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Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
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Published in final edited form as:

Nat Rev Immunol. 2024 May ; 24(5): 338–357. doi:10.1038/s41577-023-00970-x.

Therapeutic induction of antigen-specific immune tolerance

Jessica E. Kenison1, Nikolas A. Stevens1, Francisco J. Quintana1,2,✉
1AnnRomney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical
School, Boston, MA, USA.
2The Broad Institute of Harvard and MIT, Cambridge, MA, USA.
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Abstract
The development of therapeutic approaches for the induction of robust, long-lasting and antigen-
specific immune tolerance remains an important unmet clinical need for the management
of autoimmunity, allergy, organ transplantation and gene therapy. Recent breakthroughs in
our understanding of immune tolerance mechanisms have opened new research avenues and
therapeutic opportunities in this area. Here, we review mechanisms of immune tolerance and novel
methods for its therapeutic induction.

Introduction
Immune system activation is vital to the control of pathogens and cancer, but regulatory
mechanisms are needed to prevent immunopathology resulting from excessive immune
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activity. Perturbations of this balance result in infections, cancer, inflammatory diseases

or allergy. Indeed, autoimmune diseases affect as much as 5–10% of the population and
are on the rise1. Similarly, inefficacious immune modulation results in graft rejection and
graft-versus-host disease (GVHD) in 20–70% of transplant recipients, and pre-existing
immunity to viral vectors limits gene therapy efficacy. The development of antigen-specific
immunotherapies is an important unmet clinical need.

Key advances have been made in our understanding of immune tolerance and its regulation.
Indeed, new technologies for antigen discovery, drug delivery and cell targeting have opened
new avenues for the development of therapies for the induction of antigen-specific tolerance.
Here we review mechanisms of immune tolerance and discuss strategies for its therapeutic
modulation.
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Mechanisms of immune tolerance

Immune tolerance is an active state of unresponsiveness towards a specific antigen, which
involves both innate and adaptive immune cells. The breakdown of self-tolerance can result

✉
fquintana@[Link] .
Author contributions
J.E.K. and N.A.S. contributed equally to all aspects of the article. All authors contributed to the writing and editing of the manuscript.
Competing interests
F.J.Q. is the Scientific Founder of AnTolRx, a company developing novel therapies for inflammatory disorders. The other authors have
no competing interests.
 Kenison et al. Page 2

in the development of autoimmune disorders, whereas dysregulated immune responses to

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foreign antigens may lead to hypersensitivity and allergic disease. Thus it is important to
define the multiple mechanisms involved in its establishment and maintenance.

Central tolerance
Central tolerance is established during T and B cell development in the thymus and bone
marrow, respectively. Bone marrow-derived CD34+ T cell progenitors home to the thymus,
where they acquire T cell receptor (TCR) expression. Random V(D)J rearrangements
generate a diverse TCR repertoire that is reactive against a wide array of antigens. T
cells harbouring TCRs that do not recognize MHC-presented self-peptides die by neglect,
whereas those with low affinity for peptide–MHC complexes differentiate into CD4+ or
CD8+ single-positive T cells. The randomness of V(D)J rearrangements inevitably generates
some TCR clones with high affinity for self-antigen–MHC complexes. High-affinity TCR
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clones are controlled by various mechanisms of central tolerance including clonal deletion
and receptor editing. Some self-reactive T cells escape deletion and leave the thymus but
show functional impairment and/or expression of molecules associated with tolerance2,
whereas others develop into self-reactive thymus-differentiated regulatory T cells (tTreg
cells), which migrate to peripheral lymphoid and nonlymphoid tissues3.

Self-antigen–MHC complexes are expressed by thymic antigen-presenting cells (APCs)

including specialized medullary thymic epithelial cells (mTECs), dendritic cells (DCs) and
B cells. The transcriptional factor autoimmune regulator (AIRE) promotes the expression
of peripheral tissue antigens by mTECs4; mutations in AIRE are linked to autoimmune
pathology. However not all tissue-specific antigens expressed by mTECs are controlled by
AIRE. Indeed recent studies identified mTECs that express transcription factors such as
FEZF2 (ref. 5) or that co-opt lineage-defining transcription factors from peripheral cell
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types, termed mimetic cells6. These AIRE+, FEZF2+ and mimetic mTECs collaborate with
thymic B cells and DCs to promote central tolerance through clonal T cell deletion and
Treg cell induction. This process is further aided by the transfer of tissue-specific antigens
from mTECs to DCs through a process termed cooperative antigen transfer7. Of note, it
was recently reported that intestinal DCs travel to the thymus to present microbiota-derived
antigens, highlighting the contribution of peripheral DCs to central tolerance8.

In the bone marrow, developing B cells acquire the expression of a B cell antigen
receptor (BCR) that randomly rearranges its V, D and J gene regions to generate a diverse
BCR repertoire. Up to 75% of early immature B cells are self-reactive9, but a third of
them undergo immunoglobulin gene rearrangements that reduce autoantigen reactivity10.
Additional self-reactive B cells are removed by clonal deletion11. However central tolerance
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does not eliminate all self-reactive clones, for example those reactive to developmentally
restricted or inducible antigens that are not expressed by the thymus or the bone marrow.
Thus, self-reactive lymphocytes escape central tolerance and are actively controlled by
peripheral tolerance mechanisms.

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Peripheral tolerance
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About 25–40% of self-reactive T cells12 and approximately 40% of autoreactive B cells9

escape central tolerance. Thus peripheral tolerance mechanisms, including anergy, deletion
and suppression by Treg cells, are crucial for the prevention of autoimmune diseases or
hypersensitivity to antigens first encountered outside the thymus or bone marrow, including
food allergens or antigens displayed during infection or pregnancy.

Three signals are required for T cell activation. Signal 1 involves the interaction of the TCR
with peptide–MHC molecules. Signal 2 involves the binding of co-stimulatory receptors to
their ligands on APCs, most commonly CD28 on T cells and CD80 or CD86 on APCs, but
also other co-stimulatory molecules, including inducible T cell co-stimulator (ICOS) and
CD40 (ref. 13). Signal 3 involves the activation of cytokine receptors. The activation of
TCR signalling (signal 1) in the absence of co-stimulation (signal 2), or strong pre-exposure
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to cytokines (signal 3) before signals 1 and 2, induces T cell anergy, a state in which
the T cell is functionally inactivated, incapable of proliferating or producing IL-2 (ref.
14). T cell anergy can also be induced by repeated antigen stimulation15, exposure to
anti-inflammatory cytokines such as IL-10 (ref. 16), or signalling via co-inhibitory receptors
such as programmed cell death 1 (PD1) and cytotoxic T lymphocyte associated protein 4
(CTLA4)17. Similarly, B cells require BCR engagement concomitant with Toll-like receptor
(TLR) signalling or interactions with T helper cells to be fully activated. High avidity
BCR interactions with antigens in the absence of TLRs or T helper cell co-stimulation
induce clonal deletion or anergy, inhibiting B cell proliferation and differentiation into
antibody-secreting cells and overall shortening B cell lifespan18.

Long-term T cell anergy is associated with epigenetic modifications that render cells more
sensitive to inhibitory signals19, while altering gene and surface marker expression and
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inducing functional changes similar to those observed in exhausted T cells induced during
chronic infection or cancer15. However T and B cell anergy is a dynamic process, and the
removal of antigen exposure can restore T or B cell functionality15,20. Furthermore, a subset
of naturally occurring anergic T cells expressing CD73 and FR4, capable of differentiating
into functional FOXP3+ Treg cells and FOXP3−IL-10+ type 1 regulatory T (TR1) cells, has
been described21,22, although it is not clear whether this process involves specific APC types
or anatomical niches.

The peripheral deletion of T and B cells through apoptosis also controls self-reactive cells.
Intrinsic T cell apoptosis largely depends on the pro-apoptotic protein BIM, upregulated
during T cell deletion, which inhibits the anti-apoptotic proteins BCL-2 and BCL-xL,
activating pro-apoptotic BAX and BAK to permeabilize the mitochondrial membrane23,24.
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Extrinsic T cell apoptosis involves FAS25 or tumour necrosis factor (TNF) receptor26
signalling, which ultimately triggers caspase activation to induce apoptosis. Signalling
through these death receptors limits self-reactive pathogenic T cell and B cell responses.
For example, central nervous system (CNS)-resident astrocytes expressing the TNF receptor
ligand TRAIL induce T cell apoptosis and limit autoimmune neuroinflammation27. Other
forms of peripheral immune cell death (necroptosis, ferroptosis and pyroptosis) also
contribute to peripheral immune tolerance28-30.

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The mechanisms determining whether self-reactive T or B cells undergo anergy versus cell
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death following TCR or BCR activation without co-stimulation are still not fully understood.
Antigen levels have been postulated to control cell fate, with higher levels triggering anergy
and lower levels triggering cell death31. In addition, checkpoint molecule signalling (for
example, through PD1, TIGIT, TIM3, LAG3 and VISTA) can induce T cell death or
dysfunction32-34.

Finally Treg cells play central roles in peripheral tolerance. Major Treg cell subtypes include
FOXP3+ cells and IL-10-producing FOXP3− TR1 cells, but additional subsets have been
linked to immune tolerance, including CD8+ Treg cells35, regulatory γδ T cells36 and
regulatory invariant natural killer T cells (iNKT cells)37.

FOXP3+ Treg cells differentiate in the thymus (FOXP3+ tTreg cells) in response to self-
antigen expression38 and then migrate to peripheral lymphoid and nonlymphoid tissues
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to limit pathogenic autoreactivity and promote tissue repair39. Some FOXP3+ Treg cells
differentiate from naive CD4+ T cells in the periphery (FOXP3+ pTreg cells), enforcing
tolerance to antigens not expressed in the thymus, including food antigens, allergens,
microbial antigens or pregnancy-linked fetal antigens40. In addition, tissue-resident Treg
cells in the skin41, muscle42, visceral adipose tissue43,44 and mucosal tissues, such
as intestine45,46 and lungs39, display specialized phenotypes and functions, as recently
reviewed47,48.

TR1 cells are IL-10+FOXP3−CD4+ T cells that were initially described following chronic
stimulation in the presence of IL-10 (ref. 49). IL-27 was later found to be a stronger TR1
cell differentiation inducer50, with IFNα51, hyaluronic acid52, ICOSL53, CD2 (ref. 54) and
CD55 (ref. 55) expression on APCs also displaying important roles (see Box 1). FOXP3+
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pTreg and TR1 cell differentiation and function are modulated by host and microbial
metabolites, such as aryl hydrocarbon receptor (AHR) agonists56. TR1 cells produce IL-10
and transforming growth factor-β (TGFβ), as well as perforin and granzyme B, which can
kill APCs57,58. TR1 cells also express the inhibitory molecules CTLA4 and PD1, enabling
contact-dependent T cell suppression, and CD39 (ref. 59), which degrades pro-inflammatory
extracellular ATP while promoting the production of anti-inflammatory adenosine.

Multiple cell types participate in central and peripheral immune tolerance. DCs play a
central role because they process and present antigen, while providing cytokines and
stimulatory or inhibitory molecules to modulate T cell differentiation or trigger anergy or
deletion. Thus, DCs are frequently targeted for the therapeutic induction of antigen-specific
immune tolerance.
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DCs as the central mediators of immune tolerance

DC subsets and their functions
DCs display phenotypic and functional heterogeneity60,61. DCs are classified into
plasmacytoid DCs (pDCs), classical (or conventional) type 1 DCs (cDC1s) and type 2
DCs (cDC2s). In addition, monocyte-derived DCs (moDCs), sometimes called TipDCs
(TNF-producing and iNOS-producing DCs), adopt a DC-like phenotype under inflammatory

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conditions62, although recent works call into question their ability to migrate to lymph
nodes and prime CD4+ and CD8+ T cells63. A DC3 subtype displaying cDC2 and moDC
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features was also identified in humans64. Additional heterogeneity within DC subsets has
been described. For example, cDC2s are classified into cDC2As and cDC2Bs controlled by
the transcription factors T-bet and RORγt, respectively65. In addition CD103 and CD11b
distinguish functional cDC subsets in mucosal tissues66.

pDCs are primarily located in the blood and lymphoid tissues but migrate to nonlymphoid
tissues during inflammation67. When activated, mainly via TLR7 or TLR9 signalling, pDCs
produce large amounts of type I interferons, including IFNα and IFNβ68. Under homeostatic
conditions, pDCs are poor activators of naive CD4+ and CD8+ T cells. However, a
subpopulation of pDCs stimulates CD4+ T helper 1 (TH1) cells during infection69. pDCs
also promote tolerance and Treg cell induction via the expression of ICOSL70, TGFβ71 and
inhibitory indoleamine 2,3-dioxygenase (IDO)72. Indeed recent findings suggest that pDC
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deficits contribute to GVHD following organ transplantation73 and that pDCs contribute to
oral tolerance induction74.

cDCs are present in both lymphoid and nonlymphoid tissues at the steady state. cDC1 and
cDC2 distribution varies in different tissues, and although both subsets migrate between
tissues and lymph nodes, cDC2s appear to have a higher migratory potential and are
enriched at mucosal-associated sites such as the lungs and intestine75. Of note, at the steady
state cDC1s, cDC2s and pDCs are detected in the CNS choroid plexus and meninges,
but they are virtually undetectable in the brain parenchyma and perivascular space76,77.
Indeed, cDC1s are the primary subtype present in the choroid plexus, whereas cDC2s
are most abundant in the leptomeninges and dura mater76,77. Under inflammation cDC1s,
cDC2s, moDCs and pDCs infiltrate the brain parenchyma and present CNS-specific antigens
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to T cells76-78. Although both cDC1s and cDC2s can present antigen to either CD4+ or
CD8+ T cells, cDC1s are better at antigen cross-presentation79 and type III interferon
production80. Within the cDC2 subset, cDC2As appear to be less pro-inflammatory than
cDC2Bs, expressing higher levels of amphiregulin and matrix metalloproteinase 9, whereas
cDC2Bs produce higher levels of TNF and IL-6 (ref. 65). Of note, cDC2s in the intestine
have been shown to promote T helper 17 (TH17) cell differentiation81,82. However, both
cDC1s and cDC2s are reported to promote the differentiation of FOXP3+ Treg cells and
IL-10+ TR1 cells83,84.

Tolerogenic DC phenotype
Activation and maturation states dictate the effects of DCs on the immune response.
Before their activation via pattern recognition receptors (PRRs), DCs reside at mucosal
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sites, lymphoid and peripheral tissues or in the blood in an immature state. Activation
by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular
patterns (DAMPs) upregulates DC expression of MHC class I and II, co-stimulatory and
adhesion molecules such as CC-chemokine receptor 7 (CCR7). These mature DCs migrate
to lymphoid tissues to promote effector T cell differentiation. Immature DCs, conversely,
exhibit low expression of MHC class I, MHC class II and co-stimulatory molecules
and are capable of inducing T cell anergy, Treg cell differentiation and effector T cell

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deletion85. It was originally postulated that tolerogenic DCs were essentially immature
DCs, but this paradigm was challenged early on86. It has since been proposed that specific
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stimuli can induce a tolerogenic DC phenotype87 and that tolerogenic DCs undergo some
level of maturation and/or activation88. Indeed, specific transcriptional programmes in
DCs drive immunogenic versus tolerogenic states87,89. For example, β-catenin signalling90
or phagocytosis of apoptotic material91 under steady-state conditions activate tolerogenic
programmes in DCs, which migrate to lymph nodes to present self-antigens and maintain
peripheral tolerance. Moreover, a tolerogenic DC phenotype can also be induced in
semimature and mature DCs92. For example, an IL-10+ DC-10 subtype was identified
in human peripheral blood and the spleen, displaying cDC and moDC surface markers
but capable of inducing CD4+ T cell hyporesponsiveness and TR1 cell expansion93 (see
Box 1); DC-10s can be induced in vitro by monocyte differentiation in the presence
of IL-10. In addition, intestinal CD103+ DCs contribute to tolerance to dietary antigens
and the induction of oral tolerance94,95. Regardless of their origin and maturation state,
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DCs contribute to immune regulation via multiple mechanisms, including co-stimulatory

molecule downregulation (CD80, CD86 and CD40), inhibitory molecule expression (PD-L1,
ICOSL and BTLA), suppression of pro-inflammatory cytokine production (IL-6, IL-12,
IL-23 and TNF) and production of anti-inflammatory cytokines (IL-10, TGFβ and IL-27)
and metabolites (IDO, retinoic acid and lactate) (Box 1 and Fig. 1).

Numerous stimuli induce a tolerogenic DC phenotype. For example, IL-10 reduces

DC expression of MHC and co-stimulatory molecules, decreases pro-inflammatory
cytokine production and promotes T cell anergy and Treg cell expansion96,97. These anti-
inflammatory effects of IL-10 on DCs are AHR dependent98, recapitulating previous reports
of the tolerogenic effects of AHR signalling in DCs99-105. Additional cytokines such as
TGFβ106, IL-27 (ref. 107) and IL-37 (ref. 108) also promote an anti-inflammatory DC
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phenotype. Similarly the exposure of monocytes or bone marrow cells to low concentrations
of granulocyte-monocyte colony-stimulating factor (GM-CSF) induces the differentiation of
DCs with a tolerogenic phenotype, whereas exposure to higher GM-CSF doses induces a
pro-inflammatory DC phenotype109,110. Moreover, commensal bacteria signalling through
certain PRRs such as TLR2 (ref. 111) promotes tolerogenic DC induction. Indeed, some
microbial metabolites induce tolerogenic DCs, for example via AHR activation99,100.
Indeed, AHR agonists inhibit nuclear factor-κB (NF-κB) activation in DCs and drive
the expression of IL-10 and IDO, while reducing the expression of MHC molecules,
co-stimulatory molecules and pro-inflammatory cytokines such as IL-6 and IL-12. These
changes in DCs result in increased FOXP3+ and IL-10+ Treg cells and the suppression of
TH1, TH17 and CD8+ effector T cells101-104.
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Additional inducers of a tolerogenic DC phenotype include vitamin A, which is metabolized

into retinoic acid, a booster of FOXP3+ Treg cell induction112 and vitamin D3 that increases
IL-10 production while decreasing IL-12 and co-stimulatory molecule expression113,114.
Moreover, lactate, produced by microbiota, activated DCs or other immune cells, regulates
DC function via a hypoxia inducible factor 1α (HIF1α)-driven increase in the expression of
NADH dehydrogenase NDUFA4L2 that ultimately limits effector T cell activation115.

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Finally the uptake of apoptotic cells induces a tolerogenic DC phenotype via mechanisms
involving AHR activation116, prostaglandin E2 production117 and signalling via scavenger
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receptors such as MARCO118. Indeed, both cDCs and pDCs express IL-10, reduce co-
stimulatory molecule expression and promote Treg cell expansion following apoptotic cell
uptake91.

These and other pathways linked to the tolerogenic DC phenotype offer opportunities for the
development of therapeutic immunomodulatory strategies, as discussed below.

Antigen-specific therapeutic strategies to induce immune tolerance

Current therapies for autoimmune diseases, transplant rejection and other pathologies driven
by dysregulated immune responses are mostly based on untargeted immunosuppression
and consequently are linked to significant side effects. Thus novel approaches to induce
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antigen-specific immune tolerance are needed, targeting improperly activated T cells but
not interfering with protective immunity to pathogens and cancer. Consequently, numerous
technologies have been developed to induce antigen-specific tolerance (Fig. 2 and Table
1). In the next section, we discuss strategies for the induction of antigen-specific immune
tolerance in autoimmunity, organ transplantation and gene therapy (Fig. 3).

Cell-based tolerogenic therapies

The identification of stimuli inducing a tolerogenic phenotype in DCs guided cellular
therapeutic approaches, commonly based on DCs generated ex vivo from peripheral blood-
derived monocytes and loaded with disease-relevant antigens. However, there is not yet
a standardized method to generate tolerogenic DCs ex vivo, and multiple protocols and
tolerogenic molecules have been explored. For example, moDCs differentiated in vitro in
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the presence of low GM-CSF concentrations, termed autologous tolerogenic DCs (ATDCs),
display an immature phenotype with a low expression of MHC class II, CD80, CD86 and
CD40 and high IL-10 and lactate production119. ATDCs were well tolerated in a phase I/IIA
clinical trial to prevent graft rejection following kidney transplantation, and additional trials
are needed to evaluate their clinical efficacy120,121. Similarly, IL-10-induced DC-10s loaded
with disease-specific antigens induce antigen-specific immune tolerance122; their clinical
efficacy remains to be evaluated.

Vitamin D3 also induces a tolerogenic DC phenotype ex vivo113,114. Autologous vitamin

D3-treated tolerogenic DCs loaded with disease-specific antigens have been tested in phase
I clinical trials, including studies focused on type 1 diabetes (T1D)123,124 and multiple
sclerosis (MS)125 (Table 2). Moreover, moDCs differentiated in the presence of vitamin D3
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and IL-10 were shown to be tolerogenic and induce IL-10-producing T cells in a nonhuman
primate alloimmune reactivity model126. Similarly, moDCs treated with dexamethasone
display a tolerogenic phenotype characterized by high IL-10 and TGFβ secretion and
low pro-inflammatory cytokine production127,128. Dexamethasone-induced tolerogenic DCs
loaded with disease-specific peptides were well tolerated in phase I clinical trials in RA, MS
and neuromyelitis optica129,130. Moreover, tolerogenic DCs induced with dexamethasone
and vitamin A were tested in a phase I trial in Crohn’s disease131.

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Alternatively, lymphocytes and red blood cells coupled with antigens ex vivo have been used
to induce antigen-specific tolerance132,133. This approach is thought to induce tolerance
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as a result of the apoptosis of the antigen-coupled cells and their subsequent uptake
by APCs, which acquire a tolerogenic phenotype following apoptotic cell uptake134. For
example, in a study by Watkins et al. antigen-conjugated erythrocytes were taken up by
BATF3+ cDC1s, inducing antigen-specific T cell dysfunction via PD1, CTLA4, LAG3 and
TOX expression135. Building on these findings, Raposo et al. developed a microfluidic
loading technique to produce antigen-loaded erythrocytes, which reduce effector T cell
trafficking into target organs136. In addition, antigen-loaded erythrocytes induced bystander
tolerance136, inhibiting effector T cell responses against the antigen loaded in erythrocytes
and also other antigens expressed in the same tissue. Bystander tolerance induction is critical
to the success of antigen-specific immunotherapies because multiple antigens, many of them
unknown, are targeted in most autoimmune disorders and different antigens may be targeted
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in different patients.

Because of their ability to traffic to inflamed tissues, suppress pathogenic T cells and
promote tissue repair39, multiple tolerance-inducing approaches rely on FOXP3+ Treg
cells or TR1 cells. Indeed, more than 25 clinical trials have tested Treg cell-based
therapies in T1D, systemic lupus erythematosus, Crohn’s disease, organ transplantation
and GVHD120,137-140 (Table 2). These therapies usually involve autologous polyclonal Treg
cells isolated from peripheral blood and expanded ex vivo in the presence of IL-2 (ref.
141). Treg cell therapies are well tolerated and Treg cells are stable in vivo. Indeed, in
one clinical trial, 25% of ex vivo-expanded autologous polyclonal Treg cells could still be
detected 1 year after transfer into patients, pointing to a surprisingly long half-life for these
cells141. However, although several studies provide early indications of clinical efficacy
of Treg cell therapies in phase I and I/II trials, larger clinical trials are still needed140.
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Moreover, concerns regarding nonspecific immunosuppression led to the development of

antigen-specific Treg cell therapies.

A further development of Treg cell-based approaches has been the engineering of

Treg cells with chimeric antigen receptors (CARs). CAR Treg cells were reported to
ameliorate GVHD142 and other immune-mediated disorders143, and myelin oligodendrocyte
glycoprotein (MOG)-targeting CAR Treg cells homed to the CNS in a mouse model of
MS144. Treg cells engineered to target pro-inflammatory molecules such as TNF recently
showed promising results in a mouse model of GVHD and may be useful when the
pathology-driving antigens are not well known or where many antigens are targeted145.
Similarly, CAR Treg cells targeting B cells suppress antibody responses in a mouse model of
haemophilia A146, pointing to the versatility of engineered T cell therapies. Importantly,
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CAR Treg cells have been shown to remain tolerogenic in highly pro-inflammatory
environments, alleviating concerns about their potential conversion into pathogenic effector
T cells147. CAR Treg cells were also shown to induce bystander tolerance147.

The widespread use of cell-based strategies to induce antigen-specific tolerance faces

important challenges, particularly related to their patient-specific production in a clinical
setup. Strategies based on gene-edited stem cells may overcome some of these challenges

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by enabling the production of off-the-shelf universal cell lines for tolerance induction in
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multiple individuals.

Synthetic particle-based delivery systems

An exciting approach for antigen-specific immunomodulation is the use of nanoparticles.
Nanoparticles offer an attractive platform for antigen-specific tolerance induction as they
do not rely on patient-derived cells, are made with safe biodegradable materials and can be
produced at large scale with little batch-to-batch variation. In addition, nanoparticles can
be targeted to specific cells of interest and deliver multiple cargos, while improving small-
molecule and antigen solubility and bioavailability. Numerous types of nanoparticles have
been used for immunomodulation, including metallic, polymeric, lipid-based and peptide–
polymer particles, each with its own advantages and limitations (Fig. 2).

Metallic nanoparticles, including gold, silver and iron oxide particles, have been used for
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simultaneous diagnostic and therapeutic purposes, for example as contrast-enhancing agents

and for the delivery of surface-conjugated cargo148. Interestingly, iron oxide nanoparticles
conjugated to MHC class II-bound peptides induce TR1 cells, which in turn induce
regulatory B cells and limit inflammation in numerous preclinical mouse models149. In
this case, TR1 cell induction depends on the high density of MHC molecules in the
nanoparticles, which induces TCR microclusters devoid of co-stimulatory molecules on
antigen-specific CD4+ T cells149,150. In addition, regulatory B and T cells in the liver
induce immunosuppressive neutrophils, limiting liver autoimmunity and fibrosis151. Metallic
nanoparticles can be modified to improve their performance, but the resulting particles
may be unstable. Indeed, surface conjugation can make metallic nanoparticles prone to
aggregation during production, limiting the type of loadable cargo and interfering with
scale-up efforts152. In addition, metal particles are not easily biodegradable and their
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accumulation in tissues may trigger adverse effects.

Conversely, polymeric particles made from carbohydrate acids, such as poly(lactic acid)
(PLA) and poly(lactic-co-glycolic acid) (PLGA) nanoparticles, are easily modifiable,
relatively simple to manufacture and quickly degraded, although some by-products
induce adverse effects153. Polymeric particles delivering disease-specific antigens showed
therapeutic effects in preclinical autoimmune disease models of MS, rheumatoid arthritis
(RA) and T1D mediated by the induction of CTLA4+PD1+ Treg cells, the reduction of
effector T cells and decreased expression of IL-12, microRNA-155 and vascular endothelial
growth factor154-158. Moreover, phase I and phase IIa clinical trials in coeliac disease
showed that PLGA particles encapsulating a gliadin antigen were well tolerated and reduced
gliadin-specific IFNγ production and effector memory T cells159. However, additional
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trials are needed to fully evaluate their therapeutic effects. Of note, PLGA particles have
shown context-specific anti-inflammatory and pro-inflammatory effects independent of their
cargo. Indeed, one of the primary degradation products of PLA and PLGA particles is L-
lactate, which inhibits DC maturation and pro-inflammatory responses via HIF1α activation
and NF-κB inhibition115,160. Conversely, PLGA particles can activate the NBD, LRR
and pyrin domain-containing protein 3 (NLRP3) inflammasome in DCs161 and polarize
macrophages towards a pro-inflammatory phenotype162. PLGA particles are also reported

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to induce effector CD8+ T cell activation and IFNγ production163 and also act as TH2 cell
adjuvants164.
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Lipid-based nanoparticles are widely used in cosmetics165, as well as US Food and

Drug Administration (FDA)-approved cancer treatments166 and mRNA coronavirus
vaccines167,168. Depending on the production method and formulation physicochemical
properties, lipid nanoparticles can be classified into various categories, including liposomes,
lipid nanoparticles and cubosomes. Owing to the amphipathic nature of fatty acids,
lipid nanoparticles can carry hydrophobic molecules intercalated in the membrane and
hydrophilic substances in an aqueous core or conjugated to the surface. Furthermore, lipids
can be engineered to be easily degraded169. Moreover, the incorporation of lipids such
as dioleoylphosphatidylethanolamine or cholesterol can modulate the fusogenic properties
of nanoliposomes to improve endosomal drug release170. Indeed, intracellular cholesterol
accumulation can induce DC tolerance via liver X receptor activation171. Lipid nanoparticles
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have been successfully used to deliver autoantigens, with therapeutic effects in numerous
preclinical models of T1D, MS, RA and myasthenia gravis linked to the induction of
tolerogenic DCs, Treg cell expansion and suppression of pathogenic effector T cells172-175.
Moreover, in a phase Ib clinical trial in patients with RA, liposomes co-encapsulating
a collagen peptide and an NF-κB inhibitor were well tolerated, inducing an increase in
circulating collagen-specific PD1+ T cells and a decrease in disease activity176.

Protein-based nanoparticles offer a biodegradable, nontoxic and stable delivery platform but
are rarely used for antigen-specific tolerance induction because of their highly immunogenic
nature associated with their structural similarities to virus particles177.

The physicochemical characteristics of nanoparticles, including size, charge, structure,

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hydrophobicity and rigidity influence their immunomodulatory effects and can be modified
to alter nanoparticle circulation, cell targeting and uptake, and immunomodulatory function
to maximize therapeutic activity153,178,179. In general, nanoparticle surface charge is an
important determinant of cellular uptake and immunomodulation. Nanoparticles with a
negative surface charge have been proposed to mimic tolerogenic apoptotic cells180,181 and
be preferentially taken up by phagocytic cells via scavenger receptors such as MARCO in
macrophages182. Conversely, positively charged nanoparticles are thought to interact directly
with negatively charged cell membranes and thus be taken up more rapidly by a wider
variety of cell types183, although this property is also linked to an increased potential to
disrupt lipid bilayers and cause cytotoxicity184. Positively charged nanoparticles can also
promote inflammation via CD80 and CD86 upregulation and the production of reactive
oxygen species185,186. However, widespread consensus about the effects of particle charge
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on uptake, toxicity and inflammation is still lacking.

Particle size also influences particle biodistribution, targeting, uptake and toxicity. In
general, particles of <200 nm are taken up by DCs and >500 nm by macrophages187,188.
Indeed, it was suggested that the size of antigens can dictate immune responses, promoting
TH1, TH2 or Treg cell induction189. Moreover, particle size and rigidity affect the immune
response, skewing DCs and macrophages towards pro-inflammatory or anti-inflammatory
phenotypes190,191. Polyethylene glycol is commonly used as a shielding agent to reduce

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interactions with serum proteins, decrease uptake by the reticuloendothelial system and
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increase circulation time and bioavailability. The attachment of polyethylene glycol chains
to a protein may also be critical for subcutaneous uptake, reducing complement activation
and granulocyte recruitment192. Finally, it is important to consider that manufacturing
processes used in basic research often differ from those used in FDA-approved therapies.
Consequently, charge, size and other features may be altered during nanoparticle production
scale up for clinical testing, affecting immunomodulatory activity.

Targeting of specific cell types

Most untargeted nanoparticles are taken up by DCs and macrophages via scavenger
receptors and complement factor binding. This passive targeting of DCs generally results
in the presentation of nanoparticle-delivered antigens on MHC class II molecules193. CD4+
T cell recognition of MHC class II–presented antigens in the absence of co-stimulatory
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molecules induces clonal T cell deletion and inhibition via PD-L1 and induction of FOXP3+
and IL-10+ Treg cells182,194.

Nanoparticles can also be targeted to specific cell types using antibodies or other molecules
reactive with specific cell populations (Table 1). For example, mannosylated antigens
target the mannose receptor in DCs, inducing IL-10 production and antigen-specific
tolerance195,196. Mannosylated liposomes encapsulating myelin peptide antigens reduced
pro-inflammatory cytokines in blood in a phase I clinical trial in patients with MS197, but
their therapeutic value is still unknown.

An alternative approach is to target nanoparticles based on the antigen specificity of

immune receptors in the cells they aim to modulate. For example, metallic nanoparticles
displaying peptides loaded in recombinant MHC class I molecules in the absence of
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signals 2 and 3 induce antigen-specific CD8+ effector T cell anergy and a memory-like
regulatory phenotype, which inhibits DCs via IFNγ, IDO and perforin198. Thus, targeting
nanoparticles to specific immune cells, defined by their surface molecule expression or
antigenic reactivity, is an attractive approach for targeted immunotherapy. However, the
incorporation of additional components to the therapeutic nanoparticles (for example,
surface antibodies) may interfere with their manufacturing.

Introducing immunosuppressive agents into nanoparticles

A major risk of immunomodulation is the potential exacerbation of pathogenic immune
responses. Indeed, adverse effects ranging from local reactions to anaphylactic shock and
lethality have been documented while testing immunomodulatory approaches199; clinical
trials have been interrupted because of the induction of hypersensitivity reactions200
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and autoimmune disease relapses201. These adverse reactions suggest that safe antigen-
specific immunomodulation requires the activation of tolerogenic pathways. This concept
is exemplified by a recent report on the evaluation of antigen–MHC class II complexes,
which triggered inflammation in one-third of treated mice; this pro-inflammatory effect
was abrogated by attaching dexamethasone to the antigen–MHC class II complex at doses
200-fold lower than those used in dexamethasone-alone treatment schemes193. Interestingly,
self-antigen administration using nanoparticles and nanoliposomes does not seem to trigger

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 Kenison et al. Page 12

or boost pro-inflammatory responses102-104,202, suggesting that intrinsic properties make

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some platforms safer for clinical use. However, therapeutic tolerance induction in the clinic
will probably require the activation of anti-inflammatory pathways to improve both safety
and efficacy.

One of the first attempts to combine autoantigens and immunomodulatory drugs used
liposomes to co-deliver an antigen and an NF-κB inhibitor, ameliorating experimental
arthritis in a FOXP3+ Treg cell-dependent manner203. Similarly, based on the role of
AHR in the suppression of NF-κB signalling and the control of adaptive and innate
immunity204, nanoparticles engineered to co-deliver the AHR agonist 2-(1′H-indole-3′-
carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) with disease-relevant antigens
re-established antigen-specific tolerance in preclinical models of MS and T1D102-104.
Other immunomodulatory agents co-encapsulated with antigens include IL-10 (ref. 205),
vitamin D3 (ref. 206) and the mTOR inhibitor rapamycin202,207-209, with encouraging
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results in experimental autoimmune encephalomyelitis, allergy and the suppression of

antidrug antibodies. Indeed, the co-administration of a disease-relevant antigen with multiple
immunomodulators (vitamin D3, GM-CSF or TGFβ) in T1D, RA and MS models showed
significant therapeutic effects linked to the induction of IL-10 and PD1, as well as of
regulatory T and B cells210-212.

Human autoimmune diseases usually target multiple autoantigens, which may differ
between patients and disease stages, posing a significant challenge to immunomodulatory
interventions targeting one or a few antigens or epitopes. However, approaches based on the
co-delivery of self-antigens and immunomodulatory agents are reported to induce bystander
suppression. Nanoparticle-based co-delivery of antigen and ITE induced bystander tolerance
via the induction of FOXP3+ and IL-10+ Treg cells that migrate to the site of inflammation,
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also suppressing pathology driven by local innate immune responses104. Similarly, lipid-
coated calcium phosphate nanoparticles loaded with citrullinated autoantigen and rapamycin
induced bystander tolerance in an RA model213, and liposomal co-delivery of vitamin D3
and autoantigen induced bystander tolerance in a T1D model214. Collectively, these findings
suggest that the co-administration of immunomodulatory molecules with self-antigens is
needed not only to boost the therapeutic activity of antigen-specific tolerogenic approaches
but also to prevent the unwanted exacerbation of autoimmune pathology particularly
associated with some therapeutic modalities.

Nucleic acid–based and viral particle–based approaches to antigen-specific

immunotherapy
Nucleic acid–based approaches, including those based on DNA and mRNA, are attractive
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methods for antigen-specific immunomodulation. These methods offer several advantages

over peptide-based or protein-based approaches including the ease of manufacturing and
cargo alteration (both antigen and immunomodulator) and the fact that the encoded antigens
can be posttranslationally modified in the host and have relatively low production costs215.

Viral particles provide an effective platform for antigen delivery216. Viral particles are used
as gene therapy vectors and have been used to deliver autoantigen to the liver217 and
thymus218, inducing antigen-specific Treg cell expansion, effector T cell suppression and

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 Kenison et al. Page 13

bystander tolerance epitopes219. In response to safety concerns, plant virus particles have
also been tested in preclinical models of T1D and RA220. However, risks linked to viral
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gene therapy, pre-existing antibodies against adeno-associated viruses and the induction of
antivector antibodies by repeated treatment limit the utility of virus-based approaches for
antigen-specific immunomodulation.

Nucleic acid vaccines circumvent some of the risks linked to viral-based approaches. In
pioneering work, Waisman et al. used a plasmid encoding the TCR from a pathogenic
T cell clone, depleting TCR-specific pathogenic CD4+ T cells and ameliorating disease
in a mouse model of MS221. Similar encouraging results were obtained with vaccines
encoding other antigens in preclinical models of systemic lupus erythematosus, T1D and
RA222-225. Following these initial findings, DNA vaccines encoding disease-associated
antigens were tested in MS and T1D clinical trials226-228. An important feature of the DNA
vectors used for tolerance induction was the removal of TLR9-activating CpG motifs in
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the plasmid to minimize the activation of innate immunity. Despite showing reductions in
disease-associated biomarkers and evidence of some bystander tolerance, these trials did not
meet clinical end points. Thus, although DNA vaccines represent a promising approach and
additional clinical trials are ongoing (Table 2), further developments may be needed for the
success of this approach, including the co-administration of plasmids encoding tolerogenic
immunomodulators229. It is also possible that the intrinsic immunostimulatory properties
of plasmid DNA in combination with the limited control over its half-life, biodistribution
and uptake impose unsurmountable challenges for the clinical use of antigen-encoding DNA
vaccines for immunomodulation.

mRNA is less stable than DNA, requiring appropriate delivery platforms and modifications
to prevent the activation of innate immunity230. Nanoliposomes provide a unique platform
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for controlled mRNA delivery. In addition, unlike peptide-based vaccines, nanoliposome

mRNA vaccines do not need to be extensively optimized to accommodate each nucleic acid–
encoded antigen. Moreover, mRNA is quickly degraded in vivo, diminishing concerns about
long-term detrimental effects and tumorigenesis previously linked to some DNA-based
approaches. Furthermore, mRNA vaccines offer a safer alternative for the treatment of
patients who are immunosuppressed than attenuated viral or bacterial vaccines231.

mRNA is a potent pro-inflammatory adjuvant because of its ability to activate innate

immunity via TLR3, TLR7 and other immune receptors involved in sensing viral
infection232. Consequently, vaccination with mRNA-encoded epitopes induces potent
antigen-specific CD4+ and CD8+ effector T cells233. mRNA vaccines have been developed
to induce protective immunity against pathogens such as severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2)167,168. Similar exciting results have been described in the
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context of cancer immunotherapy234,235.

Eukaryotic RNA is heavily edited, facilitating the discrimination between self and microbial
mRNAs. Thus, RNA modification has been actively pursued to minimize the activation of
innate immunity and develop tolerogenic vaccines236. For example, nanoliposome-delivered
mRNA vaccines using pseudo-UTP and encoding the myelin autoantigen MOG suppressed
disease development in MS models, inducing bystander tolerance against additional myelin

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 Kenison et al. Page 14

antigens237. Mechanistically, these therapeutic effects were linked to the PD1- and CTLA4-
dependent induction of antigen-specific Treg cells237. Of note, mRNA has also been used
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to transfect T cells with autoantigen-specific CARs, with promising effects in suppressing

pathogenic CD4+ and CD8+ effector T cells in the non-obese-diabetes mouse model238,239.
Together, these findings suggest that vaccines containing mRNA-encoded antigens may
provide efficacious platforms for the treatment of inflammatory disorders.

Conclusions, challenges and outlook

The induction of antigen-specific immune tolerance is considered the “holy grail” of disease
management for autoimmunity and organ transplantation. Decades of research have resulted
in numerous promising advances. Yet despite the encouraging preclinical results, no truly
antigen-specific immunotherapies are currently approved for the treatment of autoimmune
diseases or organ transplantation, and few approaches have been tested beyond phase I or II
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clinical trials.

One important challenge is our limited understanding of the breadth of immune

targets recognized in autoimmune diseases. Indeed, antigen targets may vary from a
single autoantigen in Graves disease240 to multiple antigens in RA and systemic lupus
erythematosus241. Epitope spreading remains a significant challenge, suggesting that
successful antigen-specific immunotherapy must either halt epitope spreading, incorporate a
method for the repeated unbiased evaluation of the specificity of the autoimmune response
and/or induce bystander tolerance. In addition, it should be kept in mind that most studies
of the therapeutic induction of antigen-specific tolerance assume that the modulation of
T cell-mediated autoimmunity results in a concomitant decrease in pathogenic B cell
responses. However, it is not clear whether the magnitude, breadth and kinetics of this
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indirect B cell modulation are enough to result in significant clinical improvement of B

cell-driven pathology. Moreover, patient-to-patient variability, stage-specific autoimmune
responses and HLA allelic diversity further complicate the design of antigen-specific
therapies. Still, significant advances have been made in immune repertoire analysis,
including the development of antigen microarrays241,242, high-throughput BCR and TCR
sequencing243,244, multiplexed monitoring with barcoded tetramers245 and bioinformatic
approaches for epitope prediction246. These methods may enable not only the identification
of candidate antigens for the induction of antigen-specific tolerance but also the monitoring
of response to therapy, providing personalized approaches like those being developed for
cancer immunotherapy234.

An additional challenge is that often immunotherapeutic interventions for autoimmune

diseases are initiated after years of subclinical and clinical disease, resulting in the
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accumulation of tissue damage, immunological memory and the triggering of local

mechanisms of inflammation and disease pathology. Thus, although developments in this
area have been made for some diseases, such as T1D247, the identification of effective
biomarkers for patient identification and stratification remains an important need for the
development of antigen-specific immunotherapy. Indeed, these limitations highlight the
challenges of translating exciting findings in preclinical models into efficacious therapies
for human diseases. In this context, the selection of autoimmune diseases in which to

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 Kenison et al. Page 15

test antigen-specific immunomodulatory approaches remains critical. Coeliac disease, for

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example, offers unique opportunities for clinical trial design, as patients on a gluten-free diet
may receive experimental antigen-specific immunotherapies before dietary challenge.

Finally, how can we identify target signalling pathways to increase the therapeutic activity
of immunomodulatory approaches while preventing adverse events? Novel platforms may
guide the identification of candidate signalling pathways for the therapeutic induction of
tolerance, including the use of new methods to study cell–cell interactions involved in the
regulation of inflammation248-250, CRISPR-based platforms to study immune regulation
in vivo251 and the use of experimental systems such as zebrafish in combination with
artificial intelligence252. These approaches have already identified novel immunoregulatory
mechanisms with therapeutic potential. In addition, recently identified populations of
tolerogenic APCs may offer additional targets for immune tolerance induction253-255.
Provided these important challenges are addressed, recent advances in methods for
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the induction of antigen-specific immune tolerance, combined with novel methods

for the identification of target antigens and regulatory pathways, will probably guide
the development of platforms for personalized antigen-specific immunomodulation in
autoimmune diseases, allergy, transplantation and gene therapy.

Acknowledgements
Research in the Quintana lab was supported by grants NS102807, ES02530, ES029136, AI126880 from the
NIH; RG4111A1 andJF2161-A-5 from the NMSS; and PA-1604-08459 from the International Progressive MS
Alliance. During the writing of this article, J.E.K. was supported by a T32 Cancer Neuroscience training grant
(T32CA27386); N.A.S. was supported by funding from the Boehringer Ingelheim Fonds.

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Box 1
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Tolerogenic dendritic cells and the induction of peripheral regulatory T

cells
Tolerogenic dendritic cells (DCs) expand the peripheral regulatory T cell (Treg cell)
compartment through multiple mechanisms. Inhibitory molecules on tolerogenic DCs
such as programmed cell death ligand 1 (PD-L1) and PD-L2 engage programmed cell
death 1 (PD1) on T cells, boosting the differentiation of FOXP3+ Treg cells through
the downregulation of phosphorylated AKT, mTOR, S6 and ERK2 and simultaneous
upregulation of the phosphatase PTEN274. Additionally, DC expression of inducible T
cell co-stimulatory ligand (ICOSL) activates its receptor ICOS on T cells, also promoting
the development of FOXP3+ Treg cells and type 1 regulatory T cells (TR1 cells), although
ICOS signalling is also critical for the polarization of T helper 1 and T helper 2 effector
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cells53,70. Finally, binding of the surface receptor B and T lymphocyte attenuator (BTLA)
expressed on DCs to herpesvirus entry mediatory (HVEM) on CD4+ T cells is reported to
upregulate CD5 and induce FOXP3 expression275,276.

Several secreted factors released by DCs promote Treg cell differentiation. Transforming
growth factor-β (TGFβ) induces FOXP3+ Treg cell differentiation but promotes T helper
17 cell development in the presence of IL-6 or IL-21 (ref. 277). In the presence of TGFβ,
IL-10 promotes FOXP3 and cytotoxic T lymphocyte associated protein 4 (CTLA4)
expression278. IL-10 was also described to induce TR1 cell differentiation96,97. IL-27
is a strong inducer of TR1 cell differentiation through the induction of MAF, aryl
hydrocarbon receptor (AHR) and IL-21 (refs. 59,279,280) and has been shown to control
specific transcriptional programmes in FOXP3+ Treg cells281. Moreover, IL-27 signalling
in DCs and T cells induces the expression of CD39, which degrades extracellular ATP,
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limiting its pro-inflammatory effects107. Besides cytokines, metabolites produced by DCs

such as kynurenine, retinoic acid and lactate have important roles in modulating T cell
responses. For example, indoleamine 2,3-dioxygenase limits T cell responses via the
production of anti-inflammatory tryptophan metabolites such as kynurenine, many of
which activate AHR to promote FOXP3+ Treg cell and TR1 cell differentiation56. Retinoic
acid promotes the development of FOXP3+ Treg cells and TR1 cells, enhancing the effects
of TGFβ and IL-10 (ref. 112). Finally, lactate produced by DCs can suppress effector T
cell differentiation115.
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Fig. 1 ∣. Mechanisms and features in pro-inflammatory dendritic cells compared with tolerogenic
dendritic cells.
Pro-inflammatory dendritic cells (DCs) can be induced via activation by pathogen-
associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)
and upregulate the expression of surface molecules including MHC molecules, CD80 and
CD86. These surface molecules, in addition to secreted pro-inflammatory cytokines, such as
IL-1β, IL-6, IL-12, IL-23, tumour necrosis factor (TNF) and type I interferons, induce the
differentiation of cytotoxic and effector T cells from naive T cells. Conversely, tolerogenic
DCs can be induced via several mechanisms, including exposure to cytokines such as
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IL-10, IL-27, IL-35, IL-37 or transforming growth factor-β (TGFβ); signalling via Toll-like
receptor 2 (TLR2), TLR4 or aryl hydrocarbon receptor (AHR); or exposure to molecules
such as vitamin D3, vitamin A or lactate. Tolerogenic DCs express lower levels of MHC
molecules, CD80 and CD86 and secrete anti-inflammatory cytokines and molecules such
as IL-10, TGFβ, IL-27, indoleamine 2,3-dioxygenase (IDO) and retinoic acid. Tolerogenic
DC interactions with T cells induce the differentiation and expansion of anti-inflammatory
regulatory T cells (Treg cells) from naive T cells and the apoptosis of cytotoxic T cells
through death receptor signalling interactions, such as between programmed cell death 1
(PD1) and PD1 ligand 1 (PD-L1) or PD-L2. CTLA4, cytotoxic T lymphocyte associated
protein 4; TCR, T cell receptor.
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 Kenison et al. Page 31
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Fig. 2 ∣. Approaches for the induction of antigen-specific immune tolerance.

Cell-based approaches include the ex vivo induction of tolerogenic dendritic cells (DCs),
apoptotic cells or regulatory T cells engineered to express chimeric antigen receptors
(CAR Treg cells), all of which can be designed to deliver antigen with or without an
immunomodulatory signal. Viral particle approaches include the delivery of DNA-encoded
or RNA-encoded antigen via adenoviruses, lentiviruses or plant viruses. Synthetic particles,
including metallic, polymeric, lipid-based (including liposomes or lipid nanoparticles),
peptide–polymer, dendrimer or polyelectrolyte particles, can be designed to co-deliver
antigens, antibodies and immunomodulators, in various combinations. Alternatively,
antigens can be delivered via toxin-bound MHC molecules to induce the death of
antigen-specific cells, and albumin, antibodies or nanoemulsions can deliver antigens and
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immunomodulators to induce antigen-specific immune tolerance. FASL, FAS ligand; PEG,

polyethylene glycol.
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 Kenison et al. Page 32
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Fig. 3 ∣. Mechanisms for the induction of antigen-specific immune tolerance.

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Tolerogenic antigen-specific antigen-presenting cells (APCs) can be induced in vivo through

the delivery of synthetic particles, viral particles or cell-based approaches, or induced in
vitro and engineered to express disease-specific antigens and an immunomodulatory signal.
Tolerogenic APCs are characterized by reduced expression of pro-inflammatory markers
including CD80, CD86 and CD40 and an increased expression or production of tolerogenic
molecules such as IL-10, FAS ligand (FASL), programmed cell death ligand 1 (PD-L1)
and prostaglandin E2 (PGE2). Tolerogenic APCs can in turn induce naive CD4+ T cells to
differentiate into regulatory T (Treg) cells or can induce effector T cell anergy and ablation.
Similarly, the induction of regulatory B (Breg) cells via synthetic particle administration or
Treg cells via in vivo delivery of particles or in vitro engineering of chimeric antigen receptor
(CAR) Treg cells results in the reduction of effector T cells by the induction of anergy or cell
death. PEG, polyethylene glycol.
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Table 1 ∣

Antigen-specific immunotherapy approaches

Material Mediator of Immunomodulator Disease model Findings Refs.

antigen specificity
Kenison et al.

Cell-based approaches
IL-10-moDCs Lentiviral vectors NOD Induction of antigen-specific T cell tolerance and prevention of disease 122

Schwann cells+ PEG–PLGA Natural PD-L1–Fc, CD86–Fc, EAE Inhibition of TH1 cell responses, leflunomide loading increased myelin 256
NPs leflunomide repair

Erythrocytes Peptide mimotope NOD Reduced trafficking of effector T cells into organs 136

Erythrocytes Recombinant antigen EAE Persistent antigen exposure via erythrocytes induced T cell exhaustion 135
and dysfunction

BAR Treg cells Retroviral ADAs ADA suppression 146

CAR Treg cells Retroviral EAE Suppression of antigen-specific and other T cells even in a pro- 147
inflammatory environment

CAR Treg cells Plasmid Allograft rejection Protection of allografts better than polyclonal Treg cells 142

CAR/FOXP3+ Treg cells CAR EAE Intranasally administered cells accessed various brain regions and 144
suppressed disease

Vitamin D3–IL-4–IL-10–GM- None CTLA4–Ig Alloimmune reactivity Only in addition with CTLA4–Ig: alloreactive Treg cell induction and 126
CSF moDCs reduction in T cell activity
Apoptotic cells Peptide CFA Arthritis Induction of antigen-specific tolerance via B cell-mediated Treg cell 134
induction

Viral-based approaches
Cowpea mosaic virus Plasmid EAE Safe and efficient gene delivery 220

Adeno-associated virus Plasmid EAE Liver-targeted expression of protein induced Treg cells, regardless of 219
epitope or HLA background

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
Lentivirus Plasmid NOD Transient expression of antigen with integrase-incompetent lentivirus 217
protected from disease

Nanoparticles without adjuvant

PLGA NPs EAE, DTH Tolerance dependent on dose and antigen loading 157

PLGA NPs Multiple proteins NOD Encapsulation of multiple epitopes broadened spectrum of induced 257
tolerance

PEMA-PLGA NPs Protein EAE Kupffer cells and LSECs induced tolerance after antigen uptake 194

Liposome mRNA EAE Bystander tolerance by induction of Treg cells 237

Modified PLGA NPs Peptide EAE, NOD, colitis Optimized NPs can be effectively loaded with a variety of antigens 258
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Material Mediator of Immunomodulator Disease model Findings Refs.

antigen specificity
PEG–PLGA NPs Peptide EAE PEGylation increased bioavailability of subcutaneously injected NPs 192

Iron oxide NPs MHC class II-bound EAE Ubiquitous liver autoantigens are involved in extrahepatic immune 259
ubiquitous antigen diseases and tolerance induction mitigates extrahepatic autoimmunity
Kenison et al.

PEGylated iron oxide NPs MHC class II-bound PBC, AIH, PSC Ubiquitous antigens are involved in autoimmune liver diseases 260
peptide

PLGA NPs Hybrid peptide NOD Effector T cell anergy induced against several epitopes 154

PLGA NPs Peptides NOD Antigen-coupled NPs induced Treg cells 155

DSPG liposomes Peptide Atherosclerosis C1q-dependent update via scavenger receptors induced tolerance 173
PLGA NPs Peptide R-EAE Uptake of antigen by APCs led to PD-L1-dependent tolerance 182
induction

CdSe-ZnS-quantum dots Protein EAE Antigen density dictates disease suppression 158

Dextran-coated or PEGylated MHC class II-bound EAE, NOD Expansion of TR1 cells 149
iron oxide peptide

PSL NPs None Atherosclerosis Apoptotic cell mimicry induced IgM and reduced inflammation 181

PLGA-PEMA NPs Protein R-EAE Surfactant modification increased efficacy of NPs, reduced CNS 156
infiltration of effector T cells

PEI NPs Plasmid Arthritis Reduction of TLR9 activation by DNA promoted IDO-mediated 261
induction of tolerance

Iron oxide NPs MHC class I-bound NOD Induction of Treg cells that suppressed APCs via IDO and perforin 198
peptide

Dendrimer branched lysine Multiple antigen EAE Non-inflammatory presentation of antigen decreased effector T cell but 262
core particles peptides not Treg cell CNS infiltration

Peptide–polymer Peptide Cholangitis LSECs presented antigen on MHC class I, decreasing liver infiltration 263
of antigen-specific CD8+ T cells

Particles with adjuvant

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
PLGA NPs Peptide ICAM1 inhibitor EAE Dual-peptide NPs have stronger inhibitory effect 264

Dual size PLGA Protein GM-CSF, TGFβ, vitamin NOD Long-lasting protection in advanced disease states 210,211
D3

Liposomes/serum-albumin– Protein Treg cell epitopes NOD Tregitopes induced tolerance via specific Treg cell activation 265
bound NPs

Lipid-coated salt NPs Citrullinated peptides Rapamycin RA Induction of immune tolerance in advanced disease 213

PEG–PLGA NPs Peptide Knockdown of CD40, NOD Expansion of antigen-specific Treg cells 266
CD80, CD86

Liposomes Mimotope Vitamin D3 NOD Co-encapsulation of vitamin D3 can mediate bystander tolerance 214
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Material Mediator of Immunomodulator Disease model Findings Refs.

antigen specificity
Liposomes Peptide ITE EAE Co-encapsulation of immunomodulator induced Treg cells and 104
bystander tolerance

Liposomes Peptide Vitamin D3 RA, Goodpasture’s Calcitriol-antigen loaded NPs increased Treg cells and suppressed 206
Kenison et al.

vasculitis effector T cells in a PD-L1-dependent manner

Liposomes Peptide CD22 ligands, rapamycin Hypersensitivity Rapamycin enhanced tolerance induction in naive but not in 208
presensitized mice

PEG–Gold NPs Peptide ITE EAE, NOD Co-encapsulation of immunomodulators expanded Treg cells and 102,103
increased efficacy

PEG–PLA NPs, PLGA NPs Peptides/drug Rapamycin EAE, DTH, ADAs Co-encapsulation of rapamycin induced durable B and T cell tolerance 202

Dextran NPs Peptide Dexamethasone EAE Immunomodulator increased effectiveness 267

PLGA NPs Peptide Recombinant IL-10 EAE NPs release antigen and immunomodulator constantly for several 205
weeks, addition of IL-10 decreased IL-17 and IFNγ

PEG–liposomes Multivalent peptide CD22 ligands ADAs Tolerance induction towards presented alloantigen 268

Liposomes Protein NF-κB inhibitors RA Co-encapsulation of immunomodulators increased efficacy of tolerance 203
induction

Particle free
Anti-MHC class II antibