/or by engineering localization of GNT-III to the upper
golgi where it can act prior to FUT8 (Ferrara et al. 2006). Galactosylation of one or
both arms of the bisected Fc glycan is performed by b-1,4-galactosyltransferase 1
(B4GALT1) and, in the presence of galactosylation, terminal sialylation is cat-
alyzed by a-2,6-sialyltransferase 1 (ST6GAL1) which preferentially adds sialic acid
to the a-1,3 arm of the biantennary glycan (Barb et al. 2009).
IgG Fc Glycosylation in Human Immunity 67

3 IgG Fc Glycans in Inflammation

Antibody-mediated inflammatory responses are central in protection against infec-

tious diseases and malignancies. While inflammatory effector functions can protect
against disease, dysregulation of the response can result in autoimmunity or other
pathologic inflammation. Balanced signaling through Type I and Type II FccRs is
required for moderation of inflammatory responses. This balance is achieved, in part,
through regulated sialylation of Fc glycans, which is the determinant of binding to
Type II FccRs; it is based on the ability to interact with sialylated immune com-
plexes that Type I and Type II FccRs are distinguished. Fc sialylation has the effect
of reducing binding to Type I FccRs while enabling engagement of Type II FccRs.
Increased Type II FccR signaling due to the presence of sialylated Fc glycans
can trigger potent anti-inflammatory activity. A classic example of this is observed
with administration of high dose intravenous immunoglobulin (IVIg) during acute
inflammatory diseases such as immune thrombocytopenia, Kawasaki disease,
chronic inflammatory demyelinating polyneuropathy and Guillain-Barre syndrome.
IVIg is pooled IgG from thousands of donors and its anti-inflammatory activity is
mediated by the minor subset of IgGs within the pool that contain sialylated Fcs
(Imbach et al. 1981; Debre et al. 1993; Kaneko et al. 2006). Anti-inflammatory
activity can be increased by enhancing the abundance of sialylated Fcs within the
IgG pool (Washburn et al. 2015). Sialylated Fcs in IVIg signal through the Type II
FccR DC-SIGN on regulatory macrophages leading to production of interleukin 33
(IL-33). IL-33, in turn, triggers release of IL-4 by basophils, resulting in increased
FccRIIb expression on effector myeloid cells, including monocytes and macro-
phages at sites of inflammation (Anthony et al. 2011; Schwab et al. 2012; Schwab
et al. 2014). A recent study of in vivo sialylation by recombinantly produced
glycosyltransferases found that increasing sialylation of endogenous IgG could treat
autoimmune sequelae in a similar pathway that required DC-SIGN and FccRIIb
(Pagan et al. 2017). Regulated expression of FccRIIb constitutes a powerful
mechanism for controlling inflammation, with increased FccRIIb expression ele-
vating the threshold of cellular activation and effectively reducing the inflammatory
response by these cells (Santiago-Raber et al. 2009; Dhodapkar et al. 2007; Kaneko
et al. 2006; Boruchov et al. 2005; Lehmann et al. 2012). In addition to the
DC-SIGN/FccRIIb pathway, IL-33 release induced by administration of sialylated
Fcs can trigger a potent regulatory T cell response that is sufficient to control T cell
mediated inflammation in the experimental autoimmune encephalomyelitis model
(Fiebiger et al. 2015). Fc glycan sialylation is thus a central regulator of inflam-
mation. Interestingly, differences in baseline Fc sialylation among people can
exceed 20% in health, a magnitude well within the range that is associated with
changes in Type II FccR-mediated processes and inflammatory activity (Wang et al.
2015; Pfeifle et al. 2016; Maamary et al. 2017; Fokkink et al. 2014). This implicates
Fc sialylation as a driver of diversity in inflammatory thresholds among people.
Another mechanism by which IgG Fc glycoforms act in homeostatic regulation
of inflammation involves the Type I FccR, FccRIIIa. This activating receptor is
68 T. T. Wang

expressed on NK cells and subsets of monocytes and macrophages where it

mediates activities including cell cytotoxicity, effector cell activation, inflammatory
cytokine production and is a minor mediator of phagocytosis (relative to FccRIIa)
(Rafiq et al. 2013; Shalova et al. 2012; Yeap et al. 2016). Dysregulated FccRIIIa
activity is associated with pathologic sequelae such as alloantibody mediated dis-
eases in neonates (Kapur et al. 2014; Sonneveld et al. 2016; Wuhrer et al. 2009) and
progression to severe disease and autoimmune pathology during dengue infection.
A critical mechanism for controlling signaling through FccRIIIa in vivo is through
regulation of Fc glycan fucosylation. Specifically, IgG1 Fc glycans lacking a core
fucose residue (afucosylated Fc glycans) have higher affinity for FccRIIIa (and
FccRIIIb) due to an unusual, stabilizing sugar–sugar interaction (Ferrara et al.
2011). While afucosylation impacts the affinity of both FccRIIIa and FccRIIIb,
FccRIIIb is expressed solely on neutrophils and lacks signaling capacity; thus, the
major role of fucosylation on antibody activity is through modulation of FccRIIIa
binding. Afucosylated IgG1 antibodies have approximately 5-fold or 2-fold
enhanced affinity for the low (F158) and high (V158) isoforms of FccRIIIa,
respectively.
The role of afucosylated Fc glycoforms in modulating inflammatory responses is
well demonstrated in dengue infections, where antibodies play a direct role in
mediating the severe forms of disease, dengue hemorrhagic fever and dengue shock
syndrome. While anti-dengue antibodies can protect, they can also trigger pro-
gression to these severe disease states that cause a majority of dengue-associated
deaths. Disease progression is largely mediated by virus particles that are bound by
IgGs that don’t neutralize the virus. Instead, the virus immune complexes increase
virus infection and modulate cytokine production in FccR-bearing cells (Halstead
2009). Risk for severe dengue is nearly doubled in individuals who produce high
levels (  10%) of afucosylated Fc glycans, implicating FccRIIIa in the patho-
genesis of severe dengue (Wang et al. 2017). In healthy adults, afucosylated of
IgG1 Fc glycans are nearly always found at levels <10% of all glycoforms but can
reach >20% in some individuals. During actue dengue infections, afucosylation of
IgG1 is elevated and can approach 30% in some individuals with severe disease
(Wang et al. 2017).
One mechanism by which afucosylated IgGs impact dengue disease is through
the activity of antibodies that cross-react between the dengue non-structural protein
1 (NS1) and a platelet antigen (Wan et al. 2016). Highly afucosylated anti-NS1
IgGs can mediate platelet loss in vivo, a defining feature of severe dengue disease.
Aside from mediating platelet loss, increased afucosylated anti-dengue IgGs during
acute infection correlate with increases in hematocrit, the hallmark of plasma leak
which defines severe dengue (Wang et al. 2017). This association suggests an
additional role for afucosylated Fc glycoforms and FccRIIIa in severe dengue
through an inflammatory cascade that results in vascular permeability. The role of
afucosylated IgGs in dengue infection is a clear example of how the basal Fc
glycoform repertoire can drive heterogeneity in human disease susceptibilities and
outcomes.
IgG Fc Glycosylation in Human Immunity 69

4 IgG Fc Glycans and Adaptive Immunity

Immune complex interactions with FccRs mediate several cellular processes

involved in the maturation of high affinity antibody responses. These include:
efficient transport of antigen to the germinal center, processing and presentation of
antigens to T cells, and selection of B cells based on affinity of the B cell receptor
(Wang et al. 2015; Regnault et al. 1999; Getahun et al. 2004; de Jong et al. 2006;
Ding et al. 2016; Hjelm et al. 2008). The role of sialylated Fc glycans within
immune complexes in B cell selection was recently discovered and involves a
mechanism whereby Type I and Type II FccRs act in cis to promote selection of B
cells with high affinity B cell receptors (BCRs) (Wang et al. 2015; Maamary et al.
2017).
The mechanism by which sialylated Fc glycans impact B cell selection relies on
the activity of FccRIIb in regulating thresholds for B cell activation. Expression of
activating Type I FccRs is nearly always coupled to expression of the inhibitory
Type I FccRIIb; this combined expression ensures balanced signaling and is
required by most leukocyte types for specificity of cellular maturation and effector
activities (Boruchov et al. 2005; Clynes et al. 1999; Dhodapkar et al. 2005;
McGaha et al. 2005). B cells represent an important exception to this rule as they
express FccRIIb throughout development, without co-expression of activating
FccRs. FccRIIb on B cells acts to moderate the cell-activating signals that are
triggered by antigen binding to the B cell receptor (BCR). Thus, a key variable in
regulation of B cell activation is the expression level of FccRIIb, which changes
with development of B cells, but is also inducible. In the absence of inhibitory
FccRIIb or with low-level expression or signaling, B cells lack appropriate acti-
vation thresholds and produce higher titer, low avidity IgGs. The activation
thresholds defined through FccRIIb signaling are in fact required for maintenance
of immune tolerance, with autoantibody production observed in FccRIIb-/- mice and
low levels of B cell FccRIIb found in patients with autoantibody mediated diseases
(McGaha et al. 2005; Fukuyama et al. 2005; Kono et al. 2005; Li et al. 2014; Su
et al. 2004; Tackenberg et al. 2009; Mackay et al. 2006; Baerenwaldt et al. 2011).
FccRIIb signaling alone on B cells, as can occur in the presence of immune
complexes formed from antigens that are not reactive with the BCR, induces
pro-apoptotic signals. This pro-apoptotic signaling is attenuated by BCR engage-
ment, ensuring preservation of cells that can respond to relevant antigens (Ono et al.
1997; Pearse et al. 1999).
As FccRIIb is a critical determinant of B cell selection, regulation of its
expression over time is essential. Temporal association of B cell FccRIIb expres-
sion with the presence of antigen is achieved, in part, by regulation of Fc sialyation
on antigen-specific IgGs. This was discovered in studies that characterized a shift
towards sialylated Fc glycan production on anti-hemagglutinin (HA) IgGs by day 7
post influenza virus vaccination (Selman et al. 2012a; Wang et al. 2015). To
determine the role of sialylated immune complexes on maturation of the vaccine
response, experiments were done which found that sialylated immune complexes
70 T. T. Wang

could act through the Type II FccR, CD23, to induce increased expression of
FccRIIb on B cells. Elevated FccRIIb, in turn, increases the threshold for activation
of B cells that occurs with BCR signaling, resulting in selection of higher affinity,
antigen-specific B cells. This mechanism can be recapitulated experimentally by
administration of sialylated immune complexes to mice, leading to increased
FccRIIb on germinal center B cells and production of higher affinity antibodies in a
CD23-dependent manner (Wang et al. 2015; Maamary et al. 2017). How other
modulations in Fc glycoforms that occur following vaccination impact maturation
of the vaccine response has yet to be discovered.

5 Heterogeneity and Regulation of Fc Glycoforms

While the regulators of Fc glycan modifications are not well understood, it is clear
that multiple mechanisms impact the Fc glycoform repertoire in humans. One type
of regulation is apparent at the level of antigen specificity. An example of this is
observed in the antibody response against the influenza HA, in which anti-HA
globular head IgGs are significantly more sialylated and fucosylated than
stalk-reactive IgGs (Wang et al. 2015). Significant differences in IgG1 Fc sialyla-
tion are also observed between IgGs that react with H1 or H3 subtype HA gly-
coproteins (Wang, unpublished observation). Total IgG Fc glycans (all subclasses)
also vary with Fab specificity when comparing IgGs reactive with different HIV
proteins in infected individuals (Mahan et al. 2016). Other examples of correlations
between Fc glycosylation and Fab specificity have been found in autoimmune
diseases such as granulomatosis with polyangiitis (GPA) and rheumatoid arthritis
(RA). These diseases are marked by the presence of autoantibodies such as anti—
PR3 and anti-ACPA, found in GPA and RA respectively. Anti-PR3 and anti-ACPA
can have reduced Fc sialylation over total IgG and may contribute to disease
pathogenesis during disease flares through increased Type I FccR-mediated cellular
activation (Wuhrer et al. 2015; Kaneko et al. 2006; Scherer et al. 2010).
Distinctions in Fc glycosylation that are linked to Fab specificity are likely, in
part, a consequence of selective glycosylation by different IgG-producing B cell
subsets. This is supported by the finding that plasmablasts and memory B cells
produce distinct amounts of ST6GAL1 and FUT8, the glycosyltransferases
responsible for Fc sialylation and fucosylation, respectively (Wang et al. 2015).
Expansion of plasmablasts, which express higher levels of ST6GAL1 and FUT8 in
the days following vaccination, is mirrored by production of highly sialylated,
fucosylated anti-HA IgGs. In contrast, anti-stalk IgGs, which are likely to be
derived from the memory B cell pool, are modified by lower levels of Fc sialylation
and fucosylation (Wang et al. 2015).
A second level of Fc glycan regulation occurs with the IgG subclass. Fc gly-
coforms are distinct depending on the IgG subclass with IgG1 trending toward
lower sialylation and fucosylation than other subclasses (Wuhrer et al. 2015; Wang,
IgG Fc Glycosylation in Human Immunity 71

unpublished observation). The activity of various Fc glycoforms on IgG2, IgG3 and

IgG4 subclasses is not yet known however, so how this differential glycosylation
contributes to immune homeostasis remains to be discovered.
In addition to differences in Fc glycosylation that occur with Fab specificity or
IgG subclass, Fc glycan repertoires vary with the physiologic compartment from
which the IgG was isolated. For example, IgGs from the cerebrospinal fluid of
patients with multiple sclerosis have reduced sialylation compared with matched
donor serum IgGs (Wuhrer et al. 2015). Another study found that Fc galactosyla-
tion, the precursor to sialylation, is reduced on synovial fluid IgGs relative to serum
IgGs in RA patients (Scherer et al. 2010). The basis of differences in Fc glycosy-
lation associated with physiologic compartments is not known but may involve
production by distinct B cell subsets or variation in the availability of saccharide
precursors.
While Fc glycan modifications are generally believed to occur within B cells, a
topic of some debate is whether any sialylation of Fc glycans occurs extracellularly,
in the absence of exogenously administered glycosyltrasferases (Jones et al. 2012,
2016). In favor of sialylation within B cells, human B cell ST6GAL1 and the
abundance of sialylation on secreted IgGs can be modulated by cytokines present in
cell culture (Selman et al. 2012a; Wang et al. 2011) or increased in vivo by estrogen
treatment in postmenopausal patients with rheumatoid arthritis (Engdahl et al.
2018). In addition, human B cell ST6GAL1 expression correlates with Fc sialy-
lation after vaccination (Wang et al. 2015). Another recent study showed that
ST6Gal1 is regulated by IL-23 in antibody producing cells (APCs). This regulation
was a consequence of IL-23 acting on TH17 cells which suppressed ST6Gal1
expression in an IL-21 and IL-22-dependent pathway (Pfeifle et al. 2016). Overall,
data supporting an extracellular mechanism in humans have not yet been described
but several factors implicate a dominant B cell intrinsic pathway.
Finally, an important variable among studies of Fc glycosylation in humans
relates to the methods used for glycan measurement. In order to interpret data for
sound hypothesis generation, the analysis should reflect the Fc domain repertoire
that would be present in relevant immune complexes. This requires that the analysis
be (1) antigen specific, (2) IgG subclass specific and (3) Fc domain specific.
Antigen specific analysis is important because of the segregation of glycans by Fab
specificity. Similarly, subclass specific analysis is critical because Fc glycoforms
vary by subclass and because the activity of different glycoforms on IgG2, IgG3
and IgG4 antibodies is unknown, as discussed above. Thus, at this time, only data
on IgG1 Fc glycosylation is truly interpretable for hypothesis generation.
Importantly, bulk IgG glycan analysis cannot be used for hypothesis generation
because of the lack of antigen and subclass specificity but also because approxi-
mately 20% of IgGs are glycosylated within their Fab domains and those glyco-
forms are entirely distinct in repertoire from those that modify the Fc (Mimura et al.
2007). Therefore, a detailed study of the methods used for glycan analysis is
required of readers who wish to interpret published studies on IgG glycosylation.
72 T. T. Wang

6 Concluding Remarks

IgG Fc glycan modifications are a key regulator of antibody activity in vivo. While
Fc sialylation has an active role in anti-inflammatory signaling and in maturation of
adaptive immune responses, fucosylation of the Fc is regulated to drive appropriate
pro-inflammatory effector cell functions. A critical question for future studies to
address is how the human Fc glycoform repertoire is regulated. Understanding this
will be necessary to shift endogenous Fc glycosylation for therapeutic benefit,
which could potentially be done to treat diseases responsive to IVIg therapy, to
enhance the activity of anti-tumor antibodies, to prevent autoantibody-mediated
tissue disease, to enhance the quality of antibodies generated during vaccination or
to reduce the mortality associated with dengue virus infections. The ability to direct
shifts in the Fc domain repertoire, in both IgG subclass distribution and Fc gly-
cosylation, is an exciting prospect for treating and preventing human diseases
through tuning of the interaction between antibodies and their receptors.

Acknowledgements Support was received from Stanford University, the Chan Zuckerberg
Biohub and the Searle Scholars Program. Research reported in this publication was supported in
part by the Bill & Melinda Gates Foundation (OPP1188461) and the National Institutes of Health
(1R01AI139119-01A1, 5K22AI12347802, 5U19AI111825-05, UL1TR001866).

References

Anthony RM et al (2011) Intravenous gammaglobulin suppresses inflammation through a novel T

(H)2 pathway. Nature 475(7354):110–113
Baerenwaldt A et al (2011) Fcc receptor IIB (FccRIIB) maintains humoral tolerance in the human
immune system in vivo. Proc Natl Acad Sci U S A 108(46):18772–18777
Barb AW, Brady EK, Prestegard JH (2009) Branch-specific sialylation of IgG-Fc glycans by
ST6Gal-I. Biochemistry 48(41):9705–9707
Boruchov AM et al (2005) Activating and inhibitory IgG Fc receptors on human DCs mediate
opposing functions. J Clin Invest 115(10):2914–2923
Clynes R et al (1999) Modulation of immune complex-induced inflammation in vivo by the
coordinate expression of activation and inhibitory Fc receptors. J Exp Med 189(1):179–185
de Jong JM et al (2006) Dendritic cells, but not macrophages or B cells, activate major
histocompatibility complex class II-restricted CD4+ T cells upon immune-complex uptake
in vivo. Immunology 119(4):499–506
Debre M et al (1993) Infusion of Fcc fragments for treatment of children with acute immune
thrombocytopenic purpura. Lancet 342(8877):945–949
Dhodapkar KM et al (2005) Selective blockade of inhibitory Fcc receptor enables human dendritic
cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells. Proc
Natl Acad Sci U S A 102(8):2910–2915
Dhodapkar KM et al (2007) Selective blockade of the inhibitory Fcc receptor (FccRIIB) in human
dendritic cells and monocytes induces a type I interferon response program. J Exp Med 204
(6):1359–1369
Ding Z et al (2016) IgE-mediated enhancement of CD4(+) T cell responses requires antigen
presentation by CD8a(-) conventional dendritic cells. Sci Rep 6:28290
IgG Fc Glycosylation in Human Immunity 73

Endy TP et al (2004) Relationship of preexisting dengue virus (DV) neutralizing antibody levels to
viremia and severity of disease in a prospective cohort study of DV infection in Thailand.
J Infect Dis 189(6):990–1000
Engdahl C et al (2018) Estrogen induces St6gal1 expression and increases IgG sialylation in mice
and patients with rheumatoid arthritis: a potential explanation for the increased risk of
rheumatoid arthritis in postmenopausal women. Arthritis Res Ther 20(1):84
Ferrara C et al (2006) Modulation of therapeutic antibody effector functions by glycosylation
engineering: influence of Golgi enzyme localization domain and co-expression of heterologous
b1, 4-N-acetylglucosaminyltransferase III and Golgi a-mannosidase II. Biotechnol Bioeng 93
(5):851–861
Ferrara C et al (2011) Unique carbohydrate–carbohydrate interactions are required for high affinity
binding between FccRIII and antibodies lacking core fucose. Proc Natl Acad Sci U S A 108
(31):12669–12674
Fiebiger BM et al (2015) Protection in antibody- and T cell-mediated autoimmune diseases by
antiinflammatory IgG Fcs requires type II FcRs. Proc Natl Acad Sci U S A 112(18):E2385–
E2394
Flynn GC et al (2010) Naturally occurring glycan forms of human immunoglobulins G1 and G2.
Mol Immunol 47(11–12):2074–2082
Fokkink WJ et al (2014) IgG Fc N-glycosylation in Guillain-Barre syndrome treated with
immunoglobulins. J Proteome Res 13(3):1722–1730
Fukuyama H, Nimmerjahn F, Ravetch JV (2005) The inhibitory Fcc receptor modulates
autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat
Immunol 6(1):99–106
Getahun A et al (2004) IgG2a-mediated enhancement of antibody and T cell responses and its
relation to inhibitory and activating Fcc receptors. J Immunol 172(9):5269–5276
Halstead SB (2009) Antibodies determine virulence in dengue. Ann N Y Acad Sci 1171(Suppl 1):
E48–E56
Hjelm F, Karlsson MC, Heyman B (2008) A novel B cell-mediated transport of IgE-immune
complexes to the follicle of the spleen. J Immunol 180(10):6604–6610
Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector
functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21(6):1644–1652
Imbach P et al (1981) High-dose intravenous gammaglobulin for idiopathic thrombocytopenic
purpura in childhood. Lancet 1(8232):1228–1231
Jones MB et al (2012) Anti-inflammatory IgG production requires functional P1 promoter in
b-galactoside a2,6-sialyltransferase 1 (ST6Gal-1) gene. J Biol Chem 287(19):15365–15370
Jones MB et al (2016) B-cell-independent sialylation of IgG. Proc Natl Acad Sci U S A 113
(26):7207–7212
Kaneko Y, Nimmerjahn F, Ravetch JV (2006a) Anti-inflammatory activity of immunoglobulin G
resulting from Fc sialylation. Science 313(5787):670–673
Kaneko Y et al (2006b) Pathology and protection in nephrotoxic nephritis is determined by
selective engagement of specific Fc receptors. J Exp Med 203(3):789–797
Kao D et al (2015) A monosaccharide residue is sufficient to maintain mouse and human IgG
subclass activity and directs IgG effector functions to cellular Fc receptors. Cell Rep 13
(11):2376–2385
Kapur R et al (2014) A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in
pregnancy. Blood 123(4):471–480
Kono H et al (2005) FccRIIB Ile232Thr transmembrane polymorphism associated with human
systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects
on B cell receptor signaling. Hum Mol Genet 14(19):2881–2892
Lehmann B et al (2012) FccRIIB: a modulator of cell activation and humoral tolerance. Expert
Rev Clin Immunol 8(3):243–254
Li F, Smith P, Ravetch JV (2014) Inhibitory Fcc receptor is required for the maintenance of
tolerance through distinct mechanisms. J Immunol 192(7):3021–3028
74 T. T. Wang

Lux A et al (2013) Impact of immune complex size and glycosylation on IgG binding to human
FccRs. J Immunol 190(8):4315–4323
Maamary J et al (2017) Increasing the breadth and potency of response to the seasonal influenza
virus vaccine by immune complex immunization. Proc Natl Acad Sci U S A
Mackay M et al (2006) Selective dysregulation of the FccIIB receptor on memory B cells in SLE.
J Exp Med 203(9):2157–2164
Mahan AE et al (2015) A method for high-throughput, sensitive analysis of IgG Fc and Fab
glycosylation by capillary electrophoresis. J Immunol Methods 417:34–44
Mahan AE et al (2016) Antigen-specific antibody glycosylation is regulated via vaccination. PLoS
Pathog 12(3):e1005456
McGaha TL, Sorrentino B, Ravetch JV (2005) Restoration of tolerance in lupus by targeted
inhibitory receptor expression. Science 307(5709):590–593
Mimura Y et al (2007) Contrasting glycosylation profiles between Fab and Fc of a human IgG
protein studied by electrospray ionization mass spectrometry. J Immunol Methods 326(1–
2):116–126
Ono M et al (1997) Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory
signaling. Cell 90(2):293–301
Pagan JD, Kitaoka M, Anthony RM (2017) Engineered sialylation of pathogenic antibodies
in vivo attenuates autoimmune disease. Cell
Pearse RN et al (1999) SHIP recruitment attenuates FccRIIB-induced B cell apoptosis. Immunity
10(6):753–760
Pfeifle R et al (2016) Regulation of autoantibody activity by the IL-23-TH17 axis determines the
onset of autoimmune disease. Nat Immunol
Rafiq S et al (2013) Comparative assessment of clinically utilized CD20-directed antibodies in
chronic lymphocytic leukemia cells reveals divergent NK cell, monocyte, and macrophage
properties. J Immunol 190(6):2702–2711
Regnault A et al (1999) Fcgamma receptor-mediated induction of dendritic cell maturation and
major histocompatibility complex class I-restricted antigen presentation after immune complex
internalization. J Exp Med 189(2):371–380
Santiago-Raber ML et al (2009) Fcc receptor-dependent expansion of a hyperactive monocyte
subset in lupus-prone mice. Arthritis Rheum 60(8):2408–2417
Scherer HU et al (2010) Glycan profiling of anti-citrullinated protein antibodies isolated from
human serum and synovial fluid. Arthritis Rheum 62(6):1620–1629
Schwab I et al (2012) IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and
SIGNR1. Eur J Immunol 42(4):826–830
Schwab I et al (2014) Broad requirement for terminal sialic acid residues and FccRIIB for the
preventive and therapeutic activity of intravenous immunoglobulins in vivo. Eur J Immunol 44
(5):1444–1453
Selman MH et al (2012a) Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and
tetanus vaccination. Mol Cell Proteomics 11(4):M111 014563
Selman MH et al (2012b) Fc specific IgG glycosylation profiling by robust nano-reverse phase
HPLC-MS using a sheath-flow ESI sprayer interface. J Proteomics 75(4):1318–1329
Shalova IN et al (2012) CD16 regulates TRIF-dependent TLR4 response in human monocytes and
their subsets. J Immunol 188(8):3584–3593
Shinkawa T et al (2003) The absence of fucose but not the presence of galactose or bisecting
N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of
enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278(5):3466–3473
Sonneveld ME et al (2016) Glycosylation pattern of anti-platelet IgG is stable during pregnancy
and predicts clinical outcome in alloimmune thrombocytopenia. Br J Haematol 174(2):310–
320
Su K et al (2004) A promoter haplotype of the immunoreceptor tyrosine-based inhibitory
motif-bearing FccRIIb alters receptor expression and associates with autoimmunity.
I. Regulatory FCGR2B polymorphisms and their association with systemic lupus erythemato-
sus. J Immunol 172(11):7186–7191
IgG Fc Glycosylation in Human Immunity 75

Tackenberg B et al (2009) Impaired inhibitory Fcc receptor IIB expression on B cells in chronic
inflammatory demyelinating polyneuropathy. Proc Natl Acad Sci U S A 106(12):4788–4792
Wan SW et al (2016) Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet
phagocytosis by macrophages. Thromb Haemost 115(3):646–656
Wang J et al (2011) Fc-glycosylation of IgG1 is modulated by B-cell stimuli. Mol Cell Proteomics
10(5):M110 004655
Wang TT et al (2015) Anti-HA glycoforms drive B cell affinity selection and determine influenza
vaccine efficacy. Cell 162(1):160–169
Wang TT et al (2017) IgG antibodies to dengue enhanced for FccRIIIA binding determine disease
severity. Science 355(6323):395–398
Washburn N et al (2015) Controlled tetra-Fc sialylation of IVIg results in a drug candidate with
consistent enhanced anti-inflammatory activity. Proc Natl Acad Sci U S A
Wuhrer M et al (2009) Regulated glycosylation patterns of IgG during alloimmune responses
against human platelet antigens. J Proteome Res 8(2):450–456
Wuhrer M et al (2015a) Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1
autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection,
galactosylation, and sialylation. J Proteome Res 14(4):1657–1665
Wuhrer M et al (2015b) Pro-inflammatory pattern of IgG1 Fc glycosylation in multiple sclerosis
cerebrospinal fluid. J Neuroinflammation 12:235
Yeap WH et al (2016) CD16 is indispensable for antibody-dependent cellular cytotoxicity by
human monocytes. Sci Rep 6:34310
IgE Glycosylation in Health and Disease

Kai-Ting Shade, Michelle E. Conroy and Robert M. Anthony

Contents

1 IgE Biology in Health and Disease .................................................................................... 77

2 IgE Structure........................................................................................................................ 82
3 IgE N394 Glycosylation Site and Oligomannose Glycan.................................................. 83
4 Other N-Linked Glycosylation Sites on IgE....................................................................... 85
5 Conclusions and Future Perspectives.................................................................................. 86
References .................................................................................................................................. 87

Abstract IgE are absolutely required for initiation of allergy reactions, which
affect over 20% of the world’s population. IgE are the least prevalent
immunoglobulins in circulation with 12-h and 2-day half-lives in mouse and human
serum, respectively, but an extended tissue half-life of 3-weeks bound to the surface
of mast cells by the high affinity IgE receptor, FceRI (Gould and Sutton 2008).
Although the importance of glycosylation to IgG biology is well established, less is
known regarding the contribution of IgE glycosylation to allergic inflammation. IgE
has seven and nine N-linked glycosylation sites distributed across human and
murine constant chains, respectively. Here we discuss studies that have analyzed
IgE glycosylation and its function, and how IgE glycosylation contributions to
health and disease.

1 IgE Biology in Health and Disease

IgE antibody induced inflammation is nearly synonymous with allergic disease,

which affects one in five individuals worldwide. IgE are central to the mechanisms
of immediate type hypersensitivity reactions. These antibodies are also somewhat
enigmatic given that a comprehensive understanding of IgE biology has yet to be

K.-T. Shade
M. E. Conroy
R. M. Anthony (&)

Massachusetts General Hospital, Center for Immunology and Inflammatory Diseases,
Harvard Medical School, 149 13th Street, Room 8.321, Boston, MA 02129, USA
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2019) 423: 77–93

https://doi.org/10.1007/82_2019_151
© Springer Nature Switzerland AG 2019
Published online: 01 March 2019
78 K.-T. Shade et al.

Fig. 1 Human IgE and its glycans. a Schematics of IgE structure and glycosylation. Predicted
N-glycosylation sites in each constant domain (Ce) are represented by closed and open circles for
complex and oligomannose glycans, respectively. b Complex glycans are composed of fucose
(red), GlcNAc (blue), mannose (green), galactose (yellow circles), and sialic acid (pink). Core
complex structure is shown in dotted box. Oligomannose glycans are composed of GlcNAc and
between 5 and 9 mannoses

established. IgE antibodies are the subclass found least in the serum compared to
IgM, IgG and IgA, with a concentration of 50–100 ng/ml, markedly lower than IgG
(5–10 mg/ml). Compared to its long tissue half life of weeks, IgE serum half life is
only 2–3 days (Vieira and Rajewsky 1988). Structurally, it differs from IgG in that
it contains four domains in the constant region (Ce1-4, Fig. 1a) compared to three
in IgG (Gould et al. 2003). In the constant domains, receptor binding occurs,
yielding IgE effector functions. The IgE receptor, FceRI, has strikingly high affinity
for the antibody, *1011 M−1, and is the source of the long tissue phase of IgE
(Chang 2000). FceRI coats the surface of cells including mast cells, basophils, as
well as dendritic cells, Langerhans cells and eosinophils in humans. Notably, there
are structural variations in FceRI present in these cell types which result in dif-
ferential effects upon binding. Mast cells and basophils express FceRI as a tetramer,
abc2, with the a unit responsible for IgE binding at the Ce3 domain. The b chain of
the receptor is mainly responsible for signal amplification and is notably absent in
the trimeric form of FceRI, ac2 present on dendritic cells, Langerhan cells, and
eosniphils. The c subunit is critical as it is the main structure responsible for
orchestrating intra-cellular signaling pathways (Kraft and Kinet 2007). Allergen
binding to IgE and subsequent cross linking of FceRI receptors activates ITAMs
and subsequent downstream signaling. Specifically, FceRI is responsible for the
canonical symptoms of immediate type hypersensitivity reactions detailed below.
IgE Glycosylation in Health and Disease 79

There is a second IgE receptor, known as both CD23 and FceRII. This is often
referred to as the “low affinity” receptor given that the affinity coefficient,
106–107 M−1, is dwarfed by that of FceRI. This immunoglobulin receptor has a
trimeric C-type lectin like globular domains extracellularly, one transmembrane
domain and a short cytoplasmic domain. Interestingly, CD23 can be either mem-
brane bound or soluble forms. CD23 is expressed across a variety of cells including
T and B lymphocytes, follicular dendritic cells, and epithelial cells (Armitage et al.
1989; Rieber 1993; Yu et al. 2003). Upon activation, membrane bound CD23 causes
suppression of IgE production by B cells. Secreted CD23 can exist in monomeric or
trimeric structures and can either down or up-regulate IgE synthesis, depending of its
oligomerization (McCloskey et al. 2007). Taken together, CD23 is a potent factor in
maintaining IgE homeostasis. Additional cells including antigen-presenting and gut
epithelial cells utilize CD23 for transcytosis cross epithelial barriers and subsequent
allergen presentation to the immune system (Palaniyandi et al. 2011).
As mentioned above, IgE antibodies are present in very low levels in the serum,
particularly compared to other immunoglobulin subclasses. This suggests that
control of IgE production is very tightly regulated. After initial and successful VDJ
recombination yields a functional B cell receptor, production of eventually IgE
occurs as a result of IgM+ and IgD+ B cell activation. Tissue Th2 cells as well as Tfh
and Th2-like cells in lymphoid organs have been identified as possible sources of
cytokines to promote B cell preferential class switch to IgE (Reinhardt et al. 2009).
Class switch recombination (CSR) to IgE is triggered upon ligation of CD40, acti-
vation of certain toll like receptors and BAFF in the context of IL-4 exposure (Geha
et al. 2003). Transcription promoters lie just upstream of DNA switch (S) regions
and are responsible for “selection” of particular S recombinations and resultant
specific antibody subclass production. In the case of IgE, ligation of the IL-4 receptor
triggers STAT6 activation which is phosphorylated and translocates to the nucleus,
binding the IgE promoter (Hebenstreit et al. 2006). For counter-regulation, Bcl-6 can
bind sites in the IgE promoter which overlap with STAT6 binding loci, leading to
inhibition of IgE class switch (Harris 1999). Notably, class switch to IgE can occur
in two possible ways- direct switch from IgM to IgE or IgM to IgG and finally IgE.
In B cells stimulated by IL4 and LPS, IgG1 was produced initially and IgE several
hours later (Mandler 1993). Wesemann et al. then showed similar sequential pro-
duction in the context of IL4 and anti-CD40 stimulation (Wesemann et al. 2011).
Preferential class switch between IgG and IgE is determined by various factors.
Intriguingly, the IgG1 switch region contains the most target sequences for AID, the
enzyme responsible for class switching, and IgE the fewest. The IgG1 switch region
may also block accessibility to the IgE S region (Misaghi et al. 2013) and accessi-
bility to the IgE S region also seems to occur with a delayed rate (Wesemann et al.
2012). Taken together, control of class switching to IgE is an important means
through which the antibody level can be tightly controlled. In addition, and critically,
sequential class switch to IgE results in high-affinity antibody, perhaps owing to the
longer timeline and persistent exposure to AID, also responsible for affinity matu-
ration. Direct class switch from IgM directly to IgE results in lower affinity anti-
bodies (He et al. 2015). This has significant functional repercussions.
80 K.-T. Shade et al.

When considering where and when IgE class switch recombination, production
and memory occur there are some puzzling curiosities. First and foremost, there
seems to be an overall lack of IgE memory B cells found in immunologic tissues
(Yang, Sullivan, and Allen 2012). Typically, in lymphoid germinal centers (GCs),
B cell proliferation occurs with concurrent affinity maturation and positive selection
via interactions with follicular dendritic cells and Tfh cells. Therefore, GC inter-
actions are critical for the eventual development of memory B cells and plasma cells
resulting in long lived antibody memory (Janeway et al. 2001). Murine studies
demonstrated while IgE+ B cells were not found in spleen or lymph nodes, plasma
cells represented the preponderance of IgE producing cells in these animals. These
cells were noted to have undergone sequential switching, with an IgG intermediate,
followed by rapid differentiation to IgE expressing plasma cells (Erazo et al. 2007).
He et al. were able to show that GC IgE+ cells do exist but that they expressed
significantly lower expression of surface antibody, weak BCR signaling and did not
populate GC light zone. This suggested that IgE+ cells in germinal centers were
dying prematurely in the maturation process (He et al. 2013). As noted previously,
sequential class switching from IgG to IgE seems to provide the most significant
source of high affinity antibody. Thus, IgG+ B cells have been hypothesized as the
source of IgE memory. B220+ CD138−IgG1+ B cells have been identified as the
cell population responsible for significant IgE memory production (He et al. 2013).
Further, the same group identified a murine IgG1 memory B cell subset which was
able to generate IgE+ plasma cells and produce high affinity, functional antibody,
able to trigger anaphylaxis in a murine model (He et al. 2017). Therefore, IgG+ B
cells serve as a main reservoir for IgE memory along with IgE+ plasma cells. It is
notable that direct class switch from IgM to IgE results in low affinity antibody
production and short lived GC IgE+ cells. Together, these cells and mechanisms are
the source of both high and low affinity IgE antibodies and maintain the functional
pool of IgE (He et al. 2015).
IgE antibodies have gained significant attention as the incidence of allergic
diseases has increased substantially. The World Allergy Organization estimates that
a staggering 30–40% of the world’s population suffers from one or more allergic
conditions (Pawankar et al. 2011). This contributes to substantial burden of illness
and the economic implications are massive, thought to cost billions of dollars in
medical visits and lost productivity (2010). Atopic diseases are considered to be one
of the principle manifestations of IgE driven pathology. As discussed above,
allergen specific IgE antibodies are bound to the surface of mast cells and basophils
via Fc binding to the FceRI receptor. Allergen cross linking of cell bound IgE
results in cellular degranulation and canonical symptoms of allergic inflammation.
Immediate release of mediators causes rapid onset of vascular dilation and per-
meability, bronchoconstriction, leukocyte extravasation and smooth muscle con-
traction. Cellular activation also results in synthetic processes which generate
mediators of “late phase” reactions (Holgate 1982). These processes result in
clinical symptoms including potentially fatal anaphylaxis. Allergen specific IgE can
be utilized for diagnostics in allergy and is a target of therapies in asthma,
environmental/food/venom allergies and atopic dermatitis.
IgE Glycosylation in Health and Disease 81

Yet, significant IgE biology extends beyond allergic inflammation and atopic
diseases. It has been implicated in protection against parasitic infection and venom
exposures. Additionally, several non-allergic diseases also have IgE implicated in
their pathogenesis. As previously described, IgE antibodies coat the surface of mast
cells via the FceRI receptor. Mast cells localize to vascularized tissues and are
especially concentrated at mucosal and skin surfaces (Gurish and Austen 2012). In the
context of parasitic infections, both parasite specific and non-specific IgE antibodies
markedly increase (Anthony et al. 2007). There is speculation that these increased IgE
levels may trigger mast cell activation at mucosal surfaces leading to various biologic
functions which ultimately lead to worm expulsion. In general, specific mechanisms
responsible for these actions are not well elucidated (Fitzsimmons et al. 2014). In fact,
in mice infected with Schistosoma mansoni, significant manipulations of IgE levels
did not specifically modulate host pathology from infection, though may have con-
tributed to liver pathology (Jankovic 1997). The specific biologic relevance of IgE
with regard to parasitic immunity remains to be deciphered.
As they are in parasitic diseases, mast cells are also felt to confer innate pro-
tection against toxic proteins such as those from insects and snakes. In murine
models, venom proteins administered in vivo trigger mast cells to release
pre-formed heparin that contribute to toxin neutralization (Mukai et al. 2016). Mast
cell proteases have also been shown to directly reduce toxicity of venoms (Metz
et al. 2006). Specific IgE antibodies against insect venoms are used as diagnostic
tools to identify persons allergic to these proteins who may be at risk of anaphylaxis
with subsequent exposures. It has therefore been presumed that anti-venom IgE
antibodies are pathogenic. However, with consideration of mast cell protective
functions in the context of toxin exposures, several groups have questioned whether
venom specific IgE may play a role in facilitating mast cell protective responses.
Mice immunized with whole bee venom generated both IgG1 and IgE venom
specific antibodies. Immunized mice were more resistant to subsequent exposure to
lethal dosing of the same venom. IgE deficient and FceRI−/− mice did not
demonstrate this same resistance. Passive immunization with bee venom antibody
containing serum also provided protection upon bee venom administration in IgE
deficient mice (Marichal et al. 2013). In mice who were administered repeated
immunization with phospholipase A2, known to be a potent allergenic component
of honeybee venom, protection was established against subsequent lethal honeybee
venom. This protection did not occur in mice who were B cell deficient or in those
with dysfunctional FceRI (Palm et al. 2013). Given that most humans with venom
specific IgE have never had anaphylaxis in the context of exposure, Mukai et al.
speculate that improved protection against venomous exposures may be an “ancient
and fundamental” function of IgE and mast cells (Mukai et al. 2016).
IgE has largely been implicated in allergic diseases. However, their presence in
diseases primarily involving immune system dysregulation have raised questions as
to a more global immunologic role for IgE antibodies. As an example, decades ago,
self-reactive IgE antibodies and IgE containing immune complexes were found in
patients with systemic lupus erythematosus (SLE) (Miyawaki S 1973). It has been
well established that self reactive immune complexes lead to production of type I
82 K.-T. Shade et al.

interferons by plasmacytoid dendritic cells in SLE (Ronnblom 2001). Recently,

Henault et al. demonstrated that the presence of DNA-IgE autoantibodies in human
SLE caused self-DNA triggered TLR9 activation in plasmacytoid dendritic cells
facilitated by the interaction of IgE/FceRI. This lead to markedly increased pro-
duction of IFN-a (Henault et al. 2016). They speculate that self dsDNA-IgE
complexes may contribute to increased loss of tolerance and exacerbation of
pathology as a result. In fact, higher levels of self-reactive dsDNA-IgE in the serum
correlated with disease severity.
Immune deficiencies provide another clinical example in which IgE is elevated in
the context of immune dysregulation. Hyper-IgE (HIES) or Job syndrome is a com-
plex disease in which IgE levels can reach levels of more than 20,000 IU/L, orders of
magnitude higher than normal (Sowerwine et al. 2012). The disease presents with a
mysterious combination of symptoms including Staph aureus abscesses, eczematous
rashes, recurrent sinopulmonary infections, chronic candida infections, eosinophilia,
fungal infections, and EBV and VZV infections (Siegel et al. 2011). Notably, patients
with HIES also present with osseous abnormalities including craniofacial dysmor-
phology, delayed tooth eruption and high arched palate (Nieminen et al. 2011).
Malignancy affects significant numbers of these people as well, involving both blood
and solid organs. These clinical manifestations are the result of STAT3 mutations on
chromosome 17. This leads to abnormalities in the IL-6 pathway (Holland et al. 2007)
and, significantly, marked inhibition of the Th17 immunologic response (Milner et al.
2008). The specific mechanisms responsible for the marked elevation in IgE levels is
not fully known. Interestingly, Liu et al. recently showed that STAT3 deletion in
keratinocytes of mice with eczema-like inflammation surprisingly lead to a marked
elevation in IgE levels (Liu 2017). Cutaneous inflammation in this model was induced
by S. aureus, which is also responsible for significant disease pathology in patients
with HIES. Other immune deficiencies including DOCK8 deficiency, TYK2 defi-
ciency, Omenn’s syndrome, Wisoctt-Aldrich syndrome, DiGeorge syndrome,
Netherton and IPEX syndromes have elevated IgE as a hallmark (Mogensen 2016).
Once again, how IgE occurs in and affects these conditions is poorly understood and
studies are ongoing to improve understanding.

2 IgE Structure

IgE and IgG are perhaps the most clinically relevant immunoglobulin classes.
Further, there are a number of superficial similarities shared between these two
molecules. Both are monomeric immunoglobulins comprised of two identical heavy
chains and two identical light chains, and exert their canonical effector functions
through interactions with Fc receptors expressed by innate cells. IgE heavy chains
have four constant domains, and a single variable domain, and light chain has single
constant and variable domains. Both IgE and IgG have been shown to adopt open
and closed structures, that result in different Fc receptor binding profiles. While a
single N-linked glycosylation site is present in all IgG, IgE is the most heavily
glycosylated monomeric antibody.
IgE Glycosylation in Health and Disease 83

Seven N-linked glycosylation sites are distributed across the constant domains of
the human IgE heavy chains, while 9 sites are found on mouse IgE (Fig. 1a). One
site, at asparagine (N) 394 on human IgE, is conserved across all mammalian IgE.
The glycan that occupies the conserved site has an oligomannose structure (Fig. 1a,
b). Indeed, this site is reported to be orthologous to the IgG glycosylation site
(Wurzburg et al. 2000). Indeed, crystal structure of IgE and IgG Fcs feature a
glycan, which is consist with glycans having a fixed position in the molecule. On
human IgE, the remaining sites barring N383, which is unoccupied, contain mono-
and di-sialylated complex biantennary glycans. Indeed, the published data on IgE
from allergic patients is currently lacking, as analysis are restricted to IgE from
serum of non-atopic (Plomp et al. 2013), hyperimmune (Arnold et al. 2004; Plomp
et al. 2013), IgE myeloma (Plomp et al. 2013), and hyper IgE (Wu et al. 2016)
patients, and recombinant sources (Shade et al. 2015). The IgE molecule adopts a
bent horseshoe structure, where the Fab and Fc domains are folded towards each
other. Further, the IgE Fab is rigid, unlike the flexible IgG hinge. It is thought the
IgE molecule can rotate around it central axis reciprocally refolding. To date, no
studies have detected the presence of O-linked glycosylation in IgE (Arnold et al.
2004; Plomp et al. 2013; Wu et al. 2016).

3 IgE N394 Glycosylation Site and Oligomannose Glycan

Important structural studies have revealed that IgE binds to FceRI at the Ce3
domains and CD23 at both Ce3 and Ce4 domains of the Fc (Garman et al. 2000;
Holdom et al. 2011). The interaction to each receptor is dependent on IgE con-
formation and is mutually exclusive, with FceRI binding IgE in an open and CD23
in a closed conformation. The changes in conformation upon binding to one
receptor disables binding to the other receptor. Over the past decade, strategies to
alleviate IgE-mediated allergic diseases have generally been devised to prevent
IgE-FceRI interaction. This includes an anti-IgE therapeutic antibody Omalizumab
that binds to an epitope overlapping FceRI and directly prevents binding of serum
free IgE to FceRI. Another engineered protein inhibitor, designed ankyrin repeat
protein (DARPin E2_79) also binds to the Ce3 and accelerates the dissociation of
IgE from FceRI on preformed complexes through an allosteric mechanism (Kim
et al. 2012). Recently, a single-domain antibody derived from llama, sdab 026, have
shown to inhibit IgE-FceRI interaction by locking IgE in a closed conformation
similar to CD23 binding and prevents the open conformation that allows the FceRI
interaction (Jabs et al. 2018).
Crystal structures of human IgE Fc also showed the glycans attached to N394 on
the Ce3 domains occupy the cavity between two Fcs. This glycosylation site is
evolutionary conserved across mammalian species and homologous to the key
glycan attached on the IgG Fc (Jabs et al. 2018; Holdom et al. 2011; Wan et al.
2002; Garman et al. 2000; Sondermann et al. 2000). Previous studies have shown
that IgG Fc glycans are critical for maintaining antibody structural conformation.
84 K.-T. Shade et al.

Removal of the Fc glycans results in a more closed conformation in the IgG Fcs and
forfeits the functional conformation important for Fcc receptor binding (Krapp et al.
2003; Feige et al. 2009). In comparison, conflicting studies between 1980 and early
2000 that used aglycosylated IgE produced from Escherichia coli or deglycosylated
IgE or IgE Fc after enzymatic treatments make it hard to determine whether gly-
cosylation modulates IgE functions.
The earliest studies using E. coli–derived recombinant IgE Fc or Ce3 domains
revealed that aglycosylated IgE binds to FceRI and can elicit IgE-mediated
inflammation (Helm et al. 1998a, b; Henry et al. 2000). However, these proteins
require additional manipulation including solubilization and refolding after recov-
ery from bacterial insoluble inclusion bodies, which may compromise the native
state of conformation. Later studies using enzymes to deglycosylate IgE Fc pro-
duced from mammalian cell lines or IgE derived from myeloma or serum showed
incoherent results. While one study showed that deglycosylated IgE Fc retained
binding to FceRI with minimal difference in a radioligand competition binding
assay (Basu et al. 1993), another showed a 4-fold lower binding affinity to FceRI by
SPR (Hunt et al. 2005). These results deviated from another study that showed
significant impairment to FceRI binding upon enzymatic deglycosylation
(Bjorklund et al. 1999, 2000). On the other hand, all studies that genetically dis-
rupted the N-linked glycosylation site N394 on IgE or IgE Fc by mutating
asparagine-394 to glutamine (N394Q) or threonine (N394T), or alternatively dis-
rupting the site by substituting the third amino acid in the Asn-X-Ser/Thr sequon
theronine-396 to alanine (T396A), all abolished FceRI binding and the subsequent
IgE-mediated degranulation (Sayers et al. 1998; Shade et al. 2015; Jabs et al. 2018).
The functional significance of the glycan on IgE Fc was further exemplified by the
in vivo work using mouse models (Shade et al. 2015) The equivalent of N394 is N384
in mouse. In mouse, genetic mutation (N384Q or T386A) or enzymatic digestion of
IgE by PNG to remove all N-glycans lost the ability to bind FceRI in vivo in ear mast
cells and in vitro in ELISA or cell-based binding assays. As expected, IgE lacking
N384 glycans were also unable to initiate immediate anaphylaxis in a passive cuta-
neous anaphylaxis mouse model. In a reciprocal experiment where all N-linked
glycosylation sites were mutated except N384 (N384 only), this IgE was still able to
elicit anaphylactic response similar to the WT, demonstrating that the glycan on the
conserved site in IgE Fc is absolutely critical for IgE effector functions.
In contrast to the complex-type glycans on IgG Fc, all studies including crystal
structure and glycopeptide mass spectrometry, uniformly identified the glycan
attached to N394 is of oligomannose structures, with 2-9 mannose residues attached
to two GlcNAc and Man5GlcNAc2 being the major form. IgE were sourced from
monoclonal myeloma-derived, recombinant from different mammalian cell lines,
serum of non-diseased donors, and serum of patients with hyperimmune conditions,
IgE myeloma, atopic dermatitis or hyper IgE syndrome (Dorrington and Bennich
1978; Wan et al. 2002; Arnold et al. 2004; Holdom et al. 2011; Plomp et al. 2013;
Shade et al. 2015; Wu et al. 2016). N-linked glycans of secreted mammalian
proteins are typically processed to an intermediate oligomannose structure before
further processed to hybrid and complex glycans in the lumen of the ER and the
IgE Glycosylation in Health and Disease 85

Golgi. As mentioned, all other N-linked sites of IgE are composed of complex
glycans. Thus, it is unique for glycans at N394 to retain the oligomannose structures
from being converted to complex type. The mouse IgE homologue also harbors
oligomannose glycans at the conserved site.
The functional significance of glycans at N394 and its exclusive composition of
oligomannose structures provide a unique therapeutic opportunity to target this
glycan. Unlike some of the other glycosidases that cleave all types of N-linked
glycans, Endoglycosidase F1 selectively hydrolyses the oligomannose and hybrid
structures and leaves complex structures unaffected. Mirroring genetically disruption
of N394 glycosylation site on IgE, EndoF1-treated recombinant or native allergic
human IgE lost its ability to bind FceRI-expressed cells and elicit degranulation in
mast cells. Mouse IgE digested with EndoF1 was also unable to initiate anaphylaxis
in vivo (Shade et al. 2015). Being buried in the interstitial space between Fcs, the
importance of N394 glycan is likely to maintain IgE protein structure as IgG glycans
(Feige et al. 2009). Circular dichroism that measures the secondary structure of
proteins revealed a shift in spectrum upon the loss of oligomannose glycans in IgE
after EndoF1 treatment, reflecting changes in IgE three-dimensional conformation
(Shade et al. 2015). This change likely explains the loss of functional protein folding
necessary to bind FceRI and initiate allergic inflammation.
In contrast to the requirement of IgE glycans in FceRI interaction, deglycosylated
myeloma IgE was reported to augment binding to CD23 on B cells (Vercelli et al.
1989). This finding reinforces the idea that IgE conformation decides the exclusive
interaction with FceRI or CD23. Perhaps the loss of glycans on IgE leads to a more
closed conformation, thus promoting CD23 and preventing FceRI binding.

4 Other N-Linked Glycosylation Sites on IgE

IgE is one of the most heavily glycosylated antibody class. Besides N394, IgE has
six other glycosylation sites, of which five are known to have complex glycans
attached (Fig. 1a, b). While oligomannose glycans are composed of only GlcNAc
and mannose residues, complex glycans have additional sugars including fucose that
attaches to the core structure and galactose or sialic acid to the terminal. To date,
studies that described complex glycans on human recombinant, myeloma-derived or
native IgE all revealed fucosylated sialylated structures to be the major complex
glycoforms (Dorrington and Bennich 1978; Arnold et al. 2004; Plomp et al. 2013;
Shade et al. 2015; Wu et al. 2016). Previous study has reported that deglycosylated
IgE has a propensity to form aggregate (Basu et al. 1993). Thus, glycans on IgE
probably contribute significantly to the water solubility of IgE and removal of these
hydrophilic, acidic glycans may increase exposure of hydrophobic regions and
results in aggregation. Various studies that mutated glycosylation sites with complex
glycans attached in IgE Fc have thus far suggested a minimal role in IgE- FceRI
interaction. IgE/IgE Fc with single or double mutations at N265 and N374 sites was
still able to bind FceRI with the same affinity as the WT and initiate degranulation
86 K.-T. Shade et al.

(Nettleton and Kochan 1995; Young et al. 1995; Sayers et al. 1998; Shade et al.
2015). Interestingly, mutation of all N-linked sites in Ce1 domain of Fab portion
showed a slight reduction in IgE-mediate degranulation (Shade et al. 2015), sug-
gesting perhaps complex glycans in Ce1 modulates IgE interaction with the antigen.
Genetic disruption of the glycosylation site removes the entire glycan structure
and disregards the roles that are dependent on the heterogeneity of the complex
glycans. Studies of the complex glycans on IgG Fc demonstrated that these glycans
are heterogeneous and can display more than 30 different glycoforms at a single site
in healthy individuals (Kaneko et al. 2006; Wuhrer et al. 2007). These glycoforms
reflect physiological processes and differ in diseases, serving as potential disease
biomarkers. For example, changes in sialylation of IgG autoantibodies have been
associated with clinical manifestation of the autoimmune and inflammatory diseases
(Matsumoto et al. 2000; van de Geijn et al. 2009; Scherer et al. 2010; Ackerman
et al. 2013a, b). Importantly, changes in glycoforms markedly modulate protein
functions. When IgG is equipped with sialic acid, IgG shifts its receptor binding
preferences and mediates anti-inflammatory activity (Kaneko et al. 2006; Anthony
et al. 2008; Sondermann et al. 2013; Pincetic et al. 2014). Further, IgG that lack
fucose has enhanced antibody-dependent cellular cytotoxicity as a result of
increased affinity to FccRIIIA by 50-fold (Shields et al. 2002; Ferrara et al. 2011).
In comparison to IgG, the role of complex glycans in IgE is not known. Studies of
the ß-galactoside-binding lectins, galectins, have perhaps suggested the involvement of
complex glycans in IgE-mediated functions. Galectin-3 has been shown to bind mouse
IgE and activate IgE-sensitized rat basophilic leukemia cells (Frigeri et al. 1993; Liu
et al. 1993; Zuberi et al. 1994). On the other hand, another galectin, galectin-9, has
been shown to reduce mast cell degranulation and passive anaphylaxis by binding to
IgE and inhibit its interaction with antigen (Niki et al. 2009). It is likely that these
galectins bind to the complex glycans on the IgE to exert its regulatory roles. However,
it is not clear how these two lectins work together in vivo, for example, whether they
bind to the same glycans on IgE and their relevance in human physiology.

5 Conclusions and Future Perspectives

Despite its discovery over 50 years ago, much remains to be discovered relating to
IgE. As mentioned above, it is not clear precisely what makes allergen-specific IgE
pathogenic in some individuals, and inert in other. However, their role in diseases
of intense public health interest mandate ongoing pursuit of a comprehensive
rendering of their function and regulation. IgE is the most heavily glycosylated
monomeric antibody, and the conserved glycan at N394 has an important role in
conformational folding and binding to FceRI. This work is in early stages and
dedication to continued unraveling of the story is critical. However, even in these
early stages of understanding, it is interesting to speculate as to how glycosylation
could be a major factor in treatment of IgE-associated diseases in the future.
As described previously, galectins may play a significant role in connecting IgE to
IgE Glycosylation in Health and Disease 87

its downstream effector functions. Recent studies have identified a role for
Galectin-3 in the regulation of the IgE receptor, FceRI. Utilizing RNAi screening
experiments, one group recently demonstrated Gal-3 as a negative regulator of
FceRI triggered mast cell degranulation. Their data suggests Gal-3 may affect
surface FceRI internalization, receptor mobility, F-actin polymerization, and FceRI
ubiquination, all of which would profoundly impact mast cell signaling
(Bambouskova et al. 2016). As altered variability in IgE glycosylation may impact
galectin binding, it is certainly notable that activating or blocking interactions could
yield substantial impact on the FceRI and mast cell function. This would have
marked impact on allergic inflammation. As a notable clinical correlate, Galectin-3
has been increasingly identified as a biomarker in various allergic diseases. A recent
study identified Gal-3 as a marker increased in eosinophilic asthma but notably
decreased in neutrophilic asthma (Gao et al. 2015). This finding was extended by
Riccio et al. demonstrating Gal-3 as a predictive marker for positive response to
anti-IgE treatment of asthma. Subjects positive for Gal-3 at baseline had more
improvement in bronchial wall thickening, eosinophilic inflammation, and smooth
muscle involvement (Riccio et al. 2017). Beyond asthma, a specific Gal-3 poly-
morphism has recently been identified as a significant genetic predictor of allergy to
beta-lactam antibiotics (Cornejo-Garcia et al. 2016). These examples provide early
clinical evidence for what is an increasing connection between IgE antibodies,
glycosylation, and co-receptors with biologic relevance.
With IgE glycosylation likely to be a significant factor in its biology and effector
function, identification of relevant patterns associated with clinical disease will be
critical. Once the specific glycan patterns associated with pathology are identified
and the basic biology elucidated, novel diagnostic and therapeutic therapies can be
extrapolated. Glycoengineering is an exciting future prospect where in vivo gener-
ation of antibody specific glycosylation could be utilized for reduction in inflam-
mation. Indeed, modulation of IgG and tumor glycosylation has been reported by a
number of groups, highlighting the therapeutic potential of these approaches (Albert
et al. 2008; Xiao et al. 2016; Pagan et al. 2018). This may be one strategy of many to
target IgE glycosylation for therapeutic benefit in the futures.

Acknowledgements This work was supported by grants from the National Institutes of Health
(DP2AR068272-01), and Food Allergy, Research, and Education (FARE) to RMA. We would like
to apologize to all our colleagues whose important work was not cited here.

References

2010 Asthma’s Impact on the Nation; Data from the CDC National Asthma Control Program.
Center for Disease Control
Ackerman ME, Crispin M, Yu X, Baruah K, Boesch AW, Harvey DJ, Dugast AS, Heizen EL,
Ercan A, Choi I, Streeck H, Nigrovic PA, Bailey-Kellogg C, Scanlan C, Alter G (2013a)
Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity.
J Clin Invest 123(5):2183–2192. https://doi.org/10.1172/JCI65708
88 K.-T. Shade et al.

Ackerman ME, Dugast AS, McAndrew EG, Tsoukas S, Licht AF, Irvine DJ, Alter G (2013b)
Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of
immunoglobulins with skewed affinity for FccR2a and FccR2b. J Virol 87(10):5468–5476.
https://doi.org/10.1128/JVI.03403-12
Albert H, Collin M, Dudziak D, Ravetch JV, Nimmerjahn F (2008) In vivo enzymatic modulation
of IgG glycosylation inhibits autoimmune disease in an IgG subclass-dependent manner. Proc
Natl Acad Sci U S A 105(39):15005–15009. https://doi.org/10.1073/pnas.0808248105
Anthony RM, Rutitzky LI, Urban JF Jr, Stadecker MJ, Gause WC (2007) Protective immune
mechanisms in helminth infection. Nat Rev Immunol 7(12):975–987. https://doi.org/10.1038/
nri2199
Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV (2008)
Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320
(5874):373–376
Armitage RJ, Goff LK, Beverley PC (1989) Expression and functional role of CD23 on T cells.
Eur J Immunol 19(1):31–35. https://doi.org/10.1002/eji.1830190106
Arnold JN, Radcliffe CM, Wormald MR, Royle L, Harvey DJ, Crispin M, Dwek RA, Sim RB,
Rudd PM (2004) The glycosylation of human serum IgD and IgE and the accessibility of
identified oligomannose structures for interaction with mannan-binding lectin. J Immunol 173
(11):6831–6840
Bambouskova M, Polakovicova I, Halova I, Goel G, Draberova L, Bugajev V, Doan A, Utekal P,
Gardet A, Xavier RJ, Draber P (2016) New regulatory roles of Galectin-3 in high-affinity IgE
receptor signaling. Mol Cell Biol 36(9):1366–1382. https://doi.org/10.1128/MCB.00064-16
Basu M, Hakimi J, Dharm E, Kondas JA, Tsien WH, Pilson RS, Lin P, Gilfillan A, Haring P,
Braswell EH et al (1993) Purification and characterization of human recombinant IgE-Fc
fragments that bind to the human high affinity IgE receptor. J Biol Chem 268(18):13118–
13127
Bjorklund JE, Karlsson T, Magnusson CG (1999) N-glycosylation influences epitope expression
and receptor binding structures in human IgE. Mol Immunol 36(3):213–221
Bjorklund JE, Schmidt M, Magnusson CG (2000) Characterisation of recombinant human IgE-Fc
fragments expressed in baculovirus-infected insect cells. Mol Immunol 37(3–4):169–177
Chang TW (2000) The pharmacological basis of anti-IgE therapy. Nat Biotechnol 18(2):157–162.
https://doi.org/10.1038/72601
Cornejo-Garcia JA, Romano A, Gueant-Rodriguez RM, Oussalah A, Blanca-Lopez N, Gaeta F,
Tramoy D, Josse T, Dona I, Torres MJ, Canto G, Blanca M, Gueant JL (2016) A
non-synonymous polymorphism in galectin-3 lectin domain is associated with allergic
reactions to beta-lactam antibiotics. Pharmacogenomics J 16(1):79–82. https://doi.org/10.1038/
tpj.2015.24
Dorrington KJ, Bennich HH (1978) Structure-function relationships in human immunoglobulin E.
Immunol Rev 41:3–25
Erazo A, Kutchukhidze N, Leung M, Christ AP, Urban JF Jr, Curotto de Lafaille MA, Lafaille JJ
(2007) Unique maturation program of the IgE response in vivo. Immunity 26(2):191–203.
https://doi.org/10.1016/j.immuni.2006.12.006
Feige MJ, Nath S, Catharino SR, Weinfurtner D, Steinbacher S, Buchner J (2009) Structure of the
murine unglycosylated IgG1 Fc fragment. J Mol Biol 391(3):599–608. S0022-2836(09)
00768-2 [pii] 10.1016/j.jmb.2009.06.048
Ferrara C, Grau S, Jager C, Sondermann P, Brunker P, Waldhauer I, Hennig M, Ruf A, Rufer AC,
Stihle M, Umana P, Benz J (2011) Unique carbohydrate-carbohydrate interactions are required
for high affinity binding between Fc{c}RIII and antibodies lacking core fucose. Proc Natl Acad
Sci U S A 108(31):12669–74. 1108455108 [pii] 10.1073/pnas.1108455108
Fitzsimmons CM, Falcone FH, Dunne DW (2014) Helminth allergens, parasite-specific IgE, and
Its protective role in human immunity. Front Immunol 5:61. https://doi.org/10.3389/fimmu.
2014.00061
IgE Glycosylation in Health and Disease 89

Frigeri LG, Zuberi RI, Liu FT (1993) Epsilon BP, a beta-galactoside-binding animal lectin,
recognizes IgE receptor (Fc epsilon RI) and activates mast cells. Biochemistry 32(30):7644–
7649
Gao P, Gibson PG, Baines KJ, Yang IA, Upham JW, Reynolds PN, Hodge S, James AL,
Jenkins C, Peters MJ, Zhang J, Simpson JL (2015) Anti-inflammatory deficiencies in
neutrophilic asthma: reduced galectin-3 and IL-1RA/IL-1b. Respir Res 16:5. https://doi.org/10.
1186/s12931-014-0163-5
Garman SC, Wurzburg BA, Tarchevskaya SS, Kinet JP, Jardetzky TS (2000) Structure of the Fc
fragment of human IgE bound to its high-affinity receptor Fc
RIa. Nature 406(6793):259–266.
https://doi.org/10.1038/35018500
Geha RS, Jabara HH, Brodeur SR (2003) The regulation of immunoglobulin E class-switch
recombination. Nat Rev Immunol 3(9):721–732. https://doi.org/10.1038/nri1181
Gould HJ, Sutton BJ (2008) IgE in allergy and asthma today. Nat Rev Immunol 8(3):205–217.
https://doi.org/10.1038/nri2273
Gould HJ, Sutton BJ, Beavil AJ, Beavil RL, McCloskey N, Coker HA, Fear D, Smurthwaite L
(2003) The biology of IGE and the basis of allergic disease. Annu Rev Immunol 21:579–628.
https://doi.org/10.1146/annurev.immunol.21.120601.141103
Gurish MF, Austen KF (2012) Developmental origin and functional specialization of mast cell
subsets. Immunity 37(1):25–33. https://doi.org/10.1016/j.immuni.2012.07.003
Harris MB, Chang CC, Berton MT, Danial NN, Zhang J, Kuehner D, Ye BH, Kvatyuk M,
Pandolfi PP, Cattoretti G, Dalla-Favera R, Rothman PB (1999) Transcriptional repression of
Stat6-dependent interleukin-4-induced genes by BCL-6: specific regulation of i
transcription
and immunoglobulin E switching. Mol Cell Bio 19:7264–7275
He JS, Meyer-Hermann M, Xiangying D, Zuan LY, Jones LA, Ramakrishna L, de Vries VC,
Dolpady J, Aina H, Joseph S, Narayanan S, Subramaniam S, Puthia M, Wong G, Xiong H,
Poidinger M, Urban JF, Lafaille JJ, Curotto de Lafaille MA (2013) The distinctive germinal
center phase of IgE+ B lymphocytes limits their contribution to the classical memory response.
J Exp Med 210(12):2755–2771. https://doi.org/10.1084/jem.20131539
He JS, Narayanan S, Subramaniam S, Ho WQ, Lafaille JJ, Curotto de Lafaille MA (2015) Biology
of IgE production: IgE cell differentiation and the memory of IgE responses. Curr Top
Microbiol Immunol 388:1–19. https://doi.org/10.1007/978-3-319-13725-4_1
He JS, Subramaniam S, Narang V, Srinivasan K, Saunders SP, Carbajo D, Wen-Shan T, Hidayah
Hamadee N, Lum J, Lee A, Chen J, Poidinger M, Zolezzi F, Lafaille JJ, Curotto de
Lafaille MA (2017) IgG1 memory B cells keep the memory of IgE responses. Nat Commun 8
(1):641. https://doi.org/10.1038/s41467-017-00723-0
Hebenstreit D, Wirnsberger G, Horejs-Hoeck J, Duschl A (2006) Signaling mechanisms,
interaction partners, and target genes of STAT6. Cytokine Growth Factor Rev 17(3):173–188.
https://doi.org/10.1016/j.cytogfr.2006.01.004
Helm BA, Sayers I, Padlan EA, McKendrick JE, Spivey AC (1998) Structure/function studies on
IgE as a basis for the development of rational IgE antagonists. Allergy 53(45 Suppl):77–82
Helm B, Marsh P, Vercelli D, Padlan E, Gould H, Geha R (1988) The mast cell binding site on
human immunoglobulin E. Nature 331(6152):180–183. https://doi.org/10.1038/331180a0
Henault J, Riggs JM, Karnell JL, Liarski VM, Li J, Shirinian L, Xu L, Casey KA, Smith MA,
Khatry DB, Izhak L, Clarke L, Herbst R, Ettinger R, Petri M, Clark MR, Mustelin T,
Kolbeck R, Sanjuan MA (2016) Self-reactive IgE exacerbates interferon responses associated
with autoimmunity. Nat Immunol 17(2):196–203. https://doi.org/10.1038/ni.3326
Henry AJ, McDonnell JM, Ghirlando R, Sutton BJ, Gould HJ (2000) Conformation of the isolated
c
3 domain of IgE and its complex with the high-affinity receptor, Fc
RI. Biochemistry 39
(25):7406–7413
Holdom MD, Davies AM, Nettleship JE, Bagby SC, Dhaliwal B, Girardi E, Hunt J, Gould HJ,
Beavil AJ, McDonnell JM, Owens RJ, Sutton BJ (2011) Conformational changes in IgE
contribute to its uniquely slow dissociation rate from receptor FceRI. Nat Struct Mol Biol 18
(5):571–576. https://doi.org/10.1038/nsmb.2044
90 K.-T. Shade et al.

Holgate ST, Church MK (1982) Control of mediator release from mast cells. Clin Allergy
12(Suppl):5–13
Holland SM, DeLeo FR, Elloumi HZ, Hsu AP, Uzel G, Brodsky N, Freeman AF, Demidowich A,
Davis J, Turner ML, Anderson VL, Darnell DN, Welch PA, Kuhns DB, Frucht DM,
Malech HL, Gallin JI, Kobayashi SD, Whitney AR, Voyich JM, Musser JM, Woellner C,
Schaffer AA, Puck JM, Grimbacher B (2007) STAT3 mutations in the hyper-IgE syndrome.
N Engl J Med 357(16):1608–1619. https://doi.org/10.1056/NEJMoa073687
Hunt J, Beavil RL, Calvert RA, Gould HJ, Sutton BJ, Beavil AJ (2005) Disulfide linkage controls
the affinity and stoichiometry of IgE Fc
3-4 binding to Fc
RI. J Biol Chem 280(17):16808–
16814. https://doi.org/10.1074/jbc.M500965200
Jabs F, Plum M, Laursen NS, Jensen RK, Molgaard B, Miehe M, Mandolesi M, Rauber MM,
Pfutzner W, Jakob T, Mobs C, Andersen GR, Spillner E (2018) Trapping IgE in a closed
conformation by mimicking CD23 binding prevents and disrupts Fc
RI interaction. Nat
Commun 9(1):7. https://doi.org/10.1038/s41467-017-02312-7
Janeway CA, Travers P, Walport M, Shlomchik M (2001) Immunobiology. In: The immune
system in health and disease, 5 edn. Garland Publishing, New York
Jankovic D, Kullberg MC, Dombrowicz D, Barbieri S, Caspar P, Wynn TA, Paul WE,
Cheever AW, Kinet JP, Sher A (1997) Fc epsilonRI-deficient mice infected with Schistosoma
mansoni mount normal Th2-type responses while displaying enhanced liver pathology.
J Immunol 159:1868–1875
Kaneko Y, Nimmerjahn F, Ravetch JV (2006) Anti-inflammatory activity of immunoglobulin G
resulting from Fc sialylation. Science 313(5787):670–673
Kim B, Eggel A, Tarchevskaya SS, Vogel M, Prinz H, Jardetzky TS (2012) Accelerated
disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor. Nature
491(7425):613–617. https://doi.org/10.1038/nature11546
Kraft S, Kinet JP (2007) New developments in Fc
RI regulation, function and inhibition. Nat Rev
Immunol 7(5):365–378. https://doi.org/10.1038/nri2072
Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P (2003) Structural analysis of human
IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol
Biol 325(5):979–989
Liu FT, Frigeri LG, Gritzmacher CA, Hsu DK, Robertson MW, Zuberi RI (1993) Expression and
function of an IgE-binding animal lectin (
BP) in mast cells. Immunopharmacology 26(3):
187–195
Liu H, Archer NK, Dillen CA, Wang Y, Ashbaugh AG, Ortines RV, Lee SK, Kao T, Miller LS
(2017) Keratinocyte-specific deletion of STAT3 promotes elevated serum IgE in response to
Staphylococcus aureus exposure: relevance to hyper-IgE syndrome. J Immunol 198:194–208
Mandler R, Finkelman FD, Levine AD, Snapper CM (1993) IL-4 induction of IgE class switching
by lipopolysaccharide-activated murine B cells occurs predominantly through sequential
switching. J Immunol 150:407–418
Marichal T, Starkl P, Reber LL, Kalesnikoff J, Oettgen HC, Tsai M, Metz M, Galli SJ (2013) A
beneficial role for immunoglobulin E in host defense against honeybee venom. Immunity 39
(5):963–975. https://doi.org/10.1016/j.immuni.2013.10.005
Matsumoto A, Shikata K, Takeuchi F, Kojima N, Mizuochi T (2000) Autoantibody activity of IgG
rheumatoid factor increases with decreasing levels of galactosylation and sialylation.
J Biochem 128(4):621–628
McCloskey N, Hunt J, Beavil RL, Jutton MR, Grundy GJ, Girardi E, Fabiane SM, Fear DJ,
Conrad DH, Sutton BJ, Gould HJ (2007) Soluble CD23 monomers inhibit and oligomers
stimulate IGE synthesis in human B cells. J Biol Chem 282(33):24083–24091. https://doi.org/
10.1074/jbc.M703195200
Metz M, Piliponsky AM, Chen CC, Lammel V, Abrink M, Pejler G, Tsai M, Galli SJ (2006) Mast
cells can enhance resistance to snake and honeybee venoms. Science 313(5786):526–530.
https://doi.org/10.1126/science.1128877
IgE Glycosylation in Health and Disease 91

Milner JD, Brenchley JM, Laurence A, Freeman AF, Hill BJ, Elias KM, Kanno Y, Spalding C,
Elloumi HZ, Paulson ML, Davis J, Hsu A, Asher AI, O’Shea J, Holland SM, Paul WE,
Douek DC (2008) Impaired T(H)17 cell differentiation in subjects with autosomal dominant
hyper-IgE syndrome. Nature 452(7188):773–776. https://doi.org/10.1038/nature06764
Misaghi S, Senger K, Sai T, Qu Y, Sun Y, Hamidzadeh K, Nguyen A, Jin Z, Zhou M, Yan D,
Lin WY, Lin Z, Lorenzo MN, Sebrell A, Ding J, Xu M, Caplazi P, Austin CD, Balazs M,
Roose-Girma M, DeForge L, Warming S, Lee WP, Dixit VM, Zarrin AA (2013) Polyclonal
hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.
Proc Natl Acad Sci U S A 110(39):15770–15775. https://doi.org/10.1073/pnas.1221661110
Miyawaki S, Ritchie RF (1973) Nucleolar antigen specfic for antinucleolar antibody in the sera of
patients with rheumatologic disease. Arthritis Rheum 16:726–736
Mogensen TH (2016) Primary immunodeficiencies with elevated IgE. Int Rev Immunol 35(1):39–
56. https://doi.org/10.3109/08830185.2015.1027820
Mukai K, Tsai M, Starkl P, Marichal T, Galli SJ (2016) IgE and mast cells in host defense against
parasites and venoms. Semin Immunopathol 38(5):581–603. https://doi.org/10.1007/s00281-
016-0565-1
Nettleton MY, Kochan JP (1995) Role of glycosylation sites in the IgE Fc molecule. Int Arch
Allergy Immunol 107(1–3):328–329
Nieminen P, Morgan NV, Fenwick AL, Parmanen S, Veistinen L, Mikkola ML, van der Spek PJ,
Giraud A, Judd L, Arte S, Brueton LA, Wall SA, Mathijssen IM, Maher ER, Wilkie AO,
Kreiborg S, Thesleff I (2011) Inactivation of IL11 signaling causes craniosynostosis, delayed
tooth eruption, and supernumerary teeth. Am J Hum Genet 89(1):67–81. https://doi.org/10.
1016/j.ajhg.2011.05.024
Niki T, Tsutsui S, Hirose S, Aradono S, Sugimoto Y, Takeshita K, Nishi N, Hirashima M (2009)
Galectin-9 is a high affinity IgE-binding lectin with anti-allergic effect by blocking IgE-antigen
complex formation. J Biol Chem 284(47):32344–32352. https://doi.org/10.1074/jbc.M109.
035196
Pagan JD, Kitaoka M, Anthony RM (2018) Engineered sialylation of pathogenic antibodies
in vivo attenuates autoimmune disease. Cell. https://doi.org/10.1016/j.cell.2017.11.041
Palaniyandi S, Tomei E, Li Z, Conrad DH, Zhu X (2011) CD23-dependent transcytosis of IgE and
immune complex across the polarized human respiratory epithelial cells. J Immunol 186(6):
3484–3496. https://doi.org/10.4049/jimmunol.1002146
Palm NW, Rosenstein RK, Yu S, Schenten DD, Florsheim E, Medzhitov R (2013) Bee venom
phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and
confers protective immunity. Immunity 39(5):976–985. https://doi.org/10.1016/j.immuni.2013.
10.006
Pawankar R, Canonica GW, Holgate S, Lockey RF (2011). WAO white book on allergy 2011–
2012: executive summary. https://www.worldallergy.org/UserFiles/file/WAO-White-Book-on-
Allergy_web.pdf
Pincetic A, Bournazos S, DiLillo DJ, Maamary J, Wang TT, Dahan R, Fiebiger BM, Ravetch JV
(2014) Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol 15
(8):707–716. https://doi.org/10.1038/ni.2939
Plomp R, Hensbergen PJ, Rombouts Y, Zauner G, Dragan I, Koeleman CA, Deelder AM,
Wuhrer M (2013) Site-specific N-glycosylation analysis of human immunoglobulin E.
J Proteome Res. https://doi.org/10.1021/pr400714w
Reinhardt RL, Liang HE, Locksley RM (2009) Cytokine-secreting follicular T cells shape the
antibody repertoire. Nat Immunol 10(4):385–393. https://doi.org/10.1038/ni.1715
Riccio AM, Mauri P, De Ferrari L, Rossi R, Di Silvestre D, Benazzi L, Chiappori A, Dal
Negro RW, Micheletto C, Canonica GW (2017) Galectin-3: an early predictive biomarker of
modulation of airway remodeling in patients with severe asthma treated with omalizumab for
36 months. Clin Transl Allergy 7:6. https://doi.org/10.1186/s13601-017-0143-1
Rieber EP, Rank G, Kohler I, Krauss S (1993) Membrane expression of Fc
RII/CD23 and release
of soluble CD23 by follicular dendritic cells. Adv Exp Med Biol 329:393–398
92 K.-T. Shade et al.

Ronnblom L, Alm GV (2001) A pivotal role for the natural interferon a-producing cells
(plasmacytoid dendritic cells) in the pathogenesis of lupus. J Exp Med 194:F59–F63
Sayers I, Cain SA, Swan JR, Pickett MA, Watt PJ, Holgate ST, Padlan EA, Schuck P, Helm BA
(1998) Amino acid residues that influence Fc
RI-mediated effector functions of human
immunoglobulin E. Biochemistry 37(46):16152–16164. https://doi.org/10.1021/bi981456k
Scherer HU, van der Woude D, Ioan-Facsinay A, el Bannoudi H, Trouw LA, Wang J, Haupl T,
Burmester GR, Deelder AM, Huizinga TW, Wuhrer M, Toes RE (2010) Glycan profiling of
anti-citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis
Rheum 62(6):1620–1629. https://doi.org/10.1002/art.27414
Shade KT, Platzer B, Washburn N, Mani V, Bartsch YC, Conroy M, Pagan JD, Bosques C,
Mempel TR, Fiebiger E, Anthony RM (2015) A single glycan on IgE is indispensable for
initiation of anaphylaxis. J Exp Med 212(4):457–467. https://doi.org/10.1084/jem.20142182
Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002)
Lack of fucose on human IgG1N-linked oligosaccharide improves binding to human Fcc RIII
and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740
Siegel AM, Heimall J, Freeman AF, Hsu AP, Brittain E, Brenchley JM, Douek DC, Fahle GH,
Cohen JI, Holland SM, Milner JD (2011) A critical role for STAT3 transcription factor
signaling in the development and maintenance of human T cell memory. Immunity 35(5):806–
818. https://doi.org/10.1016/j.immuni.2011.09.016
Sondermann P, Huber R, Oosthuizen V, Jacob U (2000) The 3.2-a crystal structure of the human
IgG1 Fc fragment-FccRIII complex. Nature 406(6793):267–273. https://doi.org/10.1038/
35018508
Sondermann P, Pincetic A, Maamary J, Lammens K, Ravetch JV (2013) General mechanism for
modulating immunoglobulin effector function. Proc Natl Acad Sci U S A. https://doi.org/10.
1073/pnas.1307864110
Sowerwine KJ, Holland SM, Freeman AF (2012) Hyper-IgE syndrome update. Ann N Y Acad Sci
1250:25–32. https://doi.org/10.1111/j.1749-6632.2011.06387.x
van de Geijn FE, Wuhrer M, Selman MH, Willemsen SP, de Man YA, Deelder AM, Hazes JM,
Dolhain RJ (2009) Immunoglobulin G galactosylation and sialylation are associated with
pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from
a large prospective cohort study. Arthritis Res Ther 11(6):R193. ar2892 [pii] 10.1186/ar2892
Vercelli D, Helm B, Marsh P, Padlan E, Geha RS, Gould H (1989) The B-cell binding site on
human immunoglobulin E. Nature 338(6217):649–651. https://doi.org/10.1038/338649a0
Vieira P, Rajewsky K (1988) The half-lives of serum immunoglobulins in adult mice. Eur J
Immunol 18(2):313–316. https://doi.org/10.1002/eji.1830180221
Wan T, Beavil RL, Fabiane SM, Beavil AJ, Sohi MK, Keown M, Young RJ, Henry AJ,
Owens RJ, Gould HJ, Sutton BJ (2002) The crystal structure of IgE Fc reveals an
asymmetrically bent conformation. Nat Immunol 3(7):681–686. https://doi.org/10.1038/ni811
Wesemann DR, Magee JM, Boboila C, Calado DP, Gallagher MP, Portuguese AJ, Manis JP,
Zhou X, Recher M, Rajewsky K, Notarangelo LD, Alt FW (2011) Immature B cells
preferentially switch to IgE with increased direct Sl to S
recombination. J Exp Med 208
(13):2733–2746. https://doi.org/10.1084/jem.20111155
Wesemann DR, Portuguese AJ, Magee JM, Gallagher MP, Zhou X, Panchakshari RA, Alt FW
(2012) Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent
stem cells. Proc Natl Acad Sci U S A 109(34):13745–13750. https://doi.org/10.1073/pnas.
1210286109
Wu G, Hitchen PG, Panico M, North SJ, Barbouche MR, Binet D, Morris HR, Dell A, Haslam SM
(2016) Glycoproteomic studies of IgE from a novel hyper IgE syndrome linked to PGM3
mutation. Glycoconj J 33(3):447–456. https://doi.org/10.1007/s10719-015-9638-y
Wuhrer M, Stam JC, van de Geijn FE, Koeleman CA, Verrips CT, Dolhain RJ, Hokke CH,
Deelder AM (2007) Glycosylation profiling of immunoglobulin G (IgG) subclasses from
human serum. Proteomics 7(22):4070–4081. https://doi.org/10.1002/pmic.200700289
IgE Glycosylation in Health and Disease 93

Wurzburg BA, Garman SC, Jardetzky TS (2000) Structure of the human IgE-Fc C epsilon 3-C
epsilon 4 reveals conformational flexibility in the antibody effector domains. Immunity 13(3):
375–385
Xiao H, Woods EC, Vukojicic P, Bertozzi CR (2016) Precision glycocalyx editing as a strategy for
cancer immunotherapy. Proc Natl Acad Sci U S A 113(37):10304–10309. https://doi.org/10.
1073/pnas.1608069113
Yang Z, Sullivan BM, Allen CD (2012) Fluorescent in vivo detection reveals that IgE(+) B cells
are restrained by an intrinsic cell fate predisposition. Immunity 36(5):857–872. https://doi.org/
10.1016/j.immuni.2012.02.009
Young RJ, Owens RJ, Mackay GA, Chan CM, Shi J, Hide M, Francis DM, Henry AJ, Sutton BJ,
Gould HJ (1995) Secretion of recombinant human IgE-Fc by mammalian cells and biological
activity of glycosylation site mutants. Protein Eng 8(2):193–199
Yu LC, Montagnac G, Yang PC, Conrad DH, Benmerah A, Perdue MH (2003) Intestinal epithelial
CD23 mediates enhanced antigen transport in allergy: evidence for novel splice forms. Am J
Physiol Gastrointest Liver Physiol 285(1):G223–G234. https://doi.org/10.1152/ajpgi.00445.
2002
Zuberi RI, Frigeri LG, Liu FT (1994) Activation of rat basophilic leukemia cells by
BP, an
IgE-binding endogenous lectin. Cell Immunol 156(1):1–12
Immune Complex Vaccination

Yu-mei Wen and Yan Shi

Contents

1 Introduction.......................................................................................................................... 96
2 How ICs Enhance Immune Responses ............................................................................... 98
2.1 Fcc Receptors in IC/Antigen Uptake, Processing and Presentation ......................... 98
2.2 Other Fcc Receptors................................................................................................... 99
2.3 Antigen Presentation................................................................................................... 99
2.4 Intracellular Targeting ................................................................................................ 101
2.5 APC Regulation.......................................................................................................... 102
3 Experimental and Clinical IC Vaccines .............................................................................. 103
3.1 IC Vaccines in Poultry Industry................................................................................. 103
3.2 IC Vaccination for Influenza Virus Infection ............................................................ 104
3.3 IC Vaccination for HIV Infection .............................................................................. 106
3.4 IC Vaccination for Viral Hepatitis B ......................................................................... 107
3.5 IC Vaccination for Tumors ........................................................................................ 109
4 Future Prospective of IC Vaccines ..................................................................................... 110
References .................................................................................................................................. 111

Y. Wen (&)
Key Laboratory of Molecular Virology, Shanghai Medical College,
School of Basic Medical Sciences, Fudan University, Shanghai, China
e-mail: [email protected]
Shanghai Medical College, Fudan University, Rm 401, Fuxing Bldg,
131 Yi Xue Yuan Rd, Shanghai 200032, China
Y. Shi
Department of Basic Medical Sciences, Center for Life Sciences,
Institute of Immunology, Tsinghua University, Beijing, China
e-mail: [email protected]
Department of Microbiology, Immunology & Infectious Diseases and Snyder Institute
for Chronic Diseases, University of Calgary, Calgary, AB, Canada
D301 Medical Sciences Bldg, Tsinghua University, Beijing 00084, China

Current Topics in Microbiology and Immunology (2019) 423: 95–118

https://doi.org/10.1007/82_2019_153
© Springer Nature Switzerland AG 2019
Published online: 22 February 2019
96 Y. Wen and Y. Shi

Abstract Antibody/antigen binding results in immune complexes (IC) that have a

variety of regulatory functions. One important feature is the enhanced host immune
activation against antigen contained in the complex. ICs play important roles at
several critical steps that lead to B and T cell activation, including antigen targeting/
retention, facilitated antigen uptake, antigen presenting cell activation and proper
balancing of positive and negative stimulatory signals. In both poultry industry and
clinical health care, ICs have been used as preventive and therapeutic vaccines.
With our deepening understanding of antibody biology, particularly in light of new
revelations of regulatory functions of Fc receptors, mechanistically more precise
engineering has spearheaded tailored use of this tool for infection control and
cancer therapy. IC-based treatment and prophylaxis have been tested to different
extents in HBV, HIV and influenza viral infection control and are actively exam-
ined as an alternative treatment for several forms of tumor. As a part of this book
series, this chapter aims to discuss the mechanistic aspects of IC signaling and their
impact on immune cells. We give samples how this old technology has been used
by practitioners over the last several decades and suggest potential paths for future
development of IC-based immune therapy.

1 Introduction

Translational applications of research on immune complexes (IC) have resulted in

the development of immune complex vaccines. In contrast to pathological outcomes
induced by IC deposition, vaccines in various forms of ICs have led to facilitated
antigen uptake by antigen presenting cells (APC), modified processing, and
enhanced humoral and cellular immune responses in disease therapies and pro-
phylaxis. In the poultry industry, IC vaccines have been highly successful in pre-
venting viral outbreaks in newborn chickens. In clinical settings, human IC
vaccines induce higher antibody titers with concomitant broader coverage. In
addition to promoting effective antibodies, IC vaccines are also capable of medi-
ating cellular immune responses that target persistently infected cells. This feature
is desired in the development of therapeutic vaccines against chronical viral
infections, such as HIV and Hepatitis viruses. With our deepening understanding of
Fc subtypes and the divergent functions of Fcc receptors, more tailored IC
preparations designed to fully utilize the specific antibody/FccR pairing have
opened up a door to an even more promising future. Here, we provide a summary of
the current experimental and clinical investigations into IC vaccines and a dis-
cussion on the mechanisms of IC’s immune-enhancing effects.
Vaccination is one of the most effective public health measures of controlling
infectious diseases, lowering disease burden and increasing life expectancy on a
demographic scale. The basic goal of preventive vaccines is to elicit host immune
responses against microbial infections, which includes facilitated recall responses of
specific antibodies and antigen-specific T cells in the case of real infection chal-
lenge by the microbes bearing the same antigen. Mechanistically, the magnitude
Immune Complex Vaccination 97

and the quality of the adaptive immune responses largely rely on the native and
more ubiquitous innate immune activation, and the use of adjuvants is the most
established method to trigger the innate immunity. To enhance the immunogenicity
of vaccines, a number of adjuvants have been developed, but only alum and few
others have been licensed for use. Even under the limited selection, the use of
adjuvants has led to development of several most successful vaccines in the history
targeting DTP (Diphtheria–Tetanus–Pertussis combination), HBV, influenza and
pneumococcal pneumonia (Kool et al. 2012; Wen and Shi 2016). With the
emerging prevalence of drug-resistant microbes, vaccination has become an even
more important tool for infection control. In recent years, together with the use of
prophylactic immunization, therapeutic vaccines and immunotherapies are also
under rapid development, targeting persistent infections and tumors. In comparison
with the anti-infectious purposes, the latter is still in its early stage exploration but is
expected to hold great promise. Regardless the purpose, in the long history of
vaccine development, the aim of researchers and manufacturers has always been to
improve vaccine efficacies and to decrease its side effects. Among the tools at hand,
immune complexes are one of them.
In vaccine preparations about a century ago, antibodies were added to antigens
such as diphtheria toxin to decrease its toxicity (Copeman et al. 1922). For the same
amount of antigen, enhanced immunity to antigen in complex with antibody was
first reported by Terres et al. They found that when bovine serum albumin was
mixed with its specific antibody the resulting complex induced a stronger antibody
response in mice, compared to those immunized with antigen alone, or complexed
to a non-relevant antibody (Terres and Wolins 1959, 1961). Using the same amount
of antigen, ICs were much more effective than alum-precipitated antigen (Morrison
and Terres 1966). After three decades, Berzofsky et al. used peptides from HIV
gp120 to rejuvenate CD4 cells in HIV carriers and found that addition of antibodies
that recognized the peptides substantially increased the proliferation index of the
CD4 T cells in the patients (Berzofsky et al. 1988). Around the same time, Celis and
Chang et al. performed a series of in vitro human HBsAg-specific T cells and
HBsAg co-culture experiments. They showed that monoclonal antibodies to
HBsAg of the IgG subclass increased the amount of HBsAg captured and inter-
nalized by APCs in vitro. In addition, mouse monoclonal antibodies when pre-
sented with HBsAg as IC enhanced the proliferation of T cell clones specific to
HBsAg by about two logs and increased the specific production of IFN-c (Celis
et al. 1984, 1987; Celis and Chang 1984). Similarly, Randall et al. reported that by
using an SMAA (solid matrix antibody-antigen complex) approach,
antigen-antibody complex attached to Staphylococcus aureus Cowan I strain via the
Fc fragment induced effective humoral and cytotoxic T cell responses to simian
virus 5 protein in a persistently infected mouse model (Randall and Young 1988).
In a follow-up report, specific CD8 T cells were found responsible for the clearance
of the persistent virus (Randall and Young 1991).
Those advancements gradually established that antigen-antibody ICs can sig-
nificantly increase the immunogenicity of antigens. However, to a large extent,
those practices were empirical in nature. In recent years, the development of Fc
98 Y. Wen and Y. Shi

receptor biology has completely revamped the research on this “old” weapon,
paving the way for much broader and more precise use of ICs in the control of acute
and chronic infections as well as several forms of tumor. As antibody-based ther-
apies (without explicit intension to induce ICs) are covered in other reviews, this
chapter will focus on the general mechanisms and clinical/industrial applications of
ICs in disease control.

2 How ICs Enhance Immune Responses

2.1 Fcc Receptors in IC/Antigen Uptake, Processing

and Presentation

FccRs are quintessential to our understanding of IC functions and are therefore

discussed here first. In the middle of the last century, reports surfaced that indicated
the possible presence of a moiety on the immune cell membrane that was essential
to IC-mediated immune effects. The Fc portion of antibodies seemed to exert
regulatory roles in addition to specificity determinants in Fabs (Berken and
Benacerraf 1966; Boyden and Sorkin 1960). In the later years, several key reports
described the presence of Fc receptors on the surface of cells of the immune system
(Ravetch and Kinet 1991). By the 1980s, most common forms of FccRs were
identified, except for mouse FccRIV which was cloned in 2005 (Nimmerjahn et al.
2005). In mice, IgG receptors are made of FccRI, IIB, III and IV. The human
counterparts include FccRIA, FccRIIA, FccRIIB. FccRIIC, FccRIIIA and
FccRIIIB. Among them, signaling mechanism of human FccRIIIB, a GPI-anchored
protein, remains elusive although it is known to induce Ca2+ flux in neutrophils
(Marois et al. 2011). Both FccRIIBs contain an intracellular ITIM (immunoreceptor
tyrosine-based inhibitory motif) signaling domain and are considered inhibitory and
the sole antagonist in the type I FccR family. All other FccRs transduce activating
signal via an ITAM (immunoreceptor tyrosine-based activation motif) present either
in the associated common c chain or intrinsic to its intracellular sequence. In mice,
IgG1, IgG2a (IgG2c in C57BL/6), IgG2b, and IgG3, and in human, IgG1–4 are
their respective ligands. Affinity wise, all FccRs are silent in the absence of
cross-linking, in other words, incapable to signal without higher orders formed in
ICs, except for FccIR. FccIR has an extra immunoglobulin domain that permits the
highest affinity and binding of IgG monomer without involving receptor
cross-linking. The rest of FccRs displays different affinities to different subtypes
IgGs. As each receptor/ligand pair affinity varies, the FccRs are exposed to multiple
IgG subtypes simultaneously, FccR signaling on a given cell type can be difficult to
predict. In addition to the affinity differences, cell type/activation stage-specific
expression, ITAM versus ITIM-based signaling and competition from type II FccRs
tuned by the different glycosylation states of Fc introduce several variables into
FccR regulation, creating the mesmerizing complexity. Up to this day, an important
Immune Complex Vaccination 99

task is to precisely map the affinity and in vivo pairing of subtypes of Fc and their
FcRs (Boesch et al. 2014). These complex interactions are expertly covered by
other reviews (Bournazos et al. 2015; Lux et al. 2013). Overall, we are just
beginning to understand how ICs interact with different FcRs (Bruhns 2012; Lux
et al. 2013), and a great deal of work is still needed before the concept of IgG/FcR
pairing can be fully utilized in the clinics.

2.2 Other Fcc Receptors

In addition to type I FccRs, type II FccRs (DC-SIGN and CD23) are lectin-based Fc
receptors that are sensitive to the glycosylation state of Fc. A prominent example is
the glycosylation extended from a glycan core at N297. A number of sugar moieties
can be attached to this structure, such as fucose, galactose and sialic acid. One way
this pattern of glycosylation can modify the FccR binding is to introduce a greater
flexibility of the antibody heavy chain. For instance, the presence of sialic acid
allows open conformation of the heavy chain that leads to its preferential binding to
type II FccRs. This switch in binding affinity therefore is immune-regulatory and is
in general considered inhibitory (Pincetic et al. 2014). Interestingly, at least in one
model, where OVA was targeted in vivo to DEC205 via an antibody without CD40
signal, IgGs produced were highly sialylated due to enhanced activity of sialyl-
transferase in the plasma cells (Oefner et al. 2012). For now, the research on the
signaling mechanism of type II FccR in the context of IgG binding has just started,
for instance, CD23 ligation induces FccRIIB expression and participates in the
affinity maturation of BCRs (Wang et al. 2015), although a lot of work remains.
A nomenclature-related structure, neonatal FcR (FcRn) is a MHC class I-related
protein associated with b2 m. This endosomal protein binds to IgG in low pH
environment (pH < 6.5) and plays a role of IgG transporter in epithelial/endothelial
cells. A chimeric antibody with Fc mutations that disrupts FcRn binding without
affecting FccR affinity shows reduced antigen presentation to CD4 T cells from the
bound OVA (Qiao et al. 2008). A Fc mutant with enhanced binding to FcRn
increases MHC class II antigen presentation, apparently via efficient lysosome
targeting (Mi et al. 2008). In addition, FcRn can be used as a tool to target antigens
to mucosal sites (Gosselin et al. 2009; Hioe et al. 2009), as exemplified by the
delivery of live simian immunodeficiency virus (SIV) vaccine to cervical epithe-
lium in rhesus monkey (Li et al. 2014).

2.3 Antigen Presentation

Vaccines are delivered to antigen presenting cells via internalization. The endosomal
maturation leads to the degradation of engulfed antigens, which are typically loaded
on the MHC class II molecules in a subset of endolysosomal vesicles called MHC
100 Y. Wen and Y. Shi

class II compartment. In cross-presentation, engulfed antigens also can be delivered

to MHC class I via several yet to be completely resolved pathways (Burgdorf et al.
2007; Joffre et al. 2012; Lizee et al. 2003). Cross-presentation was initially dis-
covered using antigens coated to latex beads or as cell-associated antigens (Bevan
1976; Rock et al. 1990). It was found that antigens contained within phagocytic
targets can induce CD8 T cell activation. Although this feature is typically associated
with phagocytosis of solid structures, the process is subject to additional regulations.
The facilitating effects of ICs were found in early attempts of antibody treatment of
tumors. EG7 cells (CD4-expressing EL4 tumor line) inoculated into the host were
killed by co-injection of anti-CD4 antibody. This cytolysis was dependent on CD8 T
cells and the common Fc receptor c chain (Vasovic et al. 1997). This suggested that
antibody binding “sensitized” the tumor cells for immune eradication. In a later
transgenic model for type I (autoimmune) diabetes, whereby a membrane-bound
OVA was driven by RIP (rat insulin promoter), injection of OVA-specific CD8 T
cells did not have any overt effect. However, if in the process OVA-specific IgG
antibody was co-administered, the mice developed severe type I diabetes (Harbers
et al. 2007). The result indicated that the antibody was needed for a robust CD8 T
cell activation. As expected, the presence of FccR was required for this facilitating
effect. The enhancement of antigen presentation by ICs appears to operate at two
levels. Measured by antigen uptake, the presence of ICs/FccRs appears to increase
the efficacy by two to three logs (Amigorena and Bonnerot 1999; Schuurhuis et al.
2002). However, if the effect was measured from the degree of T cell activation, the
magnitude is amplified by 4–5 logs (Regnault et al. 1999). This seems to indicate
that ICs/FccR interaction can facilitate T cell activation beyond antigen uptake by
APCs. Because FcR common c chain deficiency leads to FccR surface level
reduction, an ITAM sequence mutated version (NOTAM) was created which per-
mits FccR expression similar to the wild type. IC uptake was restored in the
NOTAM mice. In one report, IC-associated antigen cross-presentation was lost
(Boross et al. 2014). However, a recent paper found that antibodies fused with
antigen targeting FccRIV and FccRIIB/III showed no or minimal dependence on the
ITAM signaling (Lehmann et al. 2017). Therefore intracellular signaling emanated
from the ITAM sequence is essential for cross-presentation at least in some settings,
but in other cases, such as active binding by antibodies specific for FccRs (rather
than FcR/Fc binding), the ITAM sequence may not be essential. Although most DCs
can cross-present antigens in vitro, there is evidence that in vivo, CD8+ or CD103
+ DCs are the main cell types involved in cross-presentation in mice.
OVA-conjugated to an antibody targeting DNGR1 (a c-type lectin receptor)
expressed by these cells elicited strong response against OVA-bearing B16 tumor
(Sancho et al. 2008). However, whether these cells are particularly sensitive to ICs is
not known. It has been reported that CD8+ DCs mediated cross-presentation without
help from FccRs, while antigen delivered in the form of ICs to FccR was critical for
CD8− DC antigen presentation to CD8+ T cells (den Haan and Bevan 2002).
For MHC class II antigen presentation, exogenous soluble antigens are engulfed
via several endocytic mechanisms, such as clathrin-mediated and caveolin (lipid
rafts)-mediated endocytosis. In smaller scale, these antigens can be taken up via
Immune Complex Vaccination 101

more subtle pinocytosis or membrane ruffles. Soluble antigen endocytosis is

enhanced by receptor activities, i.e., mannose, C-type lectin and cytokine receptors
(Cendrowski et al. 2016). FccR-mediated endocytosis is highly efficient and appears
to enhance both MHC class II antigen presentation as well as cross-presentation to
CD8+ T cells, in contrast to, i.e., the sole facilitating effects for CD4 T cell activation
by mannose receptor (Berlyn et al. 2001). A mutation in the ITAM sequence that
blocks Syk binding reduced the antigen delivery to the lysosome. Syk deficiency
also dissociated the colocalization of DM molecule (critical for MHC class II peptide
exchange from invariant chain-derived CLIP sequence to bona fide epitope
sequences) with class II molecules (Le Roux et al. 2007), reducing the peptide
loading efficiency. Interestingly, while both DCs and macrophages express MHC
class II molecules and ICs can also efficiently target macrophages, only DCs can
present antigens to cognate CD4 T cells (de Jong et al. 2006). This feature may be to
a large extent due to the intracellular antigen targeting in DCs.
B cells, as a group of APCs that do not usually express activating FccRs, cannot
be activated by ICs directly. However, B cell activation can be enhanced by
FccRIIB-mediated antigen retention. Lymph node follicular DCs (FDCs) express
FccRIIB (Kosco et al. 1989; Szakal et al. 1988a, b). In vivo, the long extensions of
FDCs are often coated with ICs in a FccRIIB-dependent manner, a mechanism that
avoids clearance. This provides a steady source of antigens for B cells. In addition,
FccRIIB-mediated internalization can prolong antigen presentation by “regurgita-
tion.” Large amounts of ICs can be trapped by macrophages in the subcapsular
cavities. These ICs can be transferred to FDCs for FccRIIB-based internalization. In
this case, ICs are not routed into the degradative pathway. Instead, the complexes
are retained inside FDCs and may reappear on the cell surface and made available
to B cells (Bergtold et al. 2005; Heesters et al. 2013). This process significantly
extends the duration of antigen availability and alters lymphocyte activation
kinetics. This process is involved in BCR hypermutagenesis to give rise to high
affinity antibodies (Wu et al. 2008).

2.4 Intracellular Targeting

IC-facilitated antigen presentation simultaneously alters intracellular targeting. This

is a part of the mechanistic insight how these complexes can both qualitatively and
quantitatively impact T and B cell activation. Despite the evidence that FcR
common c chain ITAM signaling is not essential in all cases (Lehmann et al. 2017),
some studies implicating FccR signaling in regulating antigen processing come
from the fact that Syk deficiency blocks IC-mediate immune activation, yet Syk
itself is not absolutely essential for IC antigen uptake (Boross et al. 2014).
Regrettably, this finding has not led to sufficient follow up at cellular and organ-
ismic levels. IC-mediated cross-presentation appears to require TAP and is sensitive
to proteasome inhibition (Regnault et al. 1999), suggesting that FccR signaling may
lead to antigen transport from endosome to cytosol or ER-phagosome fusion-based
102 Y. Wen and Y. Shi

MHC class I antigen presentation (Joffre et al. 2012). How ICs mediate these events
are completely unknown. In some studies, ICs appear to retain antigen in the early
endocytic vesicles (Chatterjee et al. 2012; Cohn et al. 2013), avoiding the speedy
endosomal maturation that exposes antigens to the harsh acidification accompanied
by the staged chain activation of vacuolar cathepsins. In this process, Cathepsin S, a
neutral pH protease, takes up the bulk of peptide generation (Hari et al. 2015),
which favors the production of epitope peptides. According to recent studies, the
delayed antigen degradation in DCs, in comparison with macrophages and neu-
trophils, may be a critical feature that leads to the exceedingly strong antigen
presentation by DCs (Joffre et al. 2012; Savina et al. 2006). How ICs and FccR
activation amplify this feature is not known. For MHC class II antigen presentation,
ICs can help target antigens into the endolysosomal vesicles that give rise to the
MHC class II compartment. Viral antigen rabies G, free or in complex of ICs, could
enter Rab5+ vesicles (early endosomes). However, IC-associated rabies G then
appeared in Rab9+ late endosomes while free antigen is seen colocalized with
Rab11+ recycling endosome (St Pierre et al. 2011). This effect can explain the more
efficient class II antigen presentation and may be not contradictory to the “shallow
targeting” observed for class I cross-presentation. As the overall effect of ICs is to
increase antigenic epitope availability for both pathways, IC-mediated antigen
targeting may show subtle changes per cell types and antigen/FcR subtypes
involved, collectively reducing the waste of antigens in the endocytic maturation
process.

2.5 APC Regulation

In an early study, it was found that bone marrow-derived DCs (BMDCs) loaded
with OVA/OVA-specific antibody complex induced antigen-specific humoral and
CD4 and CD8 cellular responses in mice. Although total IgG was not affected by
immunization, specific anti-OVA IgG responses were significantly enhanced after
immunization with OVA/IC–loaded BMDCs. This immunization was able to
suppress a tumor bearing the same antigen (Rafiq et al. 2002). Importantly, in this
experiment, DC-specific deficiency of FccRs, TAP, b2 m or MHC class II elimi-
nated the tumor immunity, suggesting that ICs delivered to DCs alone is sufficient
to include both humoral and cellular immunity. In accordance with this observation,
ICs were later reported to empower CD8+ T cells via dual effects. First, OVA IC
complex indeed increased the antigen uptake by DCs compared to OVA plus an
control antibody. ICs alone also triggered IL-12 expression, suggesting a simulta-
neous cell activation. The specific CD8 response against the OVA epitope induced
by the IC could not be replaced by OVA and LPS, pointing to IC’s unique ability to
prime CTL activation (Schuurhuis et al. 2002). Furthermore, ICs were shown to
reprogram CD8 T cells. By using epitopes from influenza virus, Leon et al. studied
ICs effects on memory CD8 T cells in mice and showed although prolonged Ag
presentation did not alter the number of memory CD8 T cells, ICs were essential for
Immune Complex Vaccination 103

programming the capacity of these cells to proliferate, produce cytokines and

protect the host after secondary challenge. Importantly, prolonged Ag presentation
by DCs was dependent on virus-specific, isotype-switched antibodies (Abs) that
facilitated the capture and cross-presentation of viral antigens by FccR-expressing
DCs (Leon et al. 2014).
From the prospective of signaling, FccR-mediated APC activation follows a
typical immune tyrosine-based cascade common to most immune cells. The
receptor binding triggers the recruitment of Syk to the phosphorylated ITAM,
followed by activation of PI3Ks, PLCs, MAPKs and NFjb (Mocsai et al. 2010). In
addition to organizing cytoskeleton for phagocytosis, these canonical immune
activation events upregulate CD40, CD86, IL-6, IL-13 and IL-2 (Boross et al. 2014;
Matsubara et al. 2006; Perreau et al. 2008). FccR binding by ICs also sends a
maturation signal that initiates the migration of DCs from the periphery to draining
lymph nodes (Clatworthy et al. 2014). It has been suggested that the internalized
ICs can activate intracellular TRIM21 (a non-typical Fc receptor), which leads an
independent signaling chain in K63-specific Ubc13-mediated ubiquitination event.
This event triggers NFjb, AP-1 and IRF pathways for synergistic activation of
APCs (McEwan et al. 2013).
Depending on the setup, the FccR signaling can be inhibitory. IVIG, the clinical
application of purified whole blood immunoglobulins, binding to FccRIII blocks
macrophage activation in response to IFN-c (Park-Min et al. 2007). In contrast to
the belief that formation of ICs during chronic infection leads to inflammation, it
was recently reported that in LCMV clone 13 long-term infection, ICs formed
during the infection blocked the T and B cell depletion by specific antibodies and
DC activation by anti-CD40 antibody (Wieland et al. 2015; Yamada et al. 2015).
This inhibitory effect was dependent on the CD4 T cell-dependent antibody
production.

3 Experimental and Clinical IC Vaccines

3.1 IC Vaccines in Poultry Industry

Preventive IC vaccine was first tested in poultry breeding to control infectious

bursal disease (IBD), a viral infection targeting bursa of Fabricius and killing
developing B cells. IBD is one of the economically most important diseases that
affects commercially produced chickens worldwide. The obstacle of using con-
ventional live IBDV vaccines was the high levels of maternal antibodies interfering
with the efficacy of vaccines in chicks (Naqi et al. 1983). To overcome this
obstacle, an IC vaccine was developed by mixing IBDV strain 2512 with bursal
disease antibodies (BDA). One-day-old chicklings were administered IBDV-BDA,
which induced active immunity and protection against a standard IBDV challenge
(Haddad et al. 1997). Later in a field trial, 40,000 broiler eggs at 18 days of
104 Y. Wen and Y. Shi

embryonation and 40,000 one-day-old chicks were each split into two groups. IC
Vaccine (Cevac® Transmune) was either administered by in ovo, or by subcuta-
neous route. A highly virulent IBDV strain was used for challenge. A marked
protection was reached by the IC vaccine (Iván et al. 2005). Most remarkable was
the low level of bursal and splenic B cell depletion in those vaccinated with IBDV–
ICs (Jeurissen et al. 1998). Furthermore, in ovo inoculation with the IBDV–IC
vaccine induced more germinal centers in the spleen, and larger amounts of IBDV
were deposited on splenic and bursal FDCs. It was concluded that not only in ovo
IC vaccination was safe to the embryo and kept the hatchability as expected, but
also a full protection against IBD was demonstrated for the broilers’ lifespan.
Although there are a number of IBD vaccines under development (Müller et al.
2012), IBDV–ICs have been in active use by chicken breeders in several countries.
Recently, recombinant neutralizing antibodies have been developed to replace BDA
previously used for IC vaccines, and the second generation of recombinant
antibody-based experimental IBDV-IC vaccine is undergoing testing (Ignjatovic
et al. 2006).
IC applications in the prevention of Newcastle disease virus (NDV, a flu-like
infection with high death rates) have also been equally successful. Maternal IgY
antibodies can protect the chicks for a short period, however, the gap of vulnera-
bility thereafter is a window for NDV infection before the full-scale immunity is
developed in more mature birds. Injection of the IgY right after hatching extends
the protection. Whether this protection is completely dependent on IC formation is
not clear. Commercially, NDV ICs have been produced by mixing antisera from
immune chickens with the viral particles. This IC provided complete protection in
broiler chickens (Pokric et al. 1993). A later report showed that in ovo application
of the IC vaccine in maternal antibody–positive chickens, the birds were protected
from clinical disease against three highly virulent strains of NDV (Kapczynski et al.
2013). In addition, IC vaccines for infectious anemia disease virus (CAV) in
chickens are also under development (Karel A. Schat et al. 2011). In duck HBV
vaccination test, one-day-old ducklings were inoculated with duck hepatitis B virus
(DHBV), an infection that is persistent and somewhat mimics the human disease.
They were then vaccinated with an DHBsAg IC linked to SMAA. 70 and 50% of
treated ducks were cleared of viremia and antigenemia after three injections (Wen
et al. 1994). This study prompted the attempts to treat persistent human HBV
infections with ICs discussed later.

3.2 IC Vaccination for Influenza Virus Infection

Influenza virus belongs to the orthomyxoviridae, with its genome consisting of

eight segmented RNAs that are prone to mutations and reassortments, leading to
emergence of new viral strains. Due to the lack of protective antibodies to the
emerging strains in the population, numerous deaths and millions of hospitalization
following each outbreak are currently difficult to control. Each year, World Health
Immune Complex Vaccination 105

Organization collects new isolates worldwide for analysis to best predict impending
danger and to prepare vaccines for all countries. It is desirable to develop a broadly
protective flu vaccine which can induce immune responses against a number of
influenza strains.
Recently, in a study of seasonal trivalent influenza vaccine (TIV) immunization
in healthy adults, it was found that the abundance of sialylated Fc (sFc) on anti-HA
(hemagglutinin) IgGs produced during the early plasmablast response correlated
with vaccine efficacy. The mechanism of this enhanced antibody response involved
co-engagement of CD23 by sialylated Fc domains. Mechanistically, sFc engage-
ment of CD23 induced the inhibitory FccRIIB expressing, setting up a high
threshold for selecting high affinity BCRs (Wang et al. 2015). This observation was
further extended by constructing ICs with 2014–15 TIV and sialylated Fc anti-HA
IgGs that were used to immunize mice. When the sera of IC vs. HA only immu-
nized mice were compared, serum IgG obtained from animals vaccinated with TIV
complexed with sFc IgG showed significantly increased binding to the H1 from
several other strains, and H3 and even H5 from avian influenza virus. This showed
that IC elicited higher-affinity antibody responses that were enhanced for breadth
and potency of activity against influenza viruses in mice. In some settings, a Fc
point mutation F241A could function in place of the sialyation to engage CD23, ICs
produced with this antibody also demonstrated high potency in inducing neutral-
izing antibodies again HA (Maamary et al. 2017). This observation has significant
implication in practical deployment for development of an universal seasonal flu
vaccine. Also in TIV vaccination, ICs may play a role in NK cell-mediated ADCC.
In TIV recipients, plasma sera collected from those patients facilitated NK cell
degranulation in the presence of TIV, suggesting an IC-dependent cytolytic event
(Goodier et al. 2016).
Another modified version of IC was to fuse the Fc fragment with a specific
antigen. This has been explored in H5N1 vaccine, an influenza A subtype virus that
surfaced in 2003 and has been circulating in aves and spreading to humans with
severe morbidity and high mortality. Although a number of preventive vaccines
against H5N1 have been studied in mice (Biesova et al. 2009; Szecsi et al. 2009;
Tao et al. 2009), and in preclinical evaluation (Kreijtz et al. 2009), Du et al.
developed a new approach and designed two recombinant influenza vaccine can-
didates by fusing HA1 fragment of A/Anhui/1/2005(H5N1) to either Fc of human
IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc). Both proteins were able to induce high
titer of antibodies. Importantly, immunization with these two vaccine candidates,
especially HA1-Fdc, provided complete cross-clade protection against high-dose
lethal challenge of different strains of H5N1 virus covering clade 0, 1 and 2.3.4 in
mouse model. This finding suggests that fusing HA antigen with Fc could be
developed into an efficacious universal H5N1 influenza vaccine (Du et al. 2011).
ICs have been used to enhance flu-specific cytolytic T cells in special popula-
tions. CTL response in aging populations has reduced potency. In young Balb/c
mice, although NP-specific CTL response can be readily induced with either killed
H1N1 virus or in combination with a NP-specific antibody, only the latter was able
trigger strong cytolytic response in older mice. This enhanced killing was
106 Y. Wen and Y. Shi

accompanied by strong IFN-c secretion by both CD8 and CD4 T cells (Zheng et al.
2007). Although these experiments point to specific use of ICs in influenza virus
protection, the advantages demonstrated may be important in setting up larger scale
prophylactic vaccination for other viral infections.

3.3 IC Vaccination for HIV Infection

HIV, an RNA retrovirus, enters cellular nucleus for genome integration during host
cell replication cycle. Although HIV was discovered several decades ago, due to its
long latent period, several transmission modes and lifelong persistency, AIDS is
still at the top of global medical concerns. Currently, antiretroviral drugs have been
effective in prolonging the life span of AIDS patients and reducing the mortality,
yet morbidity of AIDS in some developing countries and burden of health care cost
are still great concerns. From epidemiological prospective, effective prophylactic
HIV vaccine remains the most desirable measure. Currently, DNA-based vaccines
with prime and boost strategy, new/altered delivery protocols as well as approaches
to rebuild the damaged host immune systems are under study (Chen et al. 2014).
Initial attempts of IC-based HIV vaccination were not all successful. Adenovirus
5 (Ad5) encoding HIV epitopes in complex with anti adeno antibodies have been
tested in clinical trials (Shiver and Emini 2004). In a preliminary study, adeno
vector carrying b galactosidase sequence was used in combination of antibody
targeting the vector. Although the higher antibody to virus ratio induced a transient
T cell response and little humoral response, a lower ratio was effective in triggering
both arms of immunity against the model antigen (Choi et al. 2012). Importantly,
the presence of antibody significantly reduced the toxic effect of the viral vector. In
the STEP trials, HIV gag, pol and nef gene cassettes were inserted into the adeno
vector, however individuals with preexisting immunity against the vector showed
greater risk of HIV infection following the immunization (Buchbinder et al. 2008).
Subsequent studies indicated that likely due to mucosal localization of Ad5 viral
accumulation, a T cell response characterized by high a4b7 and CCR9 expression
was detected, coupled with higher CCR5 (HIV co-receptor) level. In addition,
adeno virus-stimulated T cells were more susceptible to HIV entry, aggravating the
risk of vaccine recipients (Benlahrech et al. 2009; Perreau et al. 2008). In a rhesus
monkey model, a non-neutralizing antibody against SIV did not significantly pro-
tect the recipients from secondary challenges in comparison with the SIV immu-
nization alone (Polyanskaya et al. 2001). In fact, anti-gp41 antibody resulted in IC
that was associated with FccRIIB signaling, and in expression of anti-inflammatory
genes such as SPRED1 and COMMD1, likely to suppress immune responses.
In other attempts, IC-based vaccines for HIV were reported by scientists using
antibodies recognizing CD4-binding sites and V3 region of gp120, which has
shown to elicit neutralizing antibodies in mice (Hioe et al. 2009; Visciano et al.
2008). As the variances in gp120 are large, a monoclonal antibody (654-D) with
high affinity to a large number of V3 variants was found, and the ICs formed using
Immune Complex Vaccination 107

this antibody induced V3 neutralizing antibodies in Balb/c mice (Hioe et al. 2009).
Interestingly, binding by this antibody exposed other epitopes in the V3 that are
usually inaccessible by other mAb. Similarly, IC formed with mAb A32 that binds
to gp120 also induced neutralizing antibodies against a number of HIV strains (Liao
et al. 2004). This strategy of “induced accessibility” offers a new concept in the
clinical use of ICs as anti-viral treatments.
The concept of broadly neutralizing antibodies (bNAbs) against HIV is the most
intensely studied AIDS treatment at the moment. High-throughput screening of B
cells from HIV carriers has identified clones that are capable of targeting multiple
HIV subtypes (Walker et al. 2009; Wu et al. 2010). For AIDS treatment, the
strategy employed to reactivate the latent virus with antiretroviral treatment fol-
lowed by immune therapies is also under trial. bNAbs are usually effective in
controlling HIV viremia in mice (Horwitz et al. 2013). However, one important
concern is the viremia rebound following the termination of treatment. One way to
combat this problem is to use multiple bNAbs at the same time, i.e., TriMix
antibodies (3BNC117, anti-CD4bs; PG16, anti-V1/V2 loop region; and 10-1074,
anti-V3) that can significantly delay the rebound. The other strategy is the “shock
and kill” that involves the use of TriMix in combination with several viral inducers.
This can suppress the rebound for several months (Halper-Stromberg et al. 2014). It
is important to note that the control TriMix carrying mutated Fc that is unable to
engage FccRs has a reduced inhibition of the viremia reappearance. Fc swapping
analysis suggested that for a given Fab, mouse IgG2A Fc has the highest potency in
blocking HIV. In a mouse model whereby human FccRs were used to genetically
replace mouse counterparts, human IgG1 carrying mutations that facilitate binding
to activating FccRs (FccRIIA and FccRIIIA) was more effective comparing with
the mutations that abolish the binding (Bournazos et al. 2014).
In SIV and SHIV (Simian HIV is a chimeric virus in which HIV Env substitutes
for that of SIV) models, ICs formed with infected cells were more efficiently
captured by DCs than infected cells alone, leading to stronger functional DC
activation, potent anti-viral cytotoxic T lymphocyte and T cell memory responses
and the inhibition of the expansion of regulatory T cells (Lambour et al. 2016;
Pelegrin et al. 2015). One mouse model has drawn great interest in studying human
vertical HIV transmission in childbearing mothers. FcCasE virus-infected mice
during pregnancy can transmit the virus to the newborn. This infection causes
erythroleukemia once the offspring reach the adulthood. Neonatal injection of mAb
66 induced both antibody and T cell responses against the virus and significantly
increased the rate of long survival in comparison with the nearly total fatality in the
untreated (Michaud et al. 2010).

3.4 IC Vaccination for Viral Hepatitis B

Hepatitis B virus (HBV) belongs to hepadnaviridae, a DNA virus which replicates

via a RNA–DNA intermediate. HBV enters cell nucleus forming an episomal
108 Y. Wen and Y. Shi

closed covalent circular DNA (cccDNA). cccDNA is replenished by recycling of

the viral genome and is the template for transcription and translation of multiple
viral proteins: Hepatitis B surface antigen (HBsAg), Hepatitis core antigen
(HBcAg) and the viral polymerase. Translational modification of HBcAg generates
a secreted protein HBeAg, which is a marker for the replication of virus (Seeger and
Mason 2015). Despite the remarkable success of prophylactic hepatitis B vaccines,
there are still around 300 million HBsAg-positive carriers worldwide. Among them,
some will progress to chronic hepatitis B (CHB), liver cirrhosis and hepatocellular
carcinoma. Multiple immune defects have been revealed in chronic CHB patients
(Schmidt et al. 2013) and restoration of effective host immune responses is nec-
essary for virus clearance.
Currently, several therapeutic vaccines for CHB are in experimental or clinical
studies (Lobaina and Michel 2017). Based on our previous approach of using ICs
absorbed to SMAA for restoration of immune responses in duck hepatitis B
virus-infected immune tolerant ducks (Wen et al. 1994), a YIC (yeast-derived
recombinant HBsAg complexed to human anti-HBsAg antibody) therapeutic vac-
cine candidate for CHB patients has been developed. We hypothesized that due to
the persistence of huge amount of HBV antigens, CHB patients are immune tolerant
and cannot generate effective immune responses against the virus, as a part of
immune defects in the carriers. HBsAg complexed with its antibodies was expected
to activate APCs to uptake ICs through their FccRs, and the antigen will be pro-
cessed and presented effectively to T cells (Wen 2009). In vitro studies with
PBMCs stimulated with GM-CSF/IL-4 from CHB patients, HBsAg-anti-HBs
upregulated HLA-II, CD80, CD86 and CD40 molecules on DCs and improved
dendritic and T cell interactions as measured by IFN-c and IL-2 production (Yao
et al. 2007). One version of HBsAg ICs was able to induce antibody production in
HBsAg-transgenic mice, followed by a seroconversion to become HBsAg positive
(Celis et al. 1987; Zheng et al. 2001). HBsAg Fc fusion protein was also able to
enhance T cell cytotoxicity and to decrease serum HBsAg in HBsAg-transgenic
mice (Meng et al. 2012). These efforts were followed up by clinical trials.
Although a number of HBV vaccines are in clinical trials (Lobaina and Michel
2017), the IC-based therapeutic vaccine is leading in many aspects. In a pilot study,
HBsAg IC mixed with alum was used in HBV carriers and reached statistically
significant reduction in blood HBV DNA (Wen et al. 1995). YIC was approved by
China FDA for clinical trial in 2002 and is currently entering phase III. In phase I
trial among healthy adults, YIC induced high titer of anti-HBs and up-regulated
production of IL-2 and IFN-c (Xu et al. 2005). Subsequently, in phase II A and B
clinical trials, a decrease in serum viral load and seroconversion from serum HBeAg
positive to anti-HBe positive were observed, with cytolytic and noncytolytic
immune responses comparable to treatment with conventional IFNa. To analyze the
cellular immune responses induced by YIC, a pilot study in patients treated with this
IC and the controls (alum and saline) were studied together with an anti-HBV drug,
adefovir, as a basic treatment. Results showed that the relative number of specific
CD4 and CD8 T cells were increased with decrease of Treg cells in patients treated
with YIC, compared to those in the control groups (Zhou et al. 2017). These clinical
Immune Complex Vaccination 109

observations correlated well with results previously observed in vitro and in mice,
indicating that ICs are a promising therapeutic vaccine for treatment of CHB
patients.
Recently, several human anti-HB neutralizing antibodies for treatment of animal
models mimicking CHB were reported. One antibody (2H5-A14) that blocks viral
large envelop protein preS1 domain binding to its receptor sodium taurocholate
cotransporting polypeptide was highly neutralizing. 2H5-A14 therapeutic effect was
dependent on FccRs (Li et al. 2017). In another model, mAb E6F6 binding to a
conserved epitope on the virion resulted in formation of ICs and FccR-dependent
phagocytosis. E6F6 treatment blocked HBV infection and restored anti-HBV T cell
responses in carrier mice (Zhang et al. 2016). In an HBV hydrodynamic injected
mouse model, another broadly neutralizing human monoclonal antibody against
HBsAg, G12, was shown to decrease mouse serum HbsAg, with a potency 1000
times higher than that of the current HBIG (Wang et al. 2016). These new findings
offer more diverse approaches of applying ICs in HBV prevention and treatment.

3.5 IC Vaccination for Tumors

Antibodies have long been considered a valid approach in cancer treatment. The
most discussed and so far the most successful intervention is to use antibodies or
their derivatives to block negative signaling such as CTLA4 and PD1/PDL1 in
immune cells, better known as check point blockage. Another category of
antibody-based cancer therapy involves inhibition of growth factors such as VEGF
and EGFR (Nimmerjahn and Ravetch 2012). IC-based therapies in comparison are
at the experimental stage. However, the inherent safety and better patient toleration
make this class of treatments a worthwhile study topic.
Antibody-based immune therapies can be divided into direct cell death induction
and immune responses against tumor as consequence of immunization. Some
antibodies against tumor surface molecules can induce FccR-dependent ADCC,
leading to tumor cell apoptosis (Moalli et al. 2010). ICs in this setting have shown
greater flexibility and can be used for prophylactic and therapeutic intervention.
A well-studied mouse model is B16 melanoma expressing transfected OVA anti-
gen. For instance, in an OVA-expressing B16 tumor inoculation model, injection of
DCs pulsed with OVA ICs inhibited the tumor. In fact, it reached a 40% eradication
of the established tumor in mice. The ICs induced memory responses in mice that
were protective in secondary tumor challenges (Rafiq et al. 2002).
The IC-based anti-tumor treatment has been tested with real-life tumor antigens.
Antibodies against tumor antigens such as HER2/neu and syndecan-1 triggered strong
CD8 T cell responses against the antigen-bearing tumors. These effects were mainly
mediated via facilitated antigen presentation (Dhodapkar et al. 2002; Kim et al. 2008;
Wolpoe et al. 2003). Tumor antigen Syndecan-1-specific antibody-treated melanoma
stimulated HLA-A2.1-restricted cytolytic T cell response against some of the
best-known tumor antigens such as MAGE-3 and NY-Eso-1, suggesting targeting one
110 Y. Wen and Y. Shi

antigen can release sufficient bystander activation of T cells against epitopes otherwise
“unseen” by the immune system (Dhodapkar et al. 2002).
While there is no doubt that in the IC-based tumor therapy models FccRs were
involved both at opsonization/phagocytosis and DC maturation, an emerging
consideration is the ratio of engagement of activating and inhibitory FccRs
(Nimmerjahn and Ravetch 2005). Although historically the involvement of anti-
body subtypes and FccRs was not a carefully controlled aspect, Ravetch et al.
suggested that proper matching of Fcs with their activating and inhibitory receptors
could be used to induce preferred anti-tumor effects (Nimmerjahn and Ravetch
2005). mIgG2A subtype antibody TA99 binds to activating FccRs with high
affinity. If the binding was targeted to FccRIV, a strong anti-tumor effect was
observed (Nimmerjahn and Ravetch 2005). Their group also reported that
CD20-specific IgG2A antibody treatment led to prolonged survival of mice chal-
lenged with CD20-positive EL4 cells. In this setup, mice with a DC-specific
deletion of FccRIV were not protected by the same treatment (DiLillo and Ravetch
2015). Future efforts in precise engineering to select proper activating/inhibitory
ratio in IgG subtype/FccR pairs likely represent some of the best options in
IC-based cancer treatment.
The role of ICs in tumor therapies may be unintentionally built into some
existing protocols. Several reports have shown that the antibodies against TNF
family receptors, such as OX40 as well as CTLA4, intensely studied in check point
therapy, in fact depleted Tregs in vivo in an activating FccR-dependent manner
(Bulliard et al. 2013; Bulliard et al. 2014; Simpson et al. 2013). In another instance,
Imprime, a glucan like anti-tumor drug intended to induce innate immune
responses, required serum anti b-glucan antibodies (ABA) to achieve its effects in
patients. ABA binding to Imprime resulted in complement fixation, ROS produc-
tion and antibody-dependent phagocytosis. In patients with low existing ABA,
provision of ABA increased the innate immune responses (Chan et al. 2016). Such
“unintended” effects mediated by ICs are worthwhile topics for a variety of current
cancer treatment protocols.

4 Future Prospective of IC Vaccines

In the development of IC vaccines, the goal remains to be the induction of a broad

spectrum of neutralizing antibodies against a microbe or a tumor antigen along with
a well-developed cell-mediated immunity. Such efforts are currently seeing a great
deal of success in IC-based treatments of HIV and influenza virus infections. For
future development, it is important to obtain similar efficacies to other viruses,
particularly those causing prevalent infections in humans and livestock. Although
approaches can be multifaceted, it appears that the leading research should be
centered on bNAbs and proper matching of Fc subtypes to induce activation of a
desired ratio of inhibitory and activating FccRs.
Immune Complex Vaccination 111

Several fronts in technology development are opening up new territories for IC

vaccines. Yoshida et al. reported that FcRn is the vehicle that transports IgG across
the intestinal epithelial barrier into the lumen. This transportation delivers IgG/
antigen complex into the lamina propria for processing by DCs and presentation to
CD4+ T cells in regional lymphoid structures (Yoshida et al. 2004). This suggests
that IC vaccines could be delivered via the oral route. This possibility was con-
firmed by a pilot experiment of HBsAg IC immunization per os in mice (our
unpublished results). Another important function of FcRn is the transcytosis of IgG
in the placental syncytiotrophoblasts. As maternal vaccination becomes an
increasingly important strategy for the protection of young infants, employing ICs
via FcRn for pregnant women may be considered in low-income countries where
the burden of infant morbidity and mortality is highest (Chu and Englund 2014;
Yoshida et al. 2004). Another area that deserves particular attention is the new
technologies applied in understanding, identifying and designing most efficacious
antigen specificity determinants (Fabs) with broad coverage on intended pathogens.
Efforts are being made to introduce a set of “common principles” based on
molecular, genetic and structural knowledge to generate most cutting-edge antibody
treatment (Crowe 2017). These principles are almost certainly applicable in
IC-based therapies.
Although IC vaccination has a long history, its application in preventive and
therapeutic clinical trials is still at an early stage. Sustained exploration of mech-
anisms of IC vaccines by basic research scientists, objective and candid evaluations
on clinical trials by clinicians, ample financial support from industries, and com-
prehensive and systematic guidelines from regulatory authorities are all indis-
pensable in a joint effort to bring this technology to its fruition for public health
benefits.

Acknowledgements YM. W. has been supported by 863 Grants from China National Science
and Technology since 1988–2008, and supported by the Grand Research Program for Infectious
Diseases, China (2008ZX10002-003, 2012ZX10002002004, 2017ZX10202201002.
Y. S. is supported by the joint Peking-Tsinghua Center for Life Sciences, the National Natural
Science Foundation of China General Program (31370878), and by grants from the US NIH
(R01AI098995), the Natural Sciences and Engineering Research Council of Canada
(RGPIN-355350/396037) and the Canadian Institutes for Health Research (MOP-119295).

References

Amigorena S, Bonnerot C (1999) Fc receptors for IgG and antigen presentation on MHC class I
and class II molecules. Semin Immunol 11:385–390
Benlahrech A, Harris J, Meiser A, Papagatsias T, Hornig J, Hayes P, Lieber A, Athanasopoulos T,
Bachy V, Csomor E, Daniels R, Fisher K, Gotch F, Seymour L, Logan K, Barbagallo R,
Klavinskis L, Dickson G, Patterson S (2009) Adenovirus vector vaccination induces expansion
of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to
HIV-1. Proc Natl Acad Sci U S A 106:19940–19945
112 Y. Wen and Y. Shi

Bergtold A, Desai DD, Gavhane A, Clynes R (2005) Cell surface recycling of internalized antigen
permits dendritic cell priming of B cells. Immunity 23:503–514
Berken A, Benacerraf B (1966) Properties of antibodies cytophilic for macrophages. J Exp Med
123:119–144
Berlyn KA, Schultes B, Leveugle B, Noujaim AA, Alexander RB, Mann DL (2001) Generation of
CD4(+) and CD8(+) T lymphocyte responses by dendritic cells armed with PSA/anti-PSA
(antigen/antibody) complexes. Clin Immunol 101:276–283
Berzofsky JA, Bensussan A, Cease KB, Bourge JF, Cheynier R, Lurhuma Z, Salaun JJ, Gallo RC,
Shearer GM, Zagury D (1988) Antigenic peptides recognized by T lymphocytes from AIDS
viral envelope-immune humans. Nature 334:706–708
Bevan MJ (1976) Cross-priming for a secondary cytotoxic response to minor H antigens with H-2
congenic cells which do not cross-react in the cytotoxic assay. J Exp Med 143:1283–1288
Biesova Z, Miller MA, Schneerson R, Shiloach J, Green KY, Robbins JB, Keith JM (2009)
Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5
hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus. Vaccine
27:6234–6238
Boesch AW, Brown EP, Cheng HD, Ofori MO, Normandin E, Nigrovic PA, Alter G,
Ackerman ME (2014) Highly parallel characterization of IgG Fc binding interactions. MAbs
6:915–927
Boross P, van Montfoort N, Stapels DA, van der Poel CE, Bertens C, Meeldijk J, Jansen JH,
Verbeek JS, Ossendorp F, Wubbolts R, Leusen JH (2014) FcRgamma-chain ITAM signaling is
critically required for cross-presentation of soluble antibody-antigen complexes by dendritic
cells. J Immunol 193:5506–5514
Bournazos S, DiLillo DJ, Ravetch JV (2015) The role of Fc-FcgammaR interactions in
IgG-mediated microbial neutralization. J Exp Med 212:1361–1369
Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV (2014) Broadly
neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell
158:1243–1253
Boyden SV, Sorkin E (1960) The adsorption of antigen by spleen cells previously treated with
antiserum in vitro. Immunology 3:272–283
Bruhns P (2012) Properties of mouse and human IgG receptors and their contribution to disease
models. Blood 119:5640–5649
Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, Li D, Gilbert PB, Lama JR,
Marmor M, Del Rio C, McElrath MJ, Casimiro DR, Gottesdiener KM, Chodakewitz JA,
Corey L, Robertson MN, Step Study Protocol Team (2008) Efficacy assessment of a
cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised,
placebo-controlled, test-of-concept trial. Lancet 372:1881–1893
Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, Wilson NS, Dranoff G,
Brogdon JL (2013) Activating Fc gamma receptors contribute to the antitumor activities of
immunoregulatory receptor-targeting antibodies. J Exp Med 210:1685–1693
Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson N, Brogdon J (2014) OX40 engagement
depletes intratumoral Tregs via activating FccRs, leading to antitumor efficacy. Immunol Cell
Biol 92
Burgdorf S, Kautz A, Bohnert V, Knolle PA, Kurts C (2007) Distinct pathways of antigen uptake
and intracellular routing in CD4 and CD8 T cell activation. Science 316:612–616
Celis E, Abraham KG, Miller RW (1987) Modulation of the immunological response to hepatitis B
virus by antibodies. Hepatology 7:563–568
Celis E, Chang TW (1984) Antibodies to hepatitis B surface antigen potentiate the response of
human T lymphocyte clones to the same antigen. Science 224:297–299
Celis E, Zurawski VR Jr, Chang TW (1984) Regulation of T-cell function by antibodies:
enhancement of the response of human T-cell clones to hepatitis B surface antigen by
antigen-specific monoclonal antibodies. Proc Natl Acad Sci U S A 81:6846–6850
Cendrowski J, Maminska A, Miaczynska M (2016) Endocytic regulation of cytokine receptor
signaling. Cytokine Growth Factor Rev 32:63–73
Immune Complex Vaccination 113

Chan AS, Jonas AB, Qiu X, Ottoson NR, Walsh RM, Gorden KB, Harrison B, Maimonis PJ,
Leonardo SM, Ertelt KE, Danielson ME, Michel KS, Nelson M, Graff JR, Patchen ML, Bose N
(2016) Imprime PGG-mediated anti-cancer immune activation requires immune complex
formation. PLoS One 11:e0165909
Chatterjee B, Smed-Sorensen A, Cohn L, Chalouni C, Vandlen R, Lee BC, Widger J, Keler T,
Delamarre L, Mellman I (2012) Internalization and endosomal degradation of receptor-bound
antigens regulate the efficiency of cross presentation by human dendritic cells. Blood
120:2011–2020
Chen Y, Wang S, Lu S (2014) DNA immunization for HIV vaccine development. Vaccines
2:138–159
Choi JH, Dekker J, Schafer SC, John J, Whitfill CE, Petty CS, Haddad EE, Croyle MA (2012)
Optimized adenovirus-antibody complexes stimulate strong cellular and humoral immune
responses against an encoded antigen in naive mice and those with preexisting immunity. Clin
Vaccine Immunol 19:84–95
Chu HY, Englund JA (2014) Maternal immunization. Clin Infect Dis 59:560–568
Clatworthy MR, Aronin CE, Mathews RJ, Morgan NY, Smith KG, Germain RN (2014) Immune
complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes. Nat Med
20:1458–1463
Cohn L, Chatterjee B, Esselborn F, Smed-Sorensen A, Nakamura N, Chalouni C, Lee BC,
Vandlen R, Keler T, Lauer P, Brockstedt D, Mellman I, Delamarre L (2013) Antigen delivery
to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross
presentation. J Exp Med 210:1049–1063
Copeman SM, O’brien RA, Eagleton AJ, Glenny AT (1922) Experiences with the schick test and
active immunization against diphtheria. Brit J Exper Path 3:42
Crowe JE Jr (2017) Principles of broad and potent antiviral human antibodies: insights for vaccine
design. Cell Host Microbe 22:193–206
de Jong JM, Schuurhuis DH, Ioan-Facsinay A, Welling MM, Camps MG, van der Voort EI,
Huizinga TW, Ossendorp F, Verbeek JS, Toes RE (2006) Dendritic cells, but not macrophages
or B cells, activate major histocompatibility complex class II-restricted CD4+ T cells upon
immune-complex uptake in vivo. Immunology 119:499–506
den Haan JM, Bevan MJ (2002) Constitutive versus activation-dependent cross-presentation of
immune complexes by CD8(+) and CD8(−) dendritic cells in vivo. J Exp Med 196:817–827
Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV (2002) Antitumor monoclonal
antibodies enhance cross-presentation of cellular antigens and the generation of
myeloma-specific killer T cells by dendritic cells. J Exp Med 195:125–133
DiLillo DJ, Ravetch JV (2015) Differential Fc-receptor engagement drives an anti-tumor vaccinal
effect. Cell 161:1035–1045
Du L, Leung VH-C, Zhang X, Zhou J, Chen M, He W, Zhang H-Y, Chan CCS, Poon VK-M,
Zhao G, Sun S, Cai L, Zhou Y, Zheng B-J, Jiang S (2011) A recombinant vaccine of H5N1
HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against
divergent H5N1 viruses. PLoS ONE 6:e16555
Goodier MR, Lusa C, Sherratt S, Rodriguez-Galan A, Behrens R, Riley EM (2016) Sustained
immune complex-mediated reduction in CD16 expression after vaccination regulates NK cell
function. Front Immunol 7:384
Gosselin EJ, Bitsaktsis C, Li Y, Iglesias BV (2009) Fc receptor-targeted mucosal vaccination as a
novel strategy for the generation of enhanced immunity against mucosal and non-mucosal
pathogens. Arch Immunol Ther Exp (Warsz) 57:311–323
Haddad EE, Whitfill CE, Avakian AP, Ricks CA, Andrews PD, Thoma JA, Wakenell PS (1997)
Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.
Avian Diseases 41:882–889
Halper-Stromberg A, Lu CL, Klein F, Horwitz JA, Bournazos S, Nogueira L, Eisenreich TR,
Liu C, Gazumyan A, Schaefer U, Furze RC, Seaman MS, Prinjha R, Tarakhovsky A,
Ravetch JV, Nussenzweig MC (2014) Broadly neutralizing antibodies and viral inducers
decrease rebound from HIV-1 latent reservoirs in humanized mice. Cell 158:989–999
114 Y. Wen and Y. Shi

Harbers SO, Crocker A, Catalano G, D’Agati V, Jung S, Desai DD, Clynes R (2007)
Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance. J Clin Invest
117:1361–1369
Hari A, Ganguly A, Mu L, Davis SP, Stenner MD, Lam R, Munro F, Namet I, Alghamdi E,
Furstenhaupt T, Dong W, Detampel P, Shen LJ, Amrein MW, Yates RM, Shi Y (2015)
Redirecting soluble antigen for MHC class I cross-presentation during phagocytosis. Eur J
Immunol 45:383–395
Heesters BA, Chatterjee P, Kim YA, Gonzalez SF, Kuligowski MP, Kirchhausen T, Carroll MC
(2013) Endocytosis and recycling of immune complexes by follicular dendritic cells enhances
B cell antigen binding and activation. Immunity 38:1164–1175
Hioe CE, Visciano ML, Kumar R, Liu J, Mack EA, Simon RE, Levy DN, Tuen M (2009) The use
of immune complex vaccines to enhance antibody responses against neutralizing epitopes on
HIV-1 envelope gp120. Vaccine 28:352–360
Horwitz JA, Halper-Stromberg A, Mouquet H, Gitlin AD, Tretiakova A, Eisenreich TR,
Malbec M, Gravemann S, Billerbeck E, Dorner M, Buning H, Schwartz O, Knops E, Kaiser R,
Seaman MS, Wilson JM, Rice CM, Ploss A, Bjorkman PJ, Klein F, Nussenzweig MC (2013)
HIV-1 suppression and durable control by combining single broadly neutralizing antibodies
and antiretroviral drugs in humanized mice. Proc Natl Acad Sci U S A 110:16538–16543
Ignjatovic J, Gould G, Trinidad L, Sapats S (2006) Chicken recombinant antibodies against
infectious bursal disease virus are able to form antibody-virus immune complex. Avian Pathol
35:293–301
Iván J, Velhner M, Ursu K, Germán P, Mató T, Drén CN, Mészáros J (2005) Delayed vaccine
virus replication in chickens vaccinated subcutaneously with an immune complex infectious
bursal disease vaccine: Quantification of vaccine virus by real-time polymerase chain reaction.
Can J Vet Res 69:135–142
Jeurissen SH, Janse EM, Lehrbach PR, Haddad EE, Avakian A, Whitfill CE (1998) The working
mechanism of an immune complex vaccine that protects chickens against infectious bursal
disease. Immunology 95:494–500
Joffre OP, Segura E, Savina A, Amigorena S (2012) Cross-presentation by dendritic cells. Nat Rev
Immunol 12:557–569
Kapczynski DR, Afonso CL, Miller PJ (2013) Immune responses of poultry to Newcastle disease
virus. Dev Comp Immunol 41:447–453
Kim PS, Armstrong TD, Song H, Wolpoe ME, Weiss V, Manning EA, Huang LQ, Murata S,
Sgouros G, Emens LA, Reilly RT, Jaffee EM (2008) Antibody association with HER-2/
neu-targeted vaccine enhances CD8 T cell responses in mice through Fc-mediated activation of
DCs. J Clin Invest 118:1700–1711
Kool M, Fierens K, Lambrecht BN (2012) Alum adjuvant: some of the tricks of the oldest
adjuvant. J Med Microbiol 61:927–934
Kosco MH, Burton GF, Kapasi ZF, Szakal AK, Tew JG (1989) Antibody-forming cell induction
during an early phase of germinal centre development and its delay with ageing. Immunology
68:312–318
Kreijtz JH, Suezer Y, de Mutsert G, van den Brand JM, van Amerongen G, Schnierle BS,
Kuiken T, Fouchier RA, Lower J, Osterhaus AD, Sutter G, Rimmelzwaan GF (2009)
Preclinical evaluation of a modified vaccinia virus Ankara (MVA)-based vaccine against
influenza A/H5N1 viruses. Vaccine 27:6296–6299
Lambour J, Naranjo-Gomez M, Piechaczyk M, Pelegrin M (2016) Converting monoclonal
antibody-based immunotherapies from passive to active: bringing immune complexes into
play. Emer Microbes & Infect 5:e92
Le Roux D, Lankar D, Yuseff MI, Vascotto F, Yokozeki T, Faure-Andre G, Mougneau E,
Glaichenhaus N, Manoury B, Bonnerot C, Lennon-Dumenil AM (2007) Syk-dependent actin
dynamics regulate endocytic trafficking and processing of antigens internalized through the
B-cell receptor. Mol Biol Cell 18:3451–3462
Lehmann CHK, Baranska A, Heidkamp GF, Heger L, Neubert K, Lühr JJ, Hoffmann A,
Reimer KC, Brückner C, Beck S, Seeling M, Kießling M, Soulat D, Krug AB, Ravetch JV,
Immune Complex Vaccination 115

Leusen JHW, Nimmerjahn F, Dudziak D (2017) DC subset–specific induction of T cell

responses upon antigen uptake via Fcc receptors in vivo. J Exper Med 214:1509–1528
Leon B, Ballesteros-Tato A, Randall TD, Lund FE (2014) Prolonged antigen presentation by
immune complex-binding dendritic cells programs the proliferative capacity of memory CD8 T
cells. J Exp Med 211:1637–1655
Li D, He W, Liu X, Zheng S, Qi Y, Li H, Mao F, Liu J, Sun Y, Pan L, Du K, Ye K, Li W, Sui J
(2017) A potent human neutralizing antibody Fc-dependently reduces established HBV
infections. eLife 6:e26738
Li Q, Zeng M, Duan L, Voss JE, Smith AJ, Pambuccian S, Shang L, Wietgrefe S, Southern PJ,
Reilly CS, Skinner PJ, Zupancic ML, Carlis JV, Piatak M Jr, Waterman D, Reeves RK,
Masek-Hammerman K, Derdeyn CA, Alpert MD, Evans DT, Kohler H, Muller S, Robinson J,
Lifson JD, Burton DR, Johnson RP, Haase AT (2014) Live simian immunodeficiency virus
vaccine correlate of protection: local antibody production and concentration on the path of
virus entry. J Immunol 193:3113–3125
Liao HX, Alam SM, Mascola JR, Robinson J, Ma B, Montefiori DC, Rhein M, Sutherland LL,
Scearce R, Haynes BF (2004) Immunogenicity of constrained monoclonal antibody
A32-human immunodeficiency virus (HIV) Env gp120 complexes compared to that of
recombinant HIV type 1 gp120 envelope glycoproteins. J Virol 78:5270–5278
Lizee G, Basha G, Tiong J, Julien JP, Tian M, Biron KE, Jefferies WA (2003) Control of dendritic
cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain.
Nat Immunol 4:1065–1073
Lobaina Y, Michel ML (2017) Chronic hepatitis B: Immunological profile and current therapeutic
vaccines in clinical trials. Vaccine 35:2308–2314
Lux A, Yu X, Scanlan CN, Nimmerjahn F (2013) Impact of immune complex size and
glycosylation on IgG binding to human FcgammaRs. J Immunol 190:4315–4323
Maamary J, Wang TT, Tan GS, Palese P, Ravetch JV (2017) Increasing the breadth and potency of
response to the seasonal influenza virus vaccine by immune complex immunization. Proc Natl
Acad Sci U S A 114:10172–10177
Marois L, Pare G, Vaillancourt M, Rollet-Labelle E, Naccache PH (2011) Fc gammaRIIIb triggers
raft-dependent calcium influx in IgG-mediated responses in human neutrophils. J Biol Chem
286:3509–3519
Matsubara S, Koya T, Takeda K, Joetham A, Miyahara N, Pine P, Masuda ES, Swasey CH,
Gelfand EW (2006) Syk activation in dendritic cells is essential for airway hyperresponsive-
ness and inflammation. Am J Respir Cell Mol Biol 34:426–433
McEwan WA, Tam JC, Watkinson RE, Bidgood SR, Mallery DL, James LC (2013) Intracellular
antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat
Immunol 14:327–336
Meng ZF, Wang HJ, Yao X, Wang XY, Wen YM, Dai JX, Xie YH, Xu JQ (2012) Immunization
with HBsAg-Fc fusion protein induces a predominant production of Th1 cytokines and reduces
HBsAg level in transgenic mice. Chin Med J (Engl) 125:3266–3272
Mi W, Wanjie S, Lo ST, Gan Z, Pickl-Herk B, Ober RJ, Ward ES (2008) Targeting the neonatal fc
receptor for antigen delivery using engineered fc fragments. J Immunol 181:7550–7561
Michaud HA, Gomard T, Gros L, Thiolon K, Nasser R, Jacquet C, Hernandez J, Piechaczyk M,
Pelegrin M (2010) A crucial role for infected-cell/antibody immune complexes in the
enhancement of endogenous antiviral immunity by short passive immunotherapy. PLoS Pathog
6:e1000948
Moalli F, Doni A, Deban L, Zelante T, Zagarella S, Bottazzi B, Romani L, Mantovani A,
Garlanda C (2010) Role of complement and Fc{gamma} receptors in the protective activity of
the long pentraxin PTX3 against Aspergillus fumigatus. Blood 116:5170–5180
Mocsai A, Ruland J, Tybulewicz VL (2010) The SYK tyrosine kinase: a crucial player in diverse
biological functions. Nat Rev Immunol 10:387–402
Morrison SL, Terres G (1966) Enhanced immunologic sensitization of mice by the simultaneous
injection of antigen and specific antiserum. II. Effect of varying the antigen-antibody ratio and
the amount of immune complex injected. J Immunol 96:901–905
116 Y. Wen and Y. Shi

Müller H, Mundt E, Eterradossi N, Islam MR (2012) Current status of vaccines against infectious
bursal disease. Avian Pathology 41:133–139
Naqi SA, Marquez B, Sahin N (1983) Maternal antibody and its effect on infectious bursal disease
immunization. Avian Dis 27:623–631
Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch JV (2005) FcgammaRIV: a novel FcR with distinct
IgG subclass specificity. Immunity 23:41–51
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin g subclass activity through
selective Fc receptor binding. Science 310:1510–1512
Nimmerjahn F, Ravetch JV (2012) Translating basic mechanisms of IgG effector activity into next
generation cancer therapies. Cancer Immun 12:13
Oefner CM, Winkler A, Hess C, Lorenz AK, Holecska V, Huxdorf M, Schommartz T, Petzold D,
Bitterling J, Schoen AL, Stoehr AD, Van Vu D, Darcan-Nikolaisen Y, Blanchard V,
Schmudde I, Laumonnier Y, Strover HA, Hegazy AN, Eiglmeier S, Schoen CT, Mertes MM,
Loddenkemper C, Lohning M, Konig P, Petersen A, Luger EO, Collin M, Kohl J, Hutloff A,
Hamelmann E, Berger M, Wardemann H, Ehlers M (2012) Tolerance induction with T
cell-dependent protein antigens induces regulatory sialylated IgGs. J Allergy Clin Immunol
129(1647–1655):e1613
Park-Min KH, Serbina NV, Yang W, Ma X, Krystal G, Neel BG, Nutt SL, Hu X, Ivashkiv LB
(2007) FcgammaRIII-dependent inhibition of interferon-gamma responses mediates suppres-
sive effects of intravenous immune globulin. Immunity 26:67–78
Pelegrin M, Naranjo-Gomez M, Piechaczyk M (2015) Antiviral monoclonal antibodies: can they
be more than simple neutralizing agents? Trends Microbiol 23:653–665
Perreau M, Pantaleo G, Kremer EJ (2008) Activation of a dendritic cell-T cell axis by Ad5
immune complexes creates an improved environment for replication of HIV in T cells. J Exp
Med 205:2717–2725
Pincetic A, Bournazos S, DiLillo DJ, Maamary J, Wang TT, Dahan R, Fiebiger BM, Ravetch JV
(2014) Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol
15:707–716
Pokric B, Sladic D, Juros S, Cajavec S (1993) Application of the immune complex for immune
protection against viral disease. Vaccine 11:655–659
Polyanskaya N, Bergmeier LA, Sharpe SA, Cook N, Leech S, Hall G, Dennis M, ten Haaft P,
Heeney J, Manca F, Lehner T, Cranage MP (2001) Mucosal exposure to subinfectious doses of
SIV primes gut-associated antibody-secreting cells and T cells: lack of enhancement by
nonneutralizing antibody. Virology 279:527–538
Qiao SW, Kobayashi K, Johansen FE, Sollid LM, Andersen JT, Milford E, Roopenian DC,
Lencer WI, Blumberg RS (2008) Dependence of antibody-mediated presentation of antigen on
FcRn. Proc Natl Acad Sci U S A 105:9337–9342
Rafiq K, Bergtold A, Clynes R (2002) Immune complex-mediated antigen presentation induces
tumor immunity. J Clin Invest 110:71–79
Randall RE, Young DF (1988) Humoral and cytotoxic T cell immune responses to internal and
external structural proteins of simian virus 5 induced by immunization with solid
matrix-antibody-antigen complexes. J Gen Virol 69(Pt 10):2505–2516
Randall RE, Young DF (1991) Solid matrix-antibody-antigen complexes induce antigen-specific
CD8+ cells that clear a persistent paramyxovirus infection. J Virol 65:719–726
Ravetch JV, Kinet JP (1991) Fc receptors. Annu Rev Immunol 9:457–492
Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S,
Bonnerot C, Ricciardi-Castagnoli P, Amigorena S (1999) Fcgamma receptor-mediated
induction of dendritic cell maturation and major histocompatibility complex class I-restricted
antigen presentation after immune complex internalization. J Exp Med 189:371–380
Rock KL, Gamble S, Rothstein L (1990) Presentation of exogenous antigen with class I major
histocompatibility complex molecules. Science 249:918–921
Sancho D, Mourao-Sa D, Joffre OP, Schulz O, Rogers NC, Pennington DJ, Carlyle JR, Reis e
Sousa C (2008) Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type
lectin. J Clin Invest 118:2098–2110
Immune Complex Vaccination 117

Savina A, Jancic C, Hugues S, Guermonprez P, Vargas P, Moura IC, Lennon-Dumenil AM,

Seabra MC, Raposo G, Amigorena S (2006) NOX2 controls phagosomal pH to regulate
antigen processing during crosspresentation by dendritic cells. Cell 126:205–218
Schat KA, da Silva Martins NR, O’Connell PH, Piepenbrink MS (2011) Immune complex
vaccines for chicken infectious anemia virus. Avian Dis 55:90–96
Schmidt J, Blum HE, Thimme R (2013) T-cell responses in hepatitis B and C virus infection:
similarities and differences. Emerg Microbes Infect 2:e15
Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, van Schip JJ, Sedlik C, Melief CJ, Verbeek JS,
Ossendorp F (2002) Antigen-antibody immune complexes empower dendritic cells to
efficiently prime specific CD8+ CTL responses in vivo. J Immunol 168:2240–2246
Seeger C, Mason WS (2015) Molecular biology of hepatitis B virus infection. Virology 0:672–686
Shiver JW, Emini EA (2004) Recent advances in the development of HIV-1 vaccines using
replication-incompetent adenovirus vectors. Annu Rev Med 55:355–372
Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY,
Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA (2013) Fc-dependent
depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4
therapy against melanoma. J Exp Med 210:1695–1710
St Pierre CA, Leonard D, Corvera S, Kurt-Jones EA, Finberg RW (2011) Antibodies to cell
surface proteins redirect intracellular trafficking pathways. Exp Mol Pathol 91:723–732
Szakal AK, Kosco MH, Tew JG (1988a) FDC-iccosome mediated antigen delivery to germinal
center B cells, antigen processing and presentation to T cells. Adv Exp Med Biol 237:197–202
Szakal AK, Kosco MH, Tew JG (1988b) A novel in vivo follicular dendritic cell-dependent
iccosome-mediated mechanism for delivery of antigen to antigen-processing cells. J Immunol
140:341–353
Szecsi J, Gabriel G, Edfeldt G, Michelet M, Klenk HD, Cosset FL (2009) DNA vaccination with a
single-plasmid construct coding for viruslike particles protects mice against infection with a
highly pathogenic avian influenza A virus. J Infect Dis 200:181–190
Tao P, Luo M, Zhu D, Qu S, Yang Z, Gao M, Guo D, Pan Z (2009) Virus-like particle vaccine
comprised of the HA, NA, and M1 proteins of an avian isolated H5N1 influenza virus induces
protective immunity against homologous and heterologous strains in mice. Viral Immunol
22:273–281
Terres G, Wolins W (1959) Enhanced sensitization in mice by simultaneous injection of antigen
and specific rabbit antiserum. Proc Soc Exp Biol Med 102:632–635
Terres G, Wolins W (1961) Enhanced immunological sensitization of mice by the simultaneous
injection of antigen and specific antiserum. I. Effect of varying the amount of antigen used
relative to the antiserum. J Immunol 86:361–368
Vasovic LV, Dyall R, Clynes RA, Ravetch JV, Nikolic-Zugic J (1997) Synergy between an
antibody and CD8+ cells in eliminating an established tumor. Eur J Immunol 27:374–382
Visciano ML, Tuen M, Gorny MK, Hioe CE (2008) In vivo alteration of humoral responses to
HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120. Virology
372:409–420
Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD,
Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW,
Protocol GPI, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR (2009) Broad
and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target.
Science 326:285–289
Wang TT, Maamary J, Tan GS, Bournazos S, Davis CW, Krammer F, Schlesinger SJ, Palese P,
Ahmed R, Ravetch JV (2015) Anti-HA glycoforms drive B cell affinity selection and determine
influenza vaccine efficacy. Cell 162:160–169
Wang W, Sun L, Li T, Ma Y, Li J, Liu Y, Li M, Wang L, Li C, Xie Y, Wen Y, Liang M, Chen L,
Tong S (2016) A human monoclonal antibody against small envelope protein of hepatitis B
virus with potent neutralization effect. MAbs 8:468–477
Wen Y, Shi Y (2016) Alum: an old dog with new tricks. Emerg Microbes Infect 5:e25
118 Y. Wen and Y. Shi

Wen YM (2009) Antigen-antibody immunogenic complex: promising novel vaccines for

microbial persistent infections. Expert Opin Biol Ther 9:285–291
Wen YM, Wu XH, Hu DC, Zhang QP, Guo SQ (1995) Hepatitis B vaccine and anti-HBs complex
as approach for vaccine therapy. Lancet 345:1575–1576
Wen YM, Xiong SD, Zhang W (1994) Solid matrix-antibody-antigen complex can clear viraemia
and antigenaemia in persistent duck hepatitis B virus infection. J Gen Virol 75(Pt 2):335–339
Wieland A, Shashidharamurthy R, Kamphorst AO, Han JH, Aubert RD, Choudhury BP,
Stowell SR, Lee J, Punkosdy GA, Shlomchik MJ, Selvaraj P, Ahmed R (2015) Antibody
effector functions mediated by Fcgamma-receptors are compromised during persistent viral
infection. Immunity 42:367–378
Wolpoe ME, Lutz ER, Ercolini AM, Murata S, Ivie SE, Garrett ES, Emens LA, Jaffee EM,
Reilly RT (2003) HER-2/neu-specific monoclonal antibodies collaborate with HER-2/
neu-targeted granulocyte macrophage colony-stimulating factor secreting whole cell vaccina-
tion to augment CD8+ T cell effector function and tumor-free survival in Her-2/neu-transgenic
mice. J Immunol 171:2161–2169
Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L,
Xu L, Longo NS, McKee K, O’Dell S, Louder MK, Wycuff DL, Feng Y, Nason M,
Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR (2010)
Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to
HIV-1. Science 329:856–861
Wu Y, Sukumar S, El Shikh ME, Best AM, Szakal AK, Tew JG (2008) Immune complex-bearing
follicular dendritic cells deliver a late antigenic signal that promotes somatic hypermutation.
J Immunol 180:281–290
Xu DZ, Huang KL, Zhao K, Xu LF, Shi N, Yuan ZH, Wen YM (2005) Vaccination with
recombinant HBsAg-HBIG complex in healthy adults. Vaccine 23:2658–2664
Yamada DH, Elsaesser H, Lux A, Timmerman JM, Morrison SL, de la Torre JC, Nimmerjahn F,
Brooks DG (2015) Suppression of Fcgamma-receptor-mediated antibody effector function
during persistent viral infection. Immunity 42:379–390
Yao X, Zheng B, Zhou J, Xu DZ, Zhao K, Sun SH, Yuan ZH, Wen YM (2007) Therapeutic effect
of hepatitis B surface antigen-antibody complex is associated with cytolytic and non-cytolytic
immune responses in hepatitis B patients. Vaccine 25:1771–1779
Yoshida M, Claypool SM, Wagner JS, Mizoguchi E, Mizoguchi A, Roopenian DC, Lencer WI,
Blumberg RS (2004) Human neonatal Fc receptor mediates transport of IgG into luminal
secretions for delivery of antigens to mucosal dendritic cells. Immunity 20:769–783
Zhang TY, Yuan Q, Zhao JH, Zhang YL, Yuan LZ, Lan Y, Lo YC, Sun CP, Wu CR, Zhang JF,
Zhang Y, Cao JL, Guo XR, Liu X, Mo XB, Luo WX, Cheng T, Chen YX, Tao MH, Shih JW,
Zhao QJ, Zhang J, Chen PJ, Yuan YA, Xia NS (2016) Prolonged suppression of HBV in mice
by a novel antibody that targets a unique epitope on hepatitis B surface antigen. Gut 65:658–
671
Zheng B, Zhang Y, He H, Marinova E, Switzer K, Wansley D, Mbawuike I, Han S (2007)
Rectification of age-associated deficiency in cytotoxic T cell response to influenza A virus by
immunization with immune complexes. J Immunol 179:6153–6159
Zheng BJ, Ng MH, He LF, Yao X, Chan KW, Yuen KY, Wen YM (2001) Therapeutic efficacy of
hepatitis B surface antigen-antibodies-recombinant DNA composite in HBsAg transgenic
mice. Vaccine 19:4219–4225
Zhou C, Li C, Gong GZ, Wang S, Zhang JM, Xu DZ, Guo LM, Ren H, Xu M, Xie Q, Pan C, Xu J,
Hu Z, Geng S, Zhou X, Wang X, Zhou X, Mi H, Zhao G, Yu W, Wen YM, Huang L,
Wang XY, Wang B (2017) Analysis of immunological mechanisms exerted by HBsAg-HBIG
therapeutic vaccine combined with Adefovir in chronic hepatitis B patients. Hum Vaccin
Immunother 13:1989–1996
Fc Receptors in Antimicrobial
Protection

Andreas Wieland and Rafi Ahmed

Contents

1 Introduction.......................................................................................................................... 120
2 FcR-Mediated Effector Functions with Direct Antimicrobial Activity .............................. 121
2.1 Antibody-Dependent Phagocytosis ............................................................................ 121
2.2 Antibody-Dependent Cellular Cytotoxicity................................................................ 124
2.3 Antibody-Dependent Intracellular Neutralization ...................................................... 126
3 FcR-Mediated Effector Functions Contributing to Antimicrobial Protection .................... 126
3.1 Modulation of the Inflammatory State....................................................................... 128
3.2 Antigen Presentation and Dendritic Cell Maturation ................................................ 128
3.3 B Cell Selection and Affinity Maturation .................................................................. 129
4 The Role of FcRs in Antimicrobial Protection................................................................... 130
4.1 Viruses ........................................................................................................................ 130
4.2 Bacteria ....................................................................................................................... 133
4.3 Fungi and Parasites..................................................................................................... 137
5 Microbial Evasion Strategies............................................................................................... 138
6 Conclusions.......................................................................................................................... 141
References .................................................................................................................................. 141

Abstract Antibodies are the key effector molecules of the humoral immune system
providing long-term protective immunity against a wide range of pathogens and
regulating immune responses. Traditionally, antibody-mediated protection against
microbes was thought to be mainly a result of neutralizing Fab–antigen interaction;
however, an increasing number of studies show the importance of proper FcR
engagement for the protective capacity of antimicrobial antibodies. In this chapter,
we review FcR-mediated effector functions contributing to antimicrobial protection
in a direct and indirect manner. Furthermore, we highlight recent findings about the
important role of Fc–FcR interactions for antimicrobial protection in vivo and
provide examples demonstrating the crucial role of proper FcR engagement for
antibody-mediated protection against viruses, bacteria, fungi, and parasites.

A. Wieland (&)
R. Ahmed

Department of Microbiology & Immunology, School of Medicine,
Emory University, 1510 Clifton Road, Atlanta, GA, USA
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2019) 423: 119–150

https://doi.org/10.1007/82_2019_154
© Springer Nature Switzerland AG 2019
Published online: 22 February 2019
120 A. Wieland and R. Ahmed

1 Introduction

Antibodies are involved in the protection against a wide range of pathogens

including viruses, bacteria, fungi, and parasites and are the correlate of protection
for the vast majority of available vaccines (Plotkin 2010). Vaccination and/or
infection can induce the generation of long-lived plasma cells residing mainly in the
bone marrow and producing high quantities of pathogen-specific antibodies con-
ferring long-term protective immunity (Manz et al. 1997; Slifka et al. 1998). The
ability to mount an antibody response against an almost unlimited number of target
structures and generate highly specific antibodies is accomplished through somatic
recombination and hypermutation of the Fab (fragment, antibody binding) domain.
While the variable Fab domain mediates antigen specificity and binds its respective
antigen, the Fc (fragment, crystallizable) domain can engage FcRs expressed on
leukocytes to mediated diverse effector functions. The interactions between the Fc
domain of IgG and FccRs as well as the resulting cellular effector functions are well
defined (Bournazos et al. 2017). Although the Fc domain of IgG is traditionally
considered to be an invariable region, the existence of different IgG subclasses with
divergent amino acid sequences and the differential composition of the biannten-
nary N-linked glycan at the conserved glycosylation site Asn297 results in sub-
stantial heterogeneity in this region. This structural heterogeneity affects the
capacity of the Fc domain to interact with type I and type II FccRs and subse-
quently modulates the effector functions elicited by IgG (Bournazos et al. 2017).
FccRs are expressed by different leukocytes of the myeloid and lymphoid lin-
eage, both in circulation as well as in tissues, and thus FccR-signaling can modulate
several immunological processes besides direct cytotoxic IgG-mediated effector
functions such as phagocytosis and cellular cytotoxicity (Pincetic et al. 2014).
Different leukocyte populations are characterized by distinct FccR expression
patterns and FccR expression levels are a function of cellular differentiation, cell
activation status as well as environmental cues (inflammation, infection, injury)
allowing to further modulate FccR-mediated effector functions in a
context-dependent manner (te Velde et al. 1992; Uciechowski et al. 1998; Anthony
et al. 2011; Dugast et al. 2011; Smith et al. 2012; Pincetic et al. 2014). While all
IgG-mediated cytotoxic effector functions critically depend on the engagement of
activating type I FccRs, the balanced engagement of activating and inhibitory type I
Fccs as well as type II FccRs by IgG can affect a wide range of immunological
processes involved in antimicrobial immunity, ranging from antigen uptake and
presentation, differentiation and activation of antigen-presenting cells, secretion of
cytokines and chemokines, modulation of T cell activation, affinity maturation of B
cells and plasma cell survival (Bournazos and Ravetch 2015).
Antibodies are the correlate of protection for the vast majority of available
vaccines and various in vitro assays such as toxin/virus neutralization, bactericidal
assay, and opsonophagocytosis are employed to quantify pathogen-specific anti-
body responses correlated with protection (Plotkin 2010). However, several studies
demonstrated that Fc–FccR interactions play an important role in the protective
Fc Receptors in Antimicrobial Protection 121

capacity of neutralizing antibodies against various pathogens and toxins in vivo that
cannot be predicted by simple in vitro experiments (Abboud et al. 2010; Bournazos
et al. 2014a; DiLillo et al. 2014; Hessell et al. 2007). These observations sparked an
enormous interest in elucidating the contributions of in vivo Fc–FccR interactions
in antimicrobial protection with the ultimate goal to further enhance FcR-mediated
antimicrobial effector functions of antibody-based therapeutics.
Here, we review FcR-mediated effector functions contributing to antimicrobial
protection, highlight the important role of Fc–FcR interactions for antimicrobial
protection in vivo, and briefly discuss microbial evasion strategies. Our review
focusses on the role of FccRs as its ligand IgG is by far the most prevalent isotype
in the serum and the role of FccRs in antimicrobial protection has been extensively
studied.

2 FcR-Mediated Effector Functions with Direct

Antimicrobial Activity

2.1 Antibody-Dependent Phagocytosis

Phagocytosis, first observed over 100 years ago, is a critical component of the
immune system capable of integrating both innate and adaptive immune responses
to combat pathogens. Although phagocytosis is traditionally defined as the cellular
ingestion of large (  0.5-µm) particles (Flannagan et al. 2012), we use the term
phagocytosis in the following indiscriminately of particle size. The engagement of
type I FccRs on phagocytic cells such as macrophages and neutrophils results in the
efficient phagocytosis of IgG-opsonized particles that can range in size from single
molecules, microbial pathogens to intact infected cells (Fig. 1). Although the
phagocytic capacity varies among different phagocytes and might also be influ-
enced by activation status and metabolic state of a cell (Pavlou et al. 2017), the
downstream signaling cascade and mechanisms induced by FccR cross-linking on
the cell surface is common to all leukocytes expressing activating type I FccRs.
The process of antibody-dependent cellular phagocytosis (ADCP) of
IgG-opsonized particles by FccRs can be subdivided into three distinct steps:
(i) binding of IgG-opsonized particles to FccRs on the cell surface of phagocytes,
(ii) clustering and oligomerization of FccRs, inducing a signaling cascade; followed
by (iii) engulfment of the opsonized particle by an actin-driven process (Flannagan
et al. 2012). The successful phagocytic uptake of an IgG-opsonized particle is an
iterative process that requires, in addition to the initial attachment event, circum-
ferential engagement of unoccupied FccRs by particle-bound IgG (Griffin et al.
1975). The circumferential interaction between membrane-bound FccRs and IgG
on the particle is thought to guide the pseudopod extensions as the phagocyte
membrane slowly zippers around the particle.
122 A. Wieland and R. Ahmed

(a) (b)
ADCP ADCC

antigen antibody
(c) ADIN
FcR cytotoxic granule

Fas Fas ligand

TRIM21 polyubiquitin

proteasome

autophagosome peptides pathogen

Fig. 1 FcR-mediated antibody effector functions with direct antimicrobial activity.

a Antibody-dependent cellular phagocytosis (ADCP). b Antibody-dependent cellular cytotoxicity
(ADCC). c Antibody-dependent intracellular neutralization (ADIN)

The binding of immune complexes to activating type I FccRs results in clus-

tering of the receptors on the cell surface followed by the phosphorylation of the
intracellular immunoreceptor tyrosine-based activation motif (ITAM) domain by
tyrosine kinases of the Src family including Lyn, Lck, Hck, and Fgr (Hamada et al.
1993; Durden et al. 1995; Pignata et al. 1993; Jouvin et al. 1994). Following the
phosphorylation of both tyrosines of the ITAM, the recruitment and activation of
Syk is essential for FccR-mediated phagocytosis as in the absence of Syk phago-
cytic cup formation is initiated but arrested early on (Crowley et al. 1997; Kiefer
et al. 1998). ITAM phosphorylation also results in the activation of f-chain sub-
units, ZAP70, and FAK regulating actin polymerization, phagocytosis and actin
polymerization (Jouvin et al. 1994; Swanson and Hoppe 2004; Hamawy et al.
1995). Phosphorylation of Syk leads to a recruitment cascade of several signaling
adaptor proteins to the activated FccR complex (Flannagan et al. 2012). Syk and
additionally recruited adaptor proteins subsequently induce activation of class I
phosphatidylinositol 3-kinase (PI3K) and phospholipase C c (PLCc), which gen-
erates the metabolites diacylglyercol (DAG), an efficient activator of protein kinase
C (PKC), and inositol-triphosphate (IP3) (Liao et al. 1992; Kanakaraj et al. 1994;
Fc Receptors in Antimicrobial Protection 123

Ninomiya et al. 1994). The above-mentioned signaling processes seem to contribute

to efficient phagocytosis in distinct ways as PKCe recruitment can enhance
phagocytosis independently of actin polymerization and PI3K activity seems to be
essential for the phagocytosis of large (  3-µm) particles by promoting maximal
pseudopod extension, but only has a minimal impact on the phagocytosis of smaller
particles (Larsen et al. 2002; Araki et al. 1996; Cox et al. 1999). In addition, the
activation of the PI3K-PKC pathway also results in intracellular Ca2+ mobilization
from the endoplasmatic reticulum and cellular activation (Bournazos and Ravetch
2015; Odin et al. 1991). Further, signaling proteins involved in FccR-mediated
phagocytosis encompass several GTPases of the Rho family such as Cdc42, Rac1,
and Rac2, which are key regulators of actin polymerization and remodeling (Hoppe
and Swanson 2004; Caron and Hall 1998; Park and Cox 2009). These GTPases
modulate the activity of the Arp2/3 nucleator complex, which supports actin
polymerization upon FccR signaling, through additional mediator proteins such as
Wiskott-Aldrich syndrome protein (May et al. 2000; Lorenzi et al. 2000).
The signaling cascade induced by the engagement of activating type I FccRs is
counterbalanced by the engagement of the inhibitory type I FccR, FccRIIb, and
thus FccR-mediated effector functions such as phagocytosis are a result of the
cumulative signaling from activating and inhibitory type I FccRs (Nimmerjahn and
Ravetch 2005). FccRIIb contains a single-tyrosine signaling domain known as
immunoreceptor tyrosine-based inhibition motif (ITIM). Following receptor clus-
tering and phosphorylation by kinases of the Src family, the ITIM of FccRIIb
recruits several phosphatases like SHIP, SHP-1, and SHP-2 (Pearse et al. 1999;
Amigorena et al. 1992a, Muta et al. 1994). SHIP is an inositol 5-phosphatase and
inhibits phagocytosis by hydrolysis of phosphatidylinositol 3,4,5-triphosphate thus
inhibiting the recruitment and activation of PLCc and BTK (Bournazos and
Ravetch 2015). SHP-1, a phosphotyrosine-specific protein phosphatase, has been
shown to inhibit FccR-mediated phagocytosis through a mechanism involving the
ubiquitin ligase Cbl (Kant et al. 2002). SHIP and SHP-1 have also been shown to
associate with the monophosphorylated ITAM of FccRs, suggesting a built-in
regulatory feedback of stimulatory receptors (Nakamura et al. 2002; Ganesan et al.
2003). The balanced signaling through activating and inhibitory FccRs is thus
supposed to ensure optimal effector activity and, at the same time, prevent excessive
pathogenic immune activation (Bournazos et al. 2017). Phagocytic activity directed
against pathogen-infected host cells can be further regulated by the engagement of
various inhibitory receptors expressed on phagocytes such as Siglec-5, Siglec-9,
and signal-regulating protein (SIRP)-a (Steevels and Meyaard 2011). The inter-
action of SIRP-a with its ubiquitously expressed ligand CD47, which is commonly
referred to as a “do not eat me signal” or “marker of self,” is well studied and shown
to increase the activation threshold required for efficient FccR-mediated phagocy-
tosis of host cells by activation of SHP-1 (Oldenborg et al. 2001; Okazawa et al.
2005).
The successful internalization of opsonized particles results in the formation of a
phagosome, which in its initial immature state is unable to mediate the destruction
of the engulfed pathogen. However, through sequential highly orchestrated
124 A. Wieland and R. Ahmed

membrane fusion and fission events, termed phagosome maturation, the composi-
tion of the phagosome changes dramatically in order to be equipped with the
necessary tools to destroy the engulfed pathogen (Desjardins et al. 1994). The
sequential fusion of the phagosome with discrete endosomal compartments results
in a stepwise acidification of the lumen. The mature phagosome/phagolysosome
generated by the fusion of late phagosomes with lysosomes is a highly microbicidal
organelle characterized by a highly acidic (pH 4.5) and oxidative environment
due to reactive oxygen species (ROS) (Flannagan et al. 2012). Furthermore, mature
phagosomes also contain an arsenal of antimicrobial peptides and more than 50
different hydrolytic enzymes such as lysozyme and cathepsins to ensure complete
digestion of complex pathogens.

2.2 Antibody-Dependent Cellular Cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) was first described about

50 years ago as the ability of nonimmune lymphoid cells—now known as natural
killer (NK) cells—to efficiently lyse antibody-coated target cells in vitro (Moller
1967). The expression of FcRs on the surface of cytotoxic effector cells is a
requirement for ADCC and multiple activating FcRs have been shown to mediate
ADCC upon binding to antibody-coated target cells and subsequent cross-linking of
FcRs. In humans, engagement of all activating type I FccRs (FccRI, FccRIIa/c, and
FccRIIIa) as well as the IgA-binding FcaRI (CD89) and the IgE-binding FceRI can
trigger ADCC (Bournazos et al. 2017; Morton et al. 1996; Fu et al. 2008). Whereas
the intracellular signals transduced upon cross-linking of activating type I FccRs are
common to all leukocytes expressing activating type I FccRs (described in 2.1), the
interpretation of these signals and thus the resulting biological response (i.e.,
ADCC, ADCP, cytokine secretion, …) varies among different leukocyte subsets
(Bournazos et al. 2017), probably representing a function of cell-type-specific
commitment and modulating environmental cues. ADCC represents a mechanism
complementary to ADCP that can mediate the destruction of pathogens exceeding
the size limitations of phagocytosis or of infected cells that cannot be readily
phagocytosed.
The main cytotoxic effector populations capable of ADCC include NK cells,
neutrophils, and eosinophils. NK cells are most studied effectors of ADCC and
freshly isolated NK cells exhibit efficient ADCC toward antibody-coated target
cells in vitro. NK cells express relatively high levels of FccRIIIa at steady state and
are the only leukocyte population expressing only an activating type I FccR but not
the inhibitory FccRIIb (Bournazos et al. 2017). However, NK cells can express
several inhibitory receptors such as killer Ig-like receptors (KIRs), CD94-NKG2,
and immunoglobulin-like receptor (LIR), which can counterbalance FccRIIIa
activation via SHP-1 and SHP-2 (Pegram et al. 2011). Circulating neutrophils
expressing FccRIIa, FccRIIb, and FccRIIIb, exhibit limited cytotoxic potential in
their normal resting state, but can be activated by several cytokines such as
Fc Receptors in Antimicrobial Protection 125

granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte

colony-stimulating factor (G-CSF) (Bournazos et al. 2017; Kato and Kitagawa
2006). Interferon (IFN)c signaling can further induce FccRI expression on neu-
trophils. Furthermore, neutrophils express FcaRI endowing them with the ability to
lyse IgA-coated target cells (Otten et al. 2005). Eosinophils express FccRIIa and
FccRIIb at a resting state and upregulate expression of FccRI and FccRIIIb upon
cytokine stimulation. Like neutrophils, eosinophils require cytokine-mediated
activation prior to exerting efficient degranulation (Fabian et al. 1992). In addition
to responding to IgG-coated targets, eosinophils can also exert potent cytolytic
activity against IgA- and IgE-coated targets through FcaRI and FceRI
(Abu-Ghazaleh et al. 1989; Dombrowicz et al. 2000).
The exact processes involved in ADCC have been best studied in NK cells and
share many similarities with cytotoxic T cells. Upon FcR-mediated activation,
effector cells release specialized granules at the lytic synapse by a
calcium-dependent polarized exocytotic process (de Saint Basile et al. 2010). The
composition of these lytic granules varies considerably between NK cells and
granulocytes and even between various granulocyte subsets. NK cells are well
equipped to lyse pathogen-infected host cells using granules containing the
pore-forming protein perforin and granzyme B, a serine protease which induces
apoptosis upon entry into the target cell and activation of caspase (Voskoboinik
et al. 2015). In addition to lysis of infected host cells, NK cells also seem to be able
to directly kill bacteria by the release of perforin and granulysin (Lu et al. 2014).
Upon FccR engagement, NK cells upregulate Fas ligand enabling them to kill target
cells expressing Fas through a granule-independent mechanism (Fig. 1) (Eischen
et al. 1996). The granules of granulocytes contain a cell-type- and
activation-state-specific mixture of preformed effector molecules and are released
upon FccR engagement (Jonsson et al. 2013). These granules typically contain
proteases (elastase, cathepsins, collagenase), antimicrobial peptides and proteins
(defensins, cathelicidins, lysozyme, lactoferrin), enzymes (peroxidase, alkaline
phosphatase), and leukotrienes which mediate inflammation and recruitment of
additional leukocytes (Bournazos et al. 2016). FccR signaling in granulocytes also
results in the generation of reactive oxygen intermediates (ROI) through the for-
mation and activation of the NADPH-dependent oxidase complex (Suh et al. 2006).
This results in the production of superoxide anions exhibiting direct cytotoxic
activity and serving as reactive intermediates to generate a plethora of reactive
oxygen species such as hydrogen peroxide (H2O2), hydroxyl radical (OH∙), and
hypochlorous acid (HOCl)(Nathan and Cunningham-Bussel 2013).
Although NK-cell-mediated ADCC of antibody-coated target cells has been
extensively studied in vitro, the exact contribution of NK-cell-mediated ADCC to
antimicrobial protection in vivo is less defined due to the overwhelming contri-
bution of phagocytic active cells to antibody-mediated protection in vivo. Future
studies employing sophisticated depletion strategies and/or genetic knockout
models will be able to address the exact role of the various cell populations capable
of ADCC for antimicrobial protection in vivo.
126 A. Wieland and R. Ahmed

2.3 Antibody-Dependent Intracellular Neutralization

The term antibody-dependent intracellular neutralization (ADIN) was coined to

describe antibody effector mechanisms mediated through the cytosolic Fc receptor
tripartite motif 21 (TRIM21). TRIM21, an IFN-inducible member of the TRIM
protein family of RING E3 ubiquitin ligases, is a broadly expressed cytosolic Fc
receptor that is widely conserved among mammals and structurally unrelated to
other classes of Fc receptors (James et al. 2007; Keeble et al. 2008; Reymond et al.
2001). TRIM21 interacts with the CH2-CH3 domain interface of the IgG Fc and is
unique in its ability to bind with high affinity to all IgG isotypes as well as, although
with lower affinity, to IgA and IgM (Mallery et al. 2010; Bidgood et al. 2014).
Pathogens such as non-enveloped viruses, that have entered the cytosol of a cell
and have antibodies bound to their capsid, can be recognized by TRIM21 (Mallery
et al. 2010; Bidgood et al. 2014). TRIM21 engagement results in autoubiquitination
mediated by the E3 activity of the RING domain and the subsequent recruitment of
the proteasomal machinery (Fig. 1) (Mallery et al. 2010). Due to size constraints of
the proteasomal barrel, TRIM21-degradation of viral particles by the proteasome
requires the additional recruitment of the AAA ATPase v97/VCP, which is capable
of extracting proteins from larger complexes and unfolding them (Hauler et al.
2012). TRIM21 has also been suggested to trigger autophagy to protect from
intracellular opsonized bacteria, which have been shown to colocalize with the
autophagosomal marker LC3 (Rakebrandt et al. 2014). In general, ADIN-mediated
protection has been shown to be a function of TRIM21 expression and the extent of
opsonization with the expression level of TRIM21 being the dominant determinant
(McEwan et al. 2012). ADIN can thus provide a last line of defense in case
neutralizing antibodies were not abundant enough to completely block pathogen
entry and classical FcR-mediated antibody effector functions were insufficient or
evaded by the pathogen. However, as ADIN has been mainly studied in vitro, its
role in pathogen control in vivo has not been addressed in detail.
In addition to ADIN, the recognition of cytosolic immunoglobulins by TRIM21
induces the activation of innate signaling pathways, resulting in the production of
several pro-inflammatory cytokines and chemokines mediating an antiviral state
which could potentially further upregulate TRIM21 in neighboring cells (McEwan
et al. 2013).

3 FcR-Mediated Effector Functions Contributing

to Antimicrobial Protection

The engagement of FcRs not only triggers direct effector mechanisms resulting in
pathogen clearance, but is also involved in the regulation of several immunological
processes ultimately contributing to antimicrobial protection (Fig. 2). In this sec-
tion, we briefly discuss the role of FcRs in modulating immunological processes
involved in antimicrobial protection.
Fc Receptors in Antimicrobial Protection 127

(a) cytokine secretion

leukocyte recruitment
cytokines
chemokines
lipid mediators

(b) antigen presentation/DC maturation

Co-stimulation
+ antigen
+ uptake/processing

CD8+ T cell
+

MHC-I antigen
presentation
MHC-II

CD4+ T cell

(c)

antigen antibody
inhibitory activating
CD23 Fc R Fc R
cytokines
Fc RIIb CD23
follicular DC chemokines
- costimulatory ligand
+

costimulatory receptor
B cell activation
MHC-I MHC-II TCR
Immune complex peptides
with sialylated IgG B cell

Fig. 2 FcR-mediated antibody effector functions indirectly contributing to antimicrobial protec-

tion. a Cytokine secretion. b Antigen presentation and maturation of dendritic cells (DC). c B cell
affinity maturation
128 A. Wieland and R. Ahmed

3.1 Modulation of the Inflammatory State

Granulocytes such as neutrophils, eosinophils, and basophils are among the first
leukocytes recruited to sites of inflammation. The engagement of activating FcRs
on granulocytes, monocytes, and polarized macrophages can trigger the release of
several pro-inflammatory cytokines, chemokines, and lipid mediators that will
result in recruitment of additional leukocyte subsets (Stone et al. 2010; Tecchio
et al. 2014; Bournazos et al. 2016). Furthermore, engagement of activating FccRs
by immune complexes in combination with toll-like receptor (TLR) stimulation has
further been shown to skew unpolarized macrophages toward a “regulatory” M2b
phenotype characterized by secretion of low levels of IL-12 and high levels of
IL-10, tumor necrosis factor, IL-1, and IL-6 (Guilliams et al. 2014). Ultimately, the
activation of these different leukocyte subsets by immune complexes is determined
by the balance of activating and inhibitory type I FccR engagement (Clynes et al.
1999). The engagement of DC-SIGN, a type II FccR expressed on regulatory
macrophages, by sialylated IgG Fc has been shown to induce expression of IL-33,
which can subsequently limit T cell- and IgG-mediated inflammation (Fiebiger
et al. 2015). Overall, these data show that engagement of FcRs (type I and type II
FccRs, FcaRI, FceRI) can exert both pro-inflammatory and anti-inflammatory
responses depending on the types of FcRs preferentially engaged by the
immunoglobulins opsonizing the pathogen.

3.2 Antigen Presentation and Dendritic Cell Maturation

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells

(APCs) capable of ingesting and processing foreign antigens for subsequent pre-
sentation to adaptive immune cells. Conventional human dendritic cells (cDCs)
express FccRIIa and FccRIIb at steady-state conditions and consist of two devel-
opmentally and functionally distinct subsets: CD172a+ cDCs are specialized to
present exogenous antigens to CD4 T cells, whereas XC-chemokine receptor 1
(XCR1)+ cDCs efficiently process and present exogenous antigens to CD8+ T cells
in a process termed cross-presentation (Guilliams et al. 2014). All type I FccRs
(except the FccRIIb1 splice variant) can mediate the internalization of
IgG-opsonized particles by endocytosis and/or phagocytosis; however, the
engagement of activating FccRs results in more potent priming of cellular
responses against exogenous antigens (Regnault et al. 1999; Dhodapkar et al. 2002;
Amigorena et al. 1992b). Antigens internalized by the inhibitory FccRIIb can be
recycled to the cell surface through a nondegradative intracellular vesicular com-
partment and subsequently stimulate B cell responses (Bergtold et al. 2005). These
data suggest that the type of FccR (activating or inhibitory) being engaged by
IgG-opsonized antigen influences the degradation pathway the antigen will be
subjected to and, ultimately, the kind of immune response being stimulated.
Fc Receptors in Antimicrobial Protection 129

The engagement of activating type I FccRs on DCs by immune complexes does

not only mediate efficient antigen uptake and subsequent antigen presentation on
MHC class I and MHC class II molecules, but also triggers a pro-inflammatory
signaling cascade resulting in DC maturation, which is characterized by increased
expression of MHC and co-stimulatory molecules such as CD80 and CD86 as well
as overall enhanced antigen presentation and is required for the efficient induction
of effector responses (Guilliams et al. 2014). Engagement of FccRs on DCs by
immune complexes alone is not sufficient to induce DC maturation and effector T
cell responses, as DCs express both inhibitory as well as activating type I FccRs
and signaling through activating type I FccRs is counteracted by the inhibitory
FccRIIb in DCs, like in other leukocyte populations. DC maturation in response to
immune complexes thus requires additional co-stimulatory signals to overcome the
inhibitory signaling through FccRIIb, which can be provided by engagement of
TLRs (Bournazos et al. 2016). The amount of additional co-stimulation required for
DC maturation in response to immune complexes depends on the amount of sig-
naling received through activating FccRs relative to inhibitory FccRIIb, which is
determined by the expression levels of activating and inhibitory type I FccRs. FccR
expression on DCs can be dramatically altered based on the local inflammatory
milieu with IFNc inducing expression of FccRI and downregulation of FccRIIb,
and IL-4 increasing FccRIIb expression (Pricop et al. 2001; Uciechowski et al.
1998; Boruchov et al. 2005). DCs can thus integrate signals from the local
inflammatory milieu to adjust the threshold required for IgG-mediated maturation.
Overall, these studies demonstrate that FccRs play an important role in the
induction of cellular immune responses to opsonized antigens by regulating antigen
uptake, presentation as well as maturation.

3.3 B Cell Selection and Affinity Maturation

B cells are the sole leukocyte population that constitutively expresses the inhibitory
FccRIIb but lacks expression of any activating type I FccR. In addition, B cells also
express the type II FccR CD23, which was initially identified as low-affinity FcR
for IgE and recently shown to bind sialylated IgG-containing immune complexes
(Sondermann et al. 2013). The role of inhibitory signaling through FccRIIb in B
cells and its role during B cell selection is well defined. Engagement of FccRIIb
without concomitant B cell receptor (BCR) engagement induces proapoptotic sig-
nals, which are attenuated upon co-aggregation with BCR resulting in B cell sur-
vival (Ono et al. 1997; Pearse et al. 1999). In the presence of IgG-complexed
antigen, this mechanism thus allows for the selective survival of B cells with high
affinity for the antigen, whereas nonspecific and low-affinity B cells are more likely
to undergo apoptosis. The expression of FccRIIb on B cells also sets an elevated
activation threshold for effective BCR engagement by IgG-containing immune
complexes and allows the generation of high-affinity antibody responses, while
preventing the induction of low-affinity and potentially auto-reactive antibody
130 A. Wieland and R. Ahmed

responses (Ono et al. 1996, 1997; Pearse et al. 1999; Bolland and Ravetch 2000;
Bolland et al. 2002). FccRIIb expression on follicular DCs further supports the
affinity maturation of B cells by promoting the retention of IgG-complexes in
germinal centers (Qin et al. 2000).
Binding of the type II FccR CD23 to sialylated IgG immune complexes has
recently been shown to drive the generation of high-affinity IgG responses with
broadly neutralizing capacity (Wang et al. 2015). The binding of sialylated IgG
immune complexes to CD23 on B cells induced the upregulation of FccRIIb, which
in turn elevated the threshold required for B cell activation and resulted in the
selection of high-affinity B cell clones. Overall, these studies demonstrate that both
type I and type II FccRs play an important role in regulating the humoral immune
response and inducing effective antimicrobial antibody responses.

4 The Role of FcRs in Antimicrobial Protection

Antibodies are the correlate of protection against a wide range of pathogens and
neutralizing antibodies can confer sterilizing immunity (Plotkin 2010). However,
commonly used in vitro assays to assess the protective capacity of antibodies such
as in vitro neutralization assays cannot efficiently predict their protective capacity
in vivo as they fail to account for FcR-mediated effector functions carried out by
various immune cell subsets. In this section, we highlight several studies demon-
strating the crucial role of Fc–FcR interactions for in vivo protection and provide
selected examples of FcR-dependent protection against viruses, bacteria, fungi, and
parasites.

4.1 Viruses

4.1.1 Influenza

Neutralizing antibodies against influenza viruses, especially against the hemag-

glutinin (HA) protein which mediates viral attachment and fusion, are considered
the main correlate of protection (Plotkin 2010). Neutralizing HA-specific antibodies
can be directed against the head region surrounding the receptor-binding site of HA
and thereby prevent viral attachment to sialic acid on target cells, or against the
stalk of HA blocking viral fusion with the target cells (Ekiert et al. 2009). The
globular head of HA is highly variable and most neutralizing head-reactive anti-
bodies are strain-specific. In contrast, the HA stalk is relatively conserved and
stalk-specific antibodies are broadly neutralizing influenza viruses across several
strains and even subtypes (Wrammert et al. 2011; Corti et al. 2011).
Several studies have recently addressed the role of Fc–FccR interactions for
the in vivo protective capacity of mAbs directed against the head and stalk of HA
Fc Receptors in Antimicrobial Protection 131

(Corti et al. 2011; DiLillo et al. 2014, 2016). While head-reactive neutralizing
antibodies were protective at low doses and independent of FccR engagement,
stalk-reactive broadly neutralizing antibodies (bnAbs) required FccR interactions to
provide optimal protection or high concentrations for partial protection in vivo
(DiLillo et al. 2014). Although bnAbs exhibit in vitro neutralizing activity, sig-
nificantly higher concentrations of bnAbs (10–1000-fold) are required to achieve
in vitro neutralization comparable to strain-specific antibodies (DiLillo et al. 2016).
The engagement of activating type I FccRs in vivo can, however, efficiently
compensate for the inferior in vitro neutralization activity. The engagement of
FccRs on NK cells by bnAbs but not head-specific antibodies bound to
influenza-infected target cells induces ADCC in vitro, suggesting that bnAbs
mediate efficient in vivo protection by triggering ADCC/ADCP in vivo (Corti et al.
2011; DiLillo et al. 2014). Furthermore, a comprehensive comparison of 13
HA-specific mAbs including strain-specific neutralizing mAbs, bnAbs against the
head and stalk of HA as well as head-reactive but not neutralizing mAbs revealed
that all broadly reactive HA-specific antibodies require the engagement of acti-
vating type I FccRs to mediate protection in vivo (DiLillo et al. 2016). It is not well
understood why strain-specific and broadly reactive HA mAbs differ in their ability
to induce FccR-mediated effector functions. Reduced antigen affinity has been
suggested to increase FccR engagement by favoring monovalent antibody–antigen
interactions resulting in a more densely opsonized target (Mazor et al. 2016).
Furthermore, a recent study also demonstrated that, in addition to FccR engage-
ment, FccR-mediated ADCC also required the receptor-binding domain of HA to
bind with sialic acid on the effector cells (Leon et al. 2016), thus providing an
explanation why neutralizing antibodies directed against the receptor-binding
domain do not efficiently mediate FccR-mediated effector functions.
Although most influenza studies focus on antibodies directed against HA due to
its abundance on the viral particle and the fact that HA inhibition titers of  1:40
are a correlate of protection, antibodies directed against neuraminidase (NA), and
the matrix protein 2 (M2) can also provide protection from lethal influenza chal-
lenge in vivo (El Bakkouri et al. 2011; DiLillo et al. 2016; Memoli et al. 2016).
Antibodies directed against M2 are not neutralizing and solely depend on the
engagement of activating type I FccRs for in vivo protection, which is mediated by
alveolar macrophages through ADCC/ADCP (El Bakkouri et al. 2011). Although
neutralizing NA-specific antibodies cannot prevent the initial infection, they can
efficiently prevent viral budding and spread. Interestingly, in accordance with
results obtained for HA-specific bnAbs and their reduced in vitro neutralization
capability, bnAbs against NA but not strain-specific NA antibodies required FccR
engagement for in vivo protection (DiLillo et al. 2016). Overall, these results
demonstrate that neutralizing antibodies with inferior in vitro activity can confer
superior protection in vivo if they can efficiently engage FccRs.
132 A. Wieland and R. Ahmed

4.1.2 Human Immunodeficiency Virus

The envelope glycoprotein (env) of human immunodeficiency virus (HIV) is the

major target for the development of neutralizing antibodies against HIV, and sev-
eral bnAbs have been developed despite its sequence diversity, glycan shield and
low abundance on the viral particle. Fc–FccR interactions play an important role
during HIV infection as patients homozygous for the low-affinity variant of FccRIIa
progress faster to AIDS (Forthal et al. 2007). BnAbs against HIV require the
efficient engagement of FccRs to provide in vivo protection as a bnAb unable to
efficiently engage FccRs failed to provide in vivo protection against SHIV chal-
lenge in nonhuman primates (Hessell et al. 2007). Mechanistic studies of several
bnAbs in mouse models subsequently showed that the engagement of activating
type I FccRs and triggering of FccR-mediated effector functions substantially
contributed to their capacity to block viral entry and suppress viremia (Bournazos
et al. 2014b). Furthermore, Fc–FccR interactions were also crucial for the thera-
peutic activity of bnAbs in a post-exposure prophylaxis setting interfering with the
establishment of a silent reservoir and reducing viremia in a therapeutic setting
(Bournazos et al. 2014b; Halper-Stromberg et al. 2014). Engagement of activating
type I FccRs by bnAbs has further shown to result in the accelerated clearance of
HIV-infected CD4+ T cells (Lu et al. 2016). The engagement of FccRs by bnAbs
thus enables the immune system to combat HIV infection at various stages of the
viral life cycle. A comprehensive analysis of the RV144 Thai Trial, the only trial
demonstrating modest vaccine efficacy to date, revealed an important role for
non-neutralizing antibodies and FccR-mediated effector functions as a correlate of
protection, further highlighting the important role of FccRs for antibody-mediated
protection irrespective of neutralization capacity (Pollara et al. 2017).

4.1.3 Respiratory Syncytial Virus

Palivizumab, a humanized IgG1 mAb recognizing a conserved neutralizing epitope

of the F glycoprotein of respiratory syncytial virus (RSV), shows potent in vitro and
in vivo activity and is now used for immunoprophylaxis in at-risk neonates
(Johnson et al. 1997). A recent study demonstrated that efficient FccR engagement
by palivizumab is crucial for its protective capacity in vivo (Hiatt et al. 2014).
Compared to the parental antibody, afucosylated palivizumab with enhanced
affinity for FccRIII resulted in about threefold lower RSV lung titers in a cotton rat
challenge model, whereas an IgG2 variant of palivizumab, unable to efficiently
engage FccRs, only showed minimal viral control in vivo.
Fc Receptors in Antimicrobial Protection 133

4.1.4 West Nile Virus

Murine mAbs directed against the nonstructural-1 (NS-1) protein of West Nile virus
(WNV), a nonstructural secreted glycoprotein absent on virions but associated with
the plasma membrane of infected cells, have been shown to mediate
FccR-dependent clearance of WNV-infected cells and protection from lethal WNV
infection (Chung et al. 2006, 2007). Protection by NS1-specific mAbs required the
engagement of activating FccRs but not the presence of NK cells suggesting that
ADCP is the main FccR-mediated effector function in this model (Chung et al.
2007). Furthermore, a mAb directed against the NS-1 protein of Yellow Fever virus
also required efficient FccR engagement to protect mice against viral encephalitis
(Schlesinger et al. 1993).

4.2 Bacteria

4.2.1 Bacillus Anthracis

Anthrax toxin neutralization assays using macrophage-like FccR-expressing cell

lines such as J774A.1 and RAW264.7 represent a unique in vitro setting to address
the role of FccR-dependent neutralization in vitro and demonstrate the important
role of FccRs for neutralization of Bacillus anthracis lethal toxin by polyclonal and
monoclonal antibody preparations (Abboud et al. 2010; Verma et al. 2009). The
in vitro and in vivo neutralizing capacity of a mAb directed against the protective
antigen component of the lethal toxin (19D9) critically depended on the engage-
ment of activating type I FccRs with mAb isotype variants exhibiting higher affinity
for activating FccRs being more potent neutralizers (Abboud et al. 2010; Verma
et al. 2009). A subsequent study generated a humanized version of 19D9 selectively
engaging activating human FccRs and performed B. anthracis challenge studies in
a mouse model recapitulating human FccR expression, which further demonstrated
the importance of optimal engagement of activating FccRs for antibody-mediated
toxin neutralization (Bournazos et al. 2014a).

4.2.2 Bordetella Pertussis

The engagement of activating FcRs by antibodies specific for Bordetella pertussis,

the causative agent of pertussis, can contribute to the clearance of bacteria from the
respiratory tract of infected mice (Hellwig et al. 2001a, b). The engagement of
activating FccRs such as FccRIII is important for bacterial clearance as mice
challenged with B. pertussis opsonized with a bispecific mAb targeting FccRII/III
showed improved bacterial control, whereas a mAb targeting the complement
receptor CD11b provided no benefit (Hellwig et al. 2001a). Furthermore, the results
obtained in a human FcaRI-transgenic mouse model using immune serum IgA or a
134 A. Wieland and R. Ahmed

bispecific Ab targeting hFcaRI suggest that engagement of FcaRI can also con-
tribute to bacterial clearance (Hellwig et al. 2001b). FcR-mediated phagocytosis of
both IgG- and IgA-opsonized B. pertussis triggered an efficient oxidative burst in
human polymorphonuclear leukocytes and simultaneous engagement of FccRs and
FcaR has been shown to result in enhanced phagocytosis (Rodriguez et al. 2001).

4.2.3 Chlamydia Trachomatis

FccR-mediated effector mechanisms can contribute to the clearance of secondary

infections with the intracellular bacterium Chlamydia muridarum, the murine
genital infection model recapitulating many aspects of Chlamydia trachomatis
infection in humans (Moore et al. 2002). Subsequent studies showed that mice
lacking FccRs mounted reduced Th1 responses upon reinfection due to limited
antigen presentation by FccR-deficient APCs and that CD4-derived IFNc was
crucial for efficient antibody-mediated pathogen clearance by neutrophils (Moore
et al. 2003; Naglak et al. 2016, 2017).

4.2.4 Legionella Pneumophila

Antibodies directed against Legionella pneumophila, the causative agent of

Legionnaires’ disease, have shown to protect mice by promoting the lysosomal
fusion of phagosomes containing opsonized bacteria in a FccR-dependent manner
(Joller et al. 2010). Phagocytic uptake of L. pneumophila by macrophages in the
absence of FccR signaling allows the pathogen to arrest phagolysosomal maturation
by secretion of bacterial effector proteins into the cytoplasm thus establishing a
replication-permissive vacuole. Signaling through activating FccRs, however,
renders host cells resistant and efficiently targets pathogens for lysosomal degra-
dation. Experiments using isotype variants of L. pneumophila-specific mAb further
demonstrated that isotypes efficiently engaging activating FccRs (e.g., mouse
IgG2c) provided improved protection compared with an isotype poorly engaging
activating FccRs (mouse IgG3) (Weber et al. 2014). Enhanced lysosomal targeting
of opsonized pathogens might also contribute to the protection against other
intracellular bacteria, which have evolved strategies to prevent phagolysosomal
maturation and establish replication-permissive vacuoles.

4.2.5 Mycobacterium Tuberculosis

FcRs have also been implicated in regulating the immune response to

Mycobacterium tuberculosis and ultimately bacterial burden. Mice lacking the
inhibitory FccRIIb showed elevated Th1 responses and reduced bacterial burden in
the lungs, whereas mice lacking activating FccRs demonstrated increased bacterial
burden and succumbed faster to infection (Maglione et al. 2008). Furthermore, data
Fc Receptors in Antimicrobial Protection 135

obtained in a human FcaRI-transgenic mouse model also suggest that engagement

of FcaRI by a M. tuberculosis-specific monoclonal IgA delivered intranasally in
combination with IFNc can reduce bacterial burden (Balu et al. 2011).

4.2.6 Rickettsia Conorii

Passive transfer of polyclonal immune serum and outer membrane protein A/

B-specific mAbs but not Fab fragments protected immunodeficient mice from fatal
infection with the intracellular gram-negative bacterium Rickettsia conorii, the
causative agent of Boutonneuse fever, suggesting the involvement of FccRs in
antibody-mediated protection (Feng et al. 2004a). Opsonization of R. conorii with
IgG diminished bacterial escape from phagosomes and triggered the generation of
reactive oxygen and nitrogen species mediating killing of internalized pathogens
(Feng et al. 2004b). Although this study did not utilize genetic models to address
the specific role of FccRs in this process, FccRs are likely preventing phagosomal
escape and mediate intracellular killing of opsonized R. conorii in a comparable
manner as described for Francisella tularensis, an intracellular gram-negative
bacterium and the causative agent of tularemia (Geier and Celli 2011).

4.2.7 Salmonella Enterica

The intracellular gram-negative bacterium Salmonella enterica serovar

Typhimurium (ST), the causative agent of typhoid disease in mouse models, has
evolved strategies allowing it to survive within phagocytic cells such as macro-
phages and DCs and establish replication-permissive vacuoles by preventing fusion
with lysosomes. However, the engagement of the activating FccRIII on DCs by
IgG-opsonized ST has been shown to prevent bacterial escape by mediating effi-
cient lysosomal targeting and subsequent degradation of bacteria (Tobar et al. 2004;
Herrada et al. 2007). Furthermore, FccRIII-mediated uptake and lysosomal tar-
geting of opsonized ST resulted in efficient antigen processing and presentation,
and could efficiently activate CD4+ and CD8+ T cells in vitro, suggesting that FccR
engagement can contribute to the generation of an antibacterial T cell response.
Interestingly, although mice deficient for FccRI-III initially exhibited higher bac-
terial loads upon immunization with a live-attenuated ST strain, they could effi-
ciently mount cellular and humoral responses toward ST but succumbed to a
subsequent high-dose challenge with a virulent strain (Menager et al. 2007).
Overall, these data show that FccRs play a crucial role in the control of ST
infection.
136 A. Wieland and R. Ahmed

4.2.8 Staphylococcus Aureus

Staphylococcus aureus is a gram-positive bacterium that can cause life-threating

infections, and methicillin-resistant S. aureus represents a major public health
concern. Neutralizing mAbs against staphylococcal enterotoxin B (SEB), one of the
most potent known superantigens, can protect mice against lethal sepsis following
intravenous challenge with S. aureus and result in a modest reduction of bacterial
burden in skin abscesses (Varshney et al. 2011, 2013). A subsequent study testing
the protective capacity of isotype switch variants (IgG1, IgG2a, and IgG2b) of a
neutralizing SEB-specific mAb showed comparable in vitro neutralization potency
but enhanced in vivo protection by the IgG2a variant, especially at lower doses,
suggesting that efficient engagement of activating FccRs is required for optimal
in vivo activity (Varshney et al. 2014).
In addition to binding IgG, human FccRs can also interact with members of the
pentraxin family such as human serum amyloid P (SAP) and C-reactive protein
(CRP), which are soluble innate pattern recognition molecules that can bind to
microbial pathogens (Lu et al. 2008). Furthermore, FcaRI has been shown to
interact with SAP and CRP (Lu et al. 2011). The engagement of FcRs by SAP and
CRP can trigger comparable downstream signaling events and cellular responses as
engagement by IgG or IgA (Lu et al. 2008,2011). SAP can bind to peptidoglycan
and promote the phagocytosis of tagO-deficient S. aureus in a FccR-dependent
manner (An et al. 2013). However, the in vivo role of pentraxin/FcR interactions for
antimicrobial protection is not well characterized.

4.2.9 Streptococcus pneumoniae

FccRs play an important role in antibody-mediated protection and regulation of the

immune response to Streptococcus pneumoniae. The expression of the inhibitory
FccRIIb is important for a balanced immune response and prevented septic shock
and death of immunized mice following high-dose S. pneumoniae challenge
(Clatworthy and Smith 2004). However, in a primary low-dose infection setting,
global FccRIIb deficiency conferred a survival advantage, whereas macrophage-
specific overexpression of FccRIIb was shown to reduce survival under these
conditions (Clatworthy and Smith 2004; Brownlie et al. 2008). Engagement of
FccRIIb is thus important to balance efficient pathogen clearance and the release of
septic shock cytokines with the ultimate goal of maximizing survival.
Engagement of FccRs is crucial for the in vivo protective capacity of mAbs
against the pneumococcal capsular polysaccharide (PPS) of S. pneumoniae (Tian
et al. 2009). Interestingly, the ability of PPS-specific mouse IgG1 mAbs to promote
S. pneumoniae killing in an in vitro opsonophagocytic killing assay (OPKA) failed
to predict the in vivo protective capacity as a nonopsonic antibody efficiently
protected mice in a FccR-dependent manner (Tian et al. 2009). Furthermore, the
requirements for antibody-mediated protection in vivo differ significantly between
these mAbs, as opsonic mAbs required the presence of neutrophils and FccRIIb,
Fc Receptors in Antimicrobial Protection 137

whereas as a nonopsonic mAb required macrophages and FccRIII (Tian et al. 2009;
Weber et al. 2012). These data further show that, although certain in vitro assays
such as OPKA rely on FccR engagement, they fail to recapitulate all
FccR-mediated effector functions contributing to in vivo protection.

4.3 Fungi and Parasites

4.3.1 Leishmania

FccRs have been shown to play a crucial role during the infection with protozoan
intracellular Leishmania parasites. Whereas early during infection Leishmania
major was predominantly phagocytosed in a complement-dependent manner by
macrophages, once the infection was established and parasite-specific IgG pro-
duction induced, DCs efficiently phagocytosed parasites in an activating
FccR-dependent manner (Woelbing et al. 2006). FccR-mediated uptake of
IgG-opsonized parasites by DCs resulted in the efficient priming of Th1 cells and
CD8+ T cells, whereas mice lacking antibodies or activating FccRs failed to mount
efficient T cell responses resulting in increased lesion progression and higher par-
asite burden (Woelbing et al. 2006). In contrast to the data obtained with mice on
the resistant C57BL6 background demonstrating a beneficial role of FccRs, sus-
ceptible BALB/c mice lacking activating FccRs have been shown to exhibit
increased parasite control suggesting that the genetic background determines the
role of activating FccRs in L. major pathogenesis (Padigel and Farrell 2005).

4.3.2 Toxoplasma Gondii

FccR-mediated processes such as ADCP are likely involved in host resistance to

infection with the intracellular protozoan parasite Toxoplasma gondii.
FccR-mediated phagocytosis of opsonized parasites has been shown to result in
efficient lysosomal targeting preventing replication of the parasite within the cell
(Joiner et al. 1990). A similar process was described and attributed to contribute to
resistance against Encephalitozoon cuniculi, an intracellular fungus (Niederkorn
and Shadduck 1980). Although the in vivo role of FccRs for resistance against
T. gondii has not been directly addressed so far, immune IgG transfer experiments
in B cell deficient mice demonstrated that antibody-mediated effector functions also
contribute to in vivo resistance (Kang et al. 2000).

4.3.3 Schistosoma Mansoni

IgE/FceRI interactions may play a central role in resistance to infections with

parasitic helminths such as Schistosoma mansoni, one of the causative agents of
138 A. Wieland and R. Ahmed

Schistosomiasis. IgE can mediate killing of S. mansoni larvae by macrophages,

eosinophils, and platelets in vitro (Joseph et al. 1983; Butterworth 1984).
IgE-mediated killing of Acanthocheilonema viteae, a filarial parasitic nematode, by
macrophages and eosinophils in vitro further supports the role of IgE as the central
mediator for ADCC against parasitic worms (Haque et al. 1981). Human macro-
phages, eosinophils, and platelets express the high-affinity IgE FceRI, which has
been shown to be important for IgE-mediated killing in vitro (Gounni et al. 1994).
However, species-specific differences in the expression pattern of FceRI have
hampered studies addressing the in vivo role of the IgE/FceRI interaction in murine
S. mansoni infection models and might thus explain conflicting results. Whereas
mice deficient for IgE were more susceptible for infection with S. mansoni and had
higher worm burden, FceRI deficiency did not affect worm burden (King et al.
1997; Jankovic et al. 1997). The use of hFceRI-transgenic mouse models reflecting
the human expression pattern of hFceRI might help to elucidate the in vivo role of
IgE/FceRI interactions during S. mansoni infection.

4.3.4 Cryptococcus Neoformans

The efficient engagement of activating FccRs by mAbs directed against the capsular
polyssacharide (cPS) has been shown to contribute to protection against
Cryptococcus neoformans, a fungus infecting mainly immunocompromised patients
and causing meningitis. The opsonic activity of cPS-specific mAbs in vitro crucially
depends on the isotype of the antibody, with mouse IgG2a being the most potent
isotype (Schlageter and Kozel 1990). Furthermore, therapeutic application of IgG2a
and IgG2b variants of a cPS-specific mAb to mice challenged with a highly
pathogenic strain of C. neoformans resulted in reduced yeast burden in spleen and
lungs; however, it failed to ultimately reduce yeast burden in brain and prevent
death (Sanford et al. 1990). Engagement of activating FccRs has been shown to be
important for the in vivo protective capacity of a cPS-specific IgG1 mAb and
resulted in enhanced survival, whereas an IgG3 variant with identical specificity
enhanced pathogenicity in vivo independent of activating FccRs (Yuan et al. 1998).
These data demonstrate that the isotype and efficient engagement of FccRs in vivo
are crucial for the protective capacity of C. neoformans-specific antibodies.

5 Microbial Evasion Strategies

Although Fc–FccR interactions can mediate effective antimicrobial protection

in vivo, a wide range of pathogens has developed sophisticated evasion mecha-
nisms. Pathogenic bacteria have evolved several evasion mechanisms including
Fc-binding proteins such as protein A/G (Foster 2005), IgG-cleaving enzymes such
as IdeS (Von Pawel-Rammingen et al. 2002), IgG-specific endoglycosidases such
as EndoS (Collin and Olsen 2001), and injection of phagocytosis inhibitors into
Fc Receptors in Antimicrobial Protection 139

phagocytic cells (Marches et al. 2008). Various viruses can also interfere with
FccR-mediated effector mechanisms through expression of viral FccRs and viral
homologs of immunoregulatory proteins on the surface of infected cells (Lubinski
et al. 2011; Cameron et al. 2005).
In addition to direct microbial evasion strategies requiring the expression of
specific evasion proteins, pathogens may also exploit dysfunctions of the immune
system that occur during persistent infections, autoimmune diseases, and cancer.
We and others have recently shown that excessive immune complex formation in
mice persistently infected with lymphocytic choriomeningitis virus (LCMV)
interferes with various FccR-mediated antibody effector functions without nega-
tively affecting the overall expression pattern of FccRs on effector cells (Fig. 3)
(Wieland et al. 2015; Yamada et al. 2015). The inhibition of FccR-mediated
antibody effector functions is due to immune complex formation and not the per-
sistent viral infection per se, as persistently infected mice in the absence of an
antiviral B cell response or with a strikingly diminished antiviral B cell response
exhibit normal FccR-mediated effector functions. During persistent LCMV infec-
tion, immune complexes inhibit several FccR-mediated effector functions such as
ADCC of infected cells, ADCP of antibody-coated target cells, antigen
cross-presentation to CD8+ T cells, and also abrogate the therapeutic activity of
depleting and agonistic antibodies relying on the efficient engagement of FccRs
(Wieland et al. 2015; Yamada et al. 2015).
Elevated levels of circulating immune complexes as well as deposition in the
kidney have been reported in various diseases ranging from persistent viral infec-
tions such as B virus, hepatitis C virus and HIV, to autoimmune diseases and cancer
(Johnson et al. 1993; Tsai et al. 1995; Nobakht et al. 2016; Lefaucheur et al. 2006;
Theofilopoulos et al. 1977). Excessive immune complex formation has been shown
to inhibit the therapeutic activity of rituximab, a chimeric anti-human CD20 widely
used for B cell malignancies and autoimmune diseases, during persistent LCMV
infection as well as in a murine lupus model (Wieland et al. 2015; Yamada et al.
2015; Ahuja et al. 2011). The proposed mechanism of reduced FccR engagement
due to excessive immune complex formation might thus explain the observed
variation in B cell depletion efficacy in systemic lupus erythematosus patients upon
rituximab treatment (Looney et al. 2004). Overall, these studies show that circulating
immune complexes occur in various diseases and can result in significant interfer-
ence with FccR-mediated antibody effector functions, potentially favoring infections
despite the presence of humoral immunity and contributing to viral persistence.
We have recently shown that endogenous immune complexes do not simply
abrogate the engagement of FccRs by depleting antibodies but rather compete with
depleting antibodies for binding to FccRs, increasing the apparent threshold
required for the efficient induction of FccR-mediated effector functions (Wieland
et al. 2018). Efficient antibody-mediated depletion in the presence of competing
endogenous immune complexes can be achieved by targeting highly expressed
surface antigens such as CD90 or by using antibodies with Fc domain modifications
such as afucosylation that are aimed at increasing the affinity to activating FccRs
(Fig. 3). Overall, our data demonstrate that excessive immune complex formation
140 A. Wieland and R. Ahmed

(a) naive host (b) persistently infected host

ADCP/
ADCP/ ADCC
ADCC

(c) highly expressed target molecule

during persistent infection
(d) afucosylated mAb during
persistent infection

ADCP/
ADCC ADCP/
ADCC

antigen
fucosylated IgG antibody

immune complex afucosylated IgG antibody

FcR

Fig. 3 Differential requirements for the efficient induction of antibody-dependent cellular

phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) in naïve and
persistently infected hosts. a Efficient ADCP/ADCC in naïve hosts. b Immune complexes prevent
efficient ADCP/ADCC in persistently infected hosts. c Highly opsonized targets can outcompete
endogenous immune complexes efficiently triggering ADCP/ADCC. d Afucosylated mAbs with
increased affinity for activating FccRs can outcompete endogenous immune complexes and
efficiently trigger ADCP/ADCC during persistent infection

can be an obstacle for therapeutic antibodies to efficiently engage FccRs but can be
overcome by selectively targeting highly abundant surface antigens or the use of
Fc-engineered monoclonal antibodies. Future experiments using co-infection
models will reveal the effects of excessive immune complex formation on
antibody-mediated antimicrobial protection in vivo. Furthermore, these experiments
will allow optimization of antibody-based antimicrobial intervention strategies.
Fc Receptors in Antimicrobial Protection 141

6 Conclusions

FcRs mediate direct antimicrobial protection by various effector mechanisms and

are involved in the regulation of a plethora of immunological processes which
indirectly contribute to antimicrobial protection. Using knockout mouse models as
well as Fc-engineered antibodies, studies in various infection models demonstrate
that many pathogen-specific antibodies rely on efficient FccR engagement for
optimal antimicrobial protection in vivo. Most notably, the initially surprising
observations that broadly neutralizing antibodies against various pathogens such as
Influenza virus and HIV need to engage activating FccRs for optimal in vivo
protection have important implications for the development of therapeutics and
vaccines. The induction of protective broadly neutralizing antibody responses may
thus require the induction of antibodies of a particular specificity and IgG isotype.
While some in vitro assays rely on Fc–FccR interactions to assess protective
antibody responses, these assays fail to recapitulate the complex landscape of
cellular FccR expression. Thus, in vivo experiments are ultimately required to
assess the role of Fc–FccR interactions for antimicrobial protection. Future studies
assessing the protective capacity of human mAbs in vivo should thus be performed
in human FccR transgenic mice that accurately reflect the cellular FccR expression
pattern in humans.

References

Abboud N, Chow SK, Saylor C, Janda A, Ravetch JV, Scharff MD, Casadevall A (2010) A
requirement for FccR in antibody-mediated bacterial toxin neutralization. J Exp Med
207:2395–2405
Abu-Ghazaleh RI, Fujisawa T, Mestecky J, Kyle RA, Gleich GJ (1989) IgA-induced eosinophil
degranulation. J Immunol 142:2393–2400
Ahuja A, Teichmann LL, Wang H, Dunn R, Kehry MR, Shlomchik MJ (2011) An acquired defect
in IgG-dependent phagocytosis explains the impairment in antibody-mediated cellular
depletion in Lupus. J Immunol 187:3888–3894
Amigorena S, Bonnerot C, Drake JR, Choquet D, Hunziker W, Guillet JG, Webster P, Sautes C,
Mellman I, Fridman WH (1992a) Cytoplasmic domain heterogeneity and functions of IgG Fc
receptors in B lymphocytes. Science 256:1808–1812
Amigorena S, Salamero J, Davoust J, Fridman WH, Bonnerot C (1992b) Tyrosine-containing
motif that transduces cell activation signals also determines internalization and antigen
presentation via type III receptors for IgG. Nature 358:337–341
An JH, Kurokawa K, Jung DJ, Kim MJ, Kim CH, Fujimoto Y, Fukase K, Coggeshall KM, Lee BL
(2013) Human SAP is a novel peptidoglycan recognition protein that induces
complement-independent phagocytosis of Staphylococcus aureus. J Immunol 191:3319–3327
Anthony RM, Kobayashi T, Wermeling F, Ravetch JV (2011) Intravenous cglobulin suppresses
inflammation through a novel T(H)2 pathway. Nature 475:110–113
Araki N, Johnson MT, Swanson JA (1996) A role for phosphoinositide 3-kinase in the completion
of macropinocytosis and phagocytosis by macrophages. J Cell Biol 135:1249–1260
142 A. Wieland and R. Ahmed

Balu S, Reljic R, Lewis MJ, Pleass RJ, McIntosh R, van Kooten C, van Egmond M,
Challacombe S, Woof JM, Ivanyi J (2011) A novel human IgA monoclonal antibody protects
against tuberculosis. J Immunol 186:3113–3119
Bergtold A, Desai DD, Gavhane A, Clynes R (2005) Cell surface recycling of internalized antigen
permits dendritic cell priming of B cells. Immunity 23:503–514
Bidgood SR, Tam JC, McEwan WA, Mallery DL, James LC (2014) Translocalized IgA mediates
neutralization and stimulates innate immunity inside infected cells. Proc Natl Acad Sci U S A
111:13463–13468
Bolland S, Ravetch JV (2000) Spontaneous autoimmune disease in Fc(c)RIIB-deficient mice
results from strain-specific epistasis. Immunity 13:277–285
Bolland S, Yim YS, Tus K, Wakeland EK, Ravetch JV (2002) Genetic modifiers of systemic lupus
erythematosus in FccRIIB(−/−) mice. J Exp Med 195:1167–1174
Boruchov AM, Heller G, Veri MC, Bonvini E, Ravetch JV, Young JW (2005) Activating and
inhibitory IgG Fc receptors on human DCs mediate opposing functions. J Clin Invest
115:2914–2923
Bournazos S, Chow SK, Abboud N, Casadevall A, Ravetch JV (2014a) Human IgG Fc domain
engineering enhances antitoxin neutralizing antibody activity. J Clin Invest 124:725–729
Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV (2014b) Broadly
neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell
158:1243–1253
Bournazos S, Ravetch JV (2015) Fcc receptor pathways during active and passive immunization.
Immunol Rev 268:88–103
Bournazos S, Wang TT, Dahan R, Maamary J, Ravetch JV (2017) Signaling by antibodies: recent
progress. Annu Rev Immunol 35:285–311
Bournazos S, Wang TT, Ravetch JV (2016) The role and function of fcc receptors on myeloid
cells. Microbiol Spectr, 4
Brownlie RJ, Lawlor KE, Niederer HA, Cutler AJ, Xiang Z, Clatworthy MR, Floto RA,
Greaves DR, Lyons PA, Smith KG (2008) Distinct cell-specific control of autoimmunity and
infection by FccRIIb. J Exp Med 205:883–895
Butterworth AE (1984) Cell-mediated damage to helminths. Adv Parasitol 23:143–235
Cameron CM, Barrett JW, Mann M, Lucas A, McFadden G (2005) Myxoma virus M128L is
expressed as a cell surface CD47-like virulence factor that contributes to the downregulation of
macrophage activation in vivo. Virology 337:55–67
Caron E, Hall A (1998) Identification of two distinct mechanisms of phagocytosis controlled by
different Rho GTPases. Science 282:1717–1721
Chung KM, Nybakken GE, Thompson BS, Engle MJ, Marri A, Fremont DH, Diamond MS (2006)
Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through
Fc c receptor-dependent and -independent mechanisms. J Virol 80:1340–1351
Chung KM, Thompson BS, Fremont DH, Diamond MS (2007) Antibody recognition of cell
surface-associated NS1 triggers Fc-c receptor-mediated phagocytosis and clearance of West
Nile Virus-infected cells. J Virol 81:9551–9555
Clatworthy MR, Smith KG (2004) FccRIIb balances efficient pathogen clearance and the
cytokine-mediated consequences of sepsis. J Exp Med 199:717–723
Clynes R, Maizes JS, Guinamard R, Ono M, Takai T, Ravetch JV (1999) Modulation of immune
complex-induced inflammation in vivo by the coordinate expression of activation and
inhibitory Fc receptors. J Exp Med 189:179–185
Collin M, Olsen A (2001) EndoS, a novel secreted protein from Streptococcus pyogenes with
endoglycosidase activity on human IgG. EMBO J 20:3046–3055
Corti D, Voss J, Gamblin SJ, Codoni G, Macagno A, Jarrossay D, Vachieri SG, Pinna D,
Minola A, Vanzetta F, Silacci C, Fernandez-Rodriguez BM, Agatic G, Bianchi S,
Giacchetto-Sasselli I, Calder L, Sallusto F, Collins P, Haire LF, Temperton N,
Langedijk JP, Skehel JJ, Lanzavecchia A (2011) A neutralizing antibody selected
from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science
333:850–856
Fc Receptors in Antimicrobial Protection 143

Cox D, Tseng CC, Bjekic G, Greenberg S (1999) A requirement for phosphatidylinositol 3-kinase
in pseudopod extension. J Biol Chem 274:1240–1247
Crowley MT, Costello PS, Fitzer-Attas CJ, Turner M, Meng F, Lowell C, Tybulewicz VL,
Defranco AL (1997) A critical role for Syk in signal transduction and phagocytosis mediated
by Fcc receptors on macrophages. J Exp Med 186:1027–1039
de Saint Basile G, Ménasché G, Fischer A (2010) Molecular mechanisms of biogenesis and
exocytosis of cytotoxic granules. Nat Rev Immunol 10(8):568
Desjardins M, Huber LA, Parton RG, Griffiths G (1994) Biogenesis of phagolysosomes proceeds
through a sequential series of interactions with the endocytic apparatus. J Cell Biol 124:677–
688
Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV (2002) Antitumor monoclonal
antibodies enhance cross-presentation of Cellular antigens and the generation of
myeloma-specific killer T cells by dendritic cells. J Exp Med 195:125–133
Dilillo DJ, Palese P, Wilson PC, Ravetch JV (2016) Broadly neutralizing anti-influenza antibodies
require Fc receptor engagement for in vivo protection. J Clin Invest 126:605–610
Dilillo DJ, Tan GS, Palese P, Ravetch JV (2014) Broadly neutralizing hemagglutinin stalk-specific
antibodies require FccR interactions for protection against influenza virus in vivo. Nat Med
20:143–151
Dombrowicz D, Quatannens B, Papin JP, Capron A, Capron M (2000) Expression of a functional
Fc epsilon RI on rat eosinophils and macrophages. J Immunol 165:1266–1271
Dugast AS, Tonelli A, Berger CT, Ackerman ME, Sciaranghella G, Liu Q, Sips M, Toth I,
Piechocka-Trocha A, Ghebremichael M, Alter G (2011) Decreased Fc receptor expression on
innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity
in chronically HIV-1 infected individuals. Virology 415:160–167
Durden DL, Kim HM, Calore B, Liu Y (1995) The Fc c RI receptor signals through the activation
of hck and MAP kinase. J Immunol 154:4039–4047
Eischen CM, Schilling JD, Lynch DH, Krammer PH, Leibson PJ (1996) Fc receptor-induced
expression of Fas ligand on activated NK cells facilitates cell-mediated cytotoxicity and
subsequent autocrine NK cell apoptosis. J Immunol 156:2693–2699
Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, Throsby M, Goudsmit J,
Wilson IA (2009) Antibody recognition of a highly conserved influenza virus epitope. Science
324:246–251
el Bakkouri K, Descamps F, de Filette M, Smet A, Festjens E, Birkett A, van Rooijen N,
Verbeek S, Fiers W, Saelens X (2011) Universal vaccine based on ectodomain of matrix
protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection. J Immunol
186:1022–1031
Fabian I, Kletter Y, Mor S, Geller-Bernstein C, Ben-Yaakov M, Volovitz B, Golde DW (1992)
Activation of human eosinophil and neutrophil functions by haematopoietic growth factors:
comparisons of IL-1, IL-3, IL-5 and GM-CSF. Br J Haematol 80:137–143
Feng HM, Whitworth T, Olano JP, Popov VL, Walker DH (2004a) Fc-dependent polyclonal
antibodies and antibodies to outer membrane proteins A and B, but not to lipopolysaccharide,
protect SCID mice against fatal Rickettsia conorii infection. Infect Immun 72:2222–2228
Feng HM, Whitworth T, Popov V, Walker DH (2004b) Effect of antibody on the rickettsia-host
cell interaction. Infect Immun 72:3524–3530
Fiebiger BM, Maamary J, Pincetic A, Ravetch JV (2015) Protection in antibody- and T
cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs. Proc
Natl Acad Sci U S A 112:E2385–E2394
Flannagan RS, Jaumouille V, Grinstein S (2012) The cell biology of phagocytosis. Annu Rev
Pathol 7:61–98
Forthal DN, Landucci G, Bream J, Jacobson LP, Phan TB, Montoya B (2007) FccRIIa genotype
predicts progression of HIV infection. J Immunol 179:7916–7923
Foster TJ (2005) Immune evasion by staphylococci. Nat Rev Microbiol 3:948–958
144 A. Wieland and R. Ahmed

Fu SL, Pierre J, Smith-Norowitz TA, Hagler M, Bowne W, Pincus MR, Mueller CM,
Zenilman ME, Bluth MH (2008) Immunoglobulin E antibodies from pancreatic cancer patients
mediate antibody-dependent cell-mediated cytotoxicity against pancreatic cancer cells. Clin
Exp Immunol 153:401–409
Ganesan LP, Fang H, Marsh CB, Tridandapani S (2003) The protein-tyrosine phosphatase SHP-1
associates with the phosphorylated immunoreceptor tyrosine-based activation motif of Fc c
RIIa to modulate signaling events in myeloid cells. J Biol Chem 278:35710–35717
Geier H, Celli J (2011) Phagocytic receptors dictate phagosomal escape and intracellular
proliferation of Francisella tularensis. Infect Immun 79:2204–2214
Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, Capron A, Kinet JP, Capron M
(1994) High-affinity IgE receptor on eosinophils is involved in defence against parasites.
Nature 367:183–186
Griffin FM, Griffin JA, Leider JE, Silverstein SC (1975) Studies on the mechanism of
phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to
specific receptors on the macrophage plasma membrane. J Exp Med 142(5):1263–1282
Guilliams M, Bruhns P, Saeys Y, Hammad H, Lambrecht BN (2014) The function of Fcc
receptors in dendritic cells and macrophages. Nat Rev Immunol 14:94–108
Halper-Stromberg A, Lu CL, Klein F, Horwitz JA, Bournazos S, Nogueira L, Eisenreich TR,
Liu C, Gazumyan A, Schaefer U, Furze RC, Seaman MS, Prinjha R, Tarakhovsky A,
Ravetch JV, Nussenzweig MC (2014) Broadly neutralizing antibodies and viral inducers
decrease rebound from HIV-1 latent reservoirs in humanized mice. Cell 158:989–999
Hamada F, Aoki M, Akiyama T, Toyoshima K (1993) Association of immunoglobulin G Fc
receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils. Proc Natl Acad Sci U S A
90:6305–6309
Hamawy MM, Minoguchi K, Swaim WD, Mergenhagen SE, Siraganian RP (1995) A 77-kDa
protein associates with pp125FAK in mast cells and becomes tyrosine-phosphorylated by high
affinity IgE receptor aggregation. J Biol Chem 270:12305–12309
Haque A, Ouaissi A, Joseph M, Capron M, Capron A (1981) IgE antibody in eosinophil- and
macrophage-mediated in vitro killing of Dipetalonema viteae microfilariae. J Immunol
127:716–725
Hauler F, Mallery DL, McEwan WA, Bidgood SR, James LC (2012) AAA ATPase p97/VCP is
essential for TRIM21-mediated virus neutralization. Proc Natl Acad Sci U S A 109:19733–
19738
Hellwig SM, van Oirschot HF, Hazenbos WL, van Spriel AB, Mooi FR, van de Winkel JG
(2001a) Targeting to Fcc receptors, but not CR3 (CD11b/CD18), increases clearance of
Bordetella pertussis. J Infect Dis 183:871–879
Hellwig SM, van Spriel AB, Schellekens JF, Mooi FR, van de Winkel JG (2001b)
Immunoglobulin A-mediated protection against Bordetella pertussis infection. Infect Immun
69:4846–4850
Herrada AA, Contreras FJ, Tobar JA, Pacheco R, Kalergis AM (2007) Immune complex-induced
enhancement of bacterial antigen presentation requires Fcc receptor III expression on dendritic
cells. Proc Natl Acad Sci U S A 104:13402–13407
Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM, Lanigan CM,
Landucci G, Forthal DN, Parren PW, Marx PA, Burton DR (2007) Fc receptor but not
complement binding is important in antibody protection against HIV. Nature 449:101–104
Hiatt A, Bohorova N, Bohorov O, Goodman C, Kim D, Pauly MH, Velasco J, Whaley KJ,
Piedra PA, Gilbert BE, Zeitlin L (2014) Glycan variants of a respiratory syncytial virus
antibody with enhanced effector function and in vivo efficacy. Proc Natl Acad Sci U S A
111:5992–5997
Hoppe AD, Swanson JA (2004) Cdc42, Rac1, and Rac2 display distinct patterns of activation
during phagocytosis. Mol Biol Cell 15:3509–3519
James LC, Keeble AH, Khan Z, Rhodes DA, Trowsdale J (2007) Structural basis for
PRYSPRY-mediated tripartite motif (TRIM) protein function. Proc Natl Acad Sci U S A
104:6200–6205
Fc Receptors in Antimicrobial Protection 145

Jankovic D, Kullberg MC, Dombrowicz D, Barbieri S, Caspar P, Wynn TA, Paul WE,
Cheever AW, Kinet JP, Sher A (1997) Fc epsilonRI-deficient mice infected with Schistosoma
mansoni mount normal Th2-type responses while displaying enhanced liver pathology.
J Immunol 159:1868–1875
Johnson RJ, Gretch DR, Yamabe H, Hart J, Bacchi CE, Hartwell P, Couser WG, Corey L,
Wener MH, Alpers CE et al (1993) Membranoproliferative glomerulonephritis associated with
hepatitis C virus infection. New Engl J Med 328(7):465–470
Johnson S, Oliver C, Prince GA, Hemming VG, Pfarr DS, Wang SC, Dormitzer M, O’Grady J,
Koenig S, Tamura JK, Woods R, Bansal G, Couchenour D, Tsao E, Hall WC, Young JF
(1997) Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro
and in vivo activity against respiratory syncytial virus. J Infect Dis 176:1215–1224
Joiner KA, Fuhrman SA, Miettinen HM, Kasper LH, Mellman I (1990) Toxoplasma gondii: fusion
competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science
249:641–646
Joller N, Weber SS, Muller AJ, Sporri R, Selchow P, Sander P, Hilbi H, Oxenius A (2010)
Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting.
Proc Natl Acad Sci U S A 107:20441–20446
Jonsson F, Mancardi DA, Albanesi M, Bruhns P (2013) Neutrophils in local and systemic
antibody-dependent inflammatory and anaphylactic reactions. J Leukoc Biol 94:643–656
Joseph M, Auriault C, Capron A, Vorng H, Viens P (1983) A new function for platelets:
IgE-dependent killing of schistosomes. Nature 303:810–812
Jouvin MH, Adamczewski M, Numerof R, Letourneur O, Valle A, Kinet JP (1994) Differential
control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity
immunoglobulin E receptor. J Biol Chem 269:5918–5925
Kanakaraj P, Duckworth B, Azzoni L, Kamoun M, Cantley LC, Perussia B (1994)
Phosphatidylinositol-3 kinase activation induced upon Fc c RIIIA-ligand interaction. J Exp
Med 179:551–558
Kang H, Remington JS, Suzuki Y (2000) Decreased resistance of B cell-deficient mice to infection
with Toxoplasma gondii despite unimpaired expression of IFN-c, TNF-alpha, and inducible
nitric oxide synthase. J Immunol 164:2629–2634
Kant AM, De P, Peng X, Yi T, Rawlings DJ, Kim JS, Durden DL (2002) SHP-1 regulates Fcc
receptor-mediated phagocytosis and the activation of RAC. Blood 100:1852–1859
Kato T, Kitagawa S (2006) Regulation of neutrophil functions by proinflammatory cytokines. Int J
Hematol 84:205–209
Keeble AH, Khan Z, Forster A, James LC (2008) TRIM21 is an IgG receptor that is structurally,
thermodynamically, and kinetically conserved. Proc Natl Acad Sci U S A 105:6045–6050
Kiefer F, Brumell J, Al-Alawi N, Latour S, Cheng A, Veillette A, Grinstein S, Pawson T (1998)
The Syk protein tyrosine kinase is essential for Fcc receptor signaling in macrophages and
neutrophils. Mol Cell Biol 18:4209–4220
King CL, Xianli J, Malhotra I, Liu S, Mahmoud AA, Oettgen HC (1997) Mice with a targeted
deletion of the IgE gene have increased worm burdens and reduced granulomatous
inflammation following primary infection with Schistosoma mansoni. J Immunol 158:294–300
Larsen EC, Ueyama T, Brannock PM, Shirai Y, Saito N, Larsson C, Loegering D, Weber PB,
Lennartz MR (2002) A role for PKC-epsilon in Fc cR-mediated phagocytosis by RAW 264.7
cells. J Cell Biol 159:939–944
Lefaucheur, C., Stengel, B., Nochy, D., Martel, P., Hill, G. S., Jacquot, C., Rossert, J. & Group,
G.-P. S (2006) Membranous nephropathy and cancer: epidemiologic evidence and determi-
nants of high-risk cancer association. Kidney Int 70:1510–1517
Leon PE, He W, Mullarkey CE, Bailey MJ, Miller MS, Krammer F, Palese P, Tan GS (2016)
Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin
antibodies requires two points of contact. Proc Natl Acad Sci U S A 113:E5944–E5951
Liao F, Shin HS, Rhee SG (1992) Tyrosine phosphorylation of phospholipase C-c 1 induced by
cross-linking of the high-affinity or low-affinity Fc receptor for IgG in U937 cells. Proc Natl
Acad Sci U S A 89:3659–3663
146 A. Wieland and R. Ahmed

Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend LJ, Sloand JA, Rosenblatt J,
Sanz I (2004) B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/
II dose-escalation trial of rituximab. Arthritis Rheum 50:2580–2589
Lorenzi R, Brickell PM, Katz DR, Kinnon C, Thrasher AJ (2000) Wiskott-Aldrich syndrome
protein is necessary for efficient IgG-mediated phagocytosis. Blood 95:2943–2946
Lu CC, Wu TS, Hsu YJ, Chang CJ, Lin CS, Chia JH, Wu TL, Huang TT, Martel J, Ojcius DM,
Young JD, Lai HC (2014) NK cells kill mycobacteria directly by releasing perforin and
granulysin. J Leukoc Biol 96:1119–1129
Lu CL, Murakowski DK, Bournazos S, Schoofs T, Sarkar D, Halper-Stromberg A, Horwitz JA,
Nogueira L, Golijanin J, Gazumyan A, Ravetch JV, Caskey M, Chakraborty AK,
Nussenzweig MC (2016) Enhanced clearance of HIV-1-infected cells by broadly neutralizing
antibodies against HIV-1 in vivo. Science 352:1001–1004
Lu J, Marjon KD, Marnell LL, Wang R, Mold C, du Clos TW, Sun P (2011) Recognition and
functional activation of the human IgA receptor (FcalphaRI) by C-reactive protein. Proc Natl
Acad Sci U S A 108:4974–4979
Lu J, Marnell LL, Marjon KD, Mold C, du Clos TW, Sun PD (2008) Structural recognition and
functional activation of FccR by innate pentraxins. Nature 456:989–992
Lubinski JM, Lazear HM, Awasthi S, Wang F, Friedman HM (2011) The herpes simplex virus 1
IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent
cellular cytotoxicity in vivo. J Virol 85:3239–3249
Maglione PJ, Xu J, Casadevall A, Chan J (2008) Fc c receptors regulate immune activation and
susceptibility during Mycobacterium tuberculosis infection. J Immunol 180:3329–3338
Mallery DL, McEwan WA, Bidgood SR, Towers GJ, Johnson CM, James LC (2010) Antibodies
mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc Natl
Acad Sci U S A 107:19985–19990
Manz RA, Thiel A, Radbruch A (1997) Lifetime of plasma cells in the bone marrow. Nature
388:133–134
Marches O, Covarelli V, Dahan S, Cougoule C, Bhatta P, Frankel G, Caron E (2008) EspJ of
enteropathogenic and enterohaemorrhagic Escherichia coli inhibits opsono-phagocytosis. Cell
Microbiol 10:1104–1115
May RC, Caron E, Hall A, Machesky LM (2000) Involvement of the Arp2/3 complex in
phagocytosis mediated by FccR or CR3. Nat Cell Biol 2:246–248
Mazor Y, Yang C, Borrok MJ, Ayriss J, Aherne K, Wu H, Dall’Acqua WF (2016) Enhancement
of immune effector functions by modulating IgG’s intrinsic affinity for target antigen.
PLoS ONE 11:e0157788
McEwan WA, Hauler F, Williams CR, Bidgood SR, Mallery DL, Crowther RA, James LC (2012)
Regulation of virus neutralization and the persistent fraction by TRIM21. J Virol 86:8482–
8491
McEwan WA, Tam JC, Watkinson RE, Bidgood SR, Mallery DL, James LC (2013) Intracellular
antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat
Immunol 14:327–336
Memoli MJ, Shaw PA, Han A, Czajkowski L, Reed S, Athota R, Bristol T, Fargis S, Risos K,
Powers JH, Davey RT, Taubenberger JK (2016) Evaluation of antihemagglutinin and
antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy
human challenge model. MBio 7(2):e00416–e00417
Menager N, Foster G, Ugrinovic S, Uppington H, Verbeek S, Mastroeni P (2007) Fcc receptors are
crucial for the expression of acquired resistance to virulent Salmonella enterica serovar
Typhimurium in vivo but are not required for the induction of humoral or T-cell-mediated
immunity. Immunology 120:424–432
Moller E (1967) Cytotoxicity by nonimmune allogeneic lymphoid cells. Specific suppression by
antibody treatment of the lymphoid cells. J Exp Med 126:395–405
Moore T, Ananaba GA, Bolier J, Bowers S, Belay T, Eko FO, Igietseme JU (2002) Fc receptor
regulation of protective immunity against Chlamydia trachomatis. Immunology 105:213–221
Fc Receptors in Antimicrobial Protection 147

Moore T, Ekworomadu CO, Eko FO, Macmillan L, Ramey K, Ananaba GA, Patrickson JW,
Nagappan PR, Lyn D, Black CM, Igietseme JU (2003) Fc receptor-mediated antibody
regulation of T cell immunity against intracellular pathogens. J Infect Dis 188:617–624
Morton HC, van Egmond M, van de Winkel JG (1996) Structure and function of human IgA Fc
receptors (Fc alpha R). Crit Rev Immunol 16:423–440
Muta T, Kurosaki T, Misulovin Z, Sanchez M, Nussenzweig MC, Ravetch JV (1994) A
13-amino-acid motif in the cytoplasmic domain of Fc c RIIB modulates B-cell receptor
signalling. Nature 368:70–73
Naglak EK, Morrison SG, Morrison RP (2016). IFNc is required for optimal antibody-mediated
immunity against genital chlamydia infection. Infec Immun IAI-00749
Naglak EK, Morrison SG, Morrison RP (2017) Neutrophils are central to antibody-mediated
protection against Genital Chlamydia. Infect Immun 85
Nakamura K, Malykhin A, Coggeshall KM (2002) The Src homology 2 domain-containing
inositol 5-phosphatase negatively regulates Fcc receptor-mediated phagocytosis through
immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors. Blood
100:3374–3382
Nathan C, Cunningham-Bussel A (2013) Beyond oxidative stress: an immunologist’s guide to
reactive oxygen species. Nat Rev Immunol 13:349–361
Niederkorn JY, Shadduck JA (1980) Role of antibody and complement in the control of
Encephalitozoon cuniculi infections by rabbit macrophages. Infect Immun 27:995–1002
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin g subclass activity through
selective Fc receptor binding. Science 310:1510–1512
Ninomiya N, Hazeki K, Fukui Y, Seya T, Okada T, Hazeki O, Ui M (1994) Involvement of
phosphatidylinositol 3-kinase in Fc c receptor signaling. J Biol Chem 269:22732–22737
Nobakht E, Cohen SD, Rosenberg AZ, Kimmel PL (2016) HIV-associated immune complex
kidney disease. Nat Rev Nephrol 12:291–300
Odin JA, Edberg JC, Painter CJ, Kimberly RP, Unkeless JC (1991) Regulation of phagocytosis
and [Ca2+]i flux by distinct regions of an Fc receptor. Science 254:1785–1788
Okazawa H, Motegi S, Ohyama N, Ohnishi H, Tomizawa T, Kaneko Y, Oldenborg PA,
Ishikawa O, Matozaki T (2005) Negative regulation of phagocytosis in macrophages by the
CD47-SHPS-1 system. J Immunol 174:2004–2011
Oldenborg PA, Gresham HD, Lindberg FP (2001) CD47-signal regulatory protein alpha
(SIRPalpha) regulates Fcc and complement receptor-mediated phagocytosis. J Exp Med
193:855–862
Ono M, Bolland S, Tempst P, Ravetch JV (1996) Role of the inositol phosphatase SHIP in
negative regulation of the immune system by the receptor Fc(c)RIIB. Nature 383:263–266
Ono M, Okada H, Bolland S, Yanagi S, Kurosaki T, Ravetch JV (1997) Deletion of SHIP or
SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90:293–301
Otten MA, Rudolph E, Dechant M, Tuk CW, Reijmers RM, Beelen RH, van de Winkel JG, van
Egmond M (2005) Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc
receptors. J Immunol 174:5472–5480
Padigel UM, Farrell JP (2005) Control of infection with Leishmania major in susceptible BALB/c
mice lacking the common c-chain for FcR is associated with reduced production of IL-10 and
TGF-beta by parasitized cells. J Immunol 174:6340–6345
Park H, Cox D (2009) Cdc42 regulates Fc c receptor-mediated phagocytosis through the activation
and phosphorylation of Wiskott-Aldrich syndrome protein (WASP) and neural-WASP. Mol
Biol Cell 20:4500–4508
Pavlou S, Wang L, Xu H, Chen M (2017) Higher phagocytic activity of thioglycollate-elicited
peritoneal macrophages is related to metabolic status of the cells. J Inflamm (Lond) 14:4
Pearse RN, Kawabe T, Bolland S, Guinamard R, Kurosaki T, Ravetch JV (1999) SHIP recruitment
attenuates Fc c RIIB-induced B cell apoptosis. Immunity 10:753–760
Pegram HJ, Andrews DM, Smyth MJ, Darcy PK, Kershaw MH (2011) Activating and inhibitory
receptors of natural killer cells. Immunol Cell Biol 89:216–224
148 A. Wieland and R. Ahmed

Pignata C, Prasad KV, Robertson MJ, Levine H, Rudd CE, Ritz J (1993) Fc c RIIIA-mediated
signaling involves src-family lck in human natural killer cells. J Immunol 151:6794–6800
Pincetic A, Bournazos S, Dilillo DJ, Maamary J, Wang TT, Dahan R, Fiebiger BM, Ravetch JV
(2014) Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol
15:707–716
Plotkin SA (2010) Correlates of protection induced by vaccination. Clin Vaccine Immunol
17:1055–1065
Pollara J, Easterhoff D, Fouda GG (2017) Lessons learned from human HIV vaccine trials. Curr
Opin HIV AIDS 12:216–221
Pricop L, Redecha P, Teillaud JL, Frey J, Fridman WH, Sautes-Fridman C, Salmon JE (2001)
Differential modulation of stimulatory and inhibitory Fc c receptors on human monocytes by
Th1 and Th2 cytokines. J Immunol 166:531–537
Qin D, Wu J, Vora KA, Ravetch JV, Szakal AK, Manser T, Tew JG (2000) Fc c receptor IIB on
follicular dendritic cells regulates the B cell recall response. J Immunol 164:6268–6275
Rakebrandt N, Lentes S, Neumann H, James LC, Neumann-Staubitz P (2014) Antibody- and
TRIM21-dependent intracellular restriction of Salmonella enterica. Pathog Dis 72:131–137
Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S,
Bonnerot C, Ricciardi-Castagnoli P, Amigorena S (1999) Fcc receptor-mediated induction of
dendritic cell maturation and major histocompatibility complex class I-restricted antigen
presentation after immune complex internalization. J Exp Med 189:371–380
Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, Riganelli D, Zanaria E, Messali S,
Cainarca S, Guffanti A, Minucci S, Pelicci PG, Ballabio A (2001) The tripartite motif family
identifies cell compartments. EMBO J 20:2140–2151
Rodriguez ME, Hellwig SM, Hozbor DF, Leusen J, van der Pol WL, van de Winkel JG (2001) Fc
receptor-mediated immunity against Bordetella pertussis. J Immunol 167:6545–6551
Sanford JE, Lupan DM, Schlageter AM, Kozel TR (1990) Passive immunization against
Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive
with cryptococcal polysaccharide. Infect Immun 58:1919–1923
Schlageter AM, Kozel TR (1990) Opsonization of Cryptococcus neoformans by a family of
isotype-switch variant antibodies specific for the capsular polysaccharide. Infect Immun
58:1914–1918
Schlesinger JJ, Foltzer M, Chapman S (1993) The Fc portion of antibody to yellow fever virus
NS1 is a determinant of protection against YF encephalitis in mice. Virology 192:132–141
Slifka MK, Antia R, Whitmire JK, Ahmed R (1998) Humoral immunity due to long-lived plasma
cells. Immunity 8:363–372
Smith P, Dilillo DJ, Bournazos S, Li F, Ravetch JV (2012) Mouse model recapitulating human Fcc
receptor structural and functional diversity. Proc Natl Acad Sci U S A 109:6181–6186
Sondermann P, Pincetic A, Maamary J, Lammens K, Ravetch JV (2013) General mechanism for
modulating immunoglobulin effector function. Proc Natl Acad Sci U S A 110:9868–9872
Steevels TA, Meyaard L (2011) Immune inhibitory receptors: essential regulators of phagocyte
function. Eur J Immunol 41:575–587
Stone KD, Prussin C, Metcalfe DD (2010) IgE, mast cells, basophils, and eosinophils. J Allergy
Clin Immunol 125:S73–S80
Suh CI, Stull ND, Li XJ, Tian W, Price MO, Grinstein S, Yaffe MB, Atkinson S, Dinauer MC
(2006) The phosphoinositide-binding protein p40phox activates the NADPH oxidase during
FccIIA receptor-induced phagocytosis. J Exp Med 203:1915–1925
Swanson JA, Hoppe AD (2004) The coordination of signaling during Fc receptor-mediated
phagocytosis. J Leukoc Biol 76:1093–1103
te Velde AA, de Waal Malefijt, RENE, Huijbens RJ, De Vries, JE, Figdor CG (1992) IL-10
stimulates monocyte FccR surface expression and cytotoxic activity. Distinct regulation of
antibody-dependent cellular cytotoxicity by IFN-c, IL-4, and IL-10. J Immunol 149(12):4048–
4052
Fc Receptors in Antimicrobial Protection 149

Tecchio C, Micheletti A, Cassatella MA (2014) Neutrophil-derived cytokines: facts beyond

expression. Front Immunol 5:508
Theofilopoulos AN, Andrews BS, Urist MM, Morton DL, Dixon FJ (1977) The nature of immune
complexes in human cancer sera. J Immunol 119:657–663
Tian H, Weber S, Thorkildson P, Kozel TR, Pirofski LA (2009) Efficacy of opsonic and
nonopsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibodies
against intranasal challenge with Streptococcus pneumoniae in mice. Infect Immun 77:1502–
1513
Tobar JA, Gonzalez PA, Kalergis AM (2004) Salmonella escape from antigen presentation can be
overcome by targeting bacteria to Fc c receptors on dendritic cells. J Immunol 173:4058–4065
Tsai JF, Margolis HS, Jeng JE, Ho MS, Chang WY, Lin ZY, Tsai JH (1995) Circulating immune
complexes in chronic hepatitis related to hepatitis C and B viruses infection. Clin Immunol
Immunopathol 75:39–44
Uciechowski P, Schwarz M, Gessner JE, Schmidt RE, Resch K, Radeke HH (1998) IFN-c induces
the high-affinity Fc receptor I for IgG (CD64) on human glomerular mesangial cells. Eur J
Immunol 28:2928–2935
Varshney AK, Wang X, Aguilar JL, Scharff MD, Fries BC (2014) Isotype switching increases
efficacy of antibody protection against staphylococcal enterotoxin B-induced lethal shock and
Staphylococcus aureus sepsis in mice. MBio 5:e01007–e01014
Varshney AK, Wang X, Cook E, Dutta K, Scharff MD, Goger MJ, Fries BC (2011) Generation,
characterization, and epitope mapping of neutralizing and protective monoclonal antibodies
against staphylococcal enterotoxin B-induced lethal shock. J Biol Chem 286:9737–9747
Varshney AK, Wang X, Scharff MD, Macintyre J, Zollner RS, Kovalenko OV, Martinez LR,
Byrne FR, Fries BC (2013) Staphylococcal Enterotoxin B-specific monoclonal antibody 20B1
successfully treats diverse Staphylococcus aureus infections. J Infect Dis 208:2058–2066
Verma A, Ngundi MM, Meade BD, de Pascalis R, Elkins KL, Burns DL (2009) Analysis of the Fc
c receptor-dependent component of neutralization measured by anthrax toxin neutralization
assays. Clin Vaccine Immunol 16:1405–1412
Von Pawel-Rammingen U, Johansson BP, Bjorck L (2002) IdeS, a novel streptococcal cysteine
proteinase with unique specificity for immunoglobulin G. EMBO J 21:1607–1615
Voskoboinik I, Whisstock JC, Trapani JA (2015) Perforin and granzymes: function, dysfunction
and human pathology. Nat Rev Immunol 15:388–400
Wang TT, Maamary J, Tan GS, Bournazos S, Davis CW, Krammer F, Schlesinger SJ, Palese P,
Ahmed R, Ravetch JV (2015) Anti-HA glycoforms drive B cell affinity selection and determine
influenza vaccine efficacy. Cell 162:160–169
Weber S, Tian H, van Rooijen N, Pirofski LA (2012) A serotype 3 pneumococcal capsular
polysaccharide-specific monoclonal antibody requires Fcc receptor III and macrophages to
mediate protection against pneumococcal pneumonia in mice. Infect Immun 80:1314–1322
Weber SS, Ducry J, Oxenius A (2014) Dissecting the contribution of IgG subclasses in restricting
airway infection with Legionella pneumophila. J Immunol 193:4053–4059
Wieland A, Kamphorst AO, Valanparambil RM, Han JH, Xu X, Choudhury BP, Ahmed R (2018)
Enhancing FccR-mediated antibody effector function during persistent viral infection. Sci
Immunol 3(27):eaao3125
Wieland A, Shashidharamurthy R, Kamphorst AO, Han JH, Aubert RD, Choudhury BP,
Stowell SR, Lee J, Punkosdy GA, Shlomchik MJ, Selvaraj P, Ahmed R (2015) Antibody
effector functions mediated by Fcc-receptors are compromised during persistent viral infection.
Immunity 42:367–378
Woelbing F, Kostka SL, Moelle K, Belkaid Y, Sunderkoetter C, Verbeek S, Waisman A, Nigg AP,
Knop J, Udey MC, von Stebut E (2006) Uptake of Leishmania major by dendritic cells is
mediated by Fcc receptors and facilitates acquisition of protective immunity. J Exp Med
203:177–188
150 A. Wieland and R. Ahmed

Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, Morrissey M, McCausland M,

Skountzou I, Hornig M, Lipkin WI, Mehta A, Razavi B, del Rio C, Zheng NY, Lee JH,
Huang M, Ali Z, Kaur K, Andrews S, Amara RR, Wang Y, Das SR, O’Donnell CD,
Yewdell JW, Subbarao K, Marasco WA, Mulligan MJ, Compans R, Ahmed R, Wilson PC
(2011) Broadly cross-reactive antibodies dominate the human B cell response against 2009
pandemic H1N1 influenza virus infection. J Exp Med 208:181–193
Yamada DH, Elsaesser H, Lux A, Timmerman JM, Morrison SL, de la Torre JC, Nimmerjahn F,
Brooks DG (2015) Suppression of Fcc-receptor-mediated antibody effector function during
persistent viral infection. Immunity 42:379–390
Yuan R, Clynes R, Oh J, Ravetch JV, Scharff MD (1998) Antibody-mediated modulation of
Cryptococcus neoformans infection is dependent on distinct Fc receptor functions and IgG
subclasses. J Exp Med 187:641–648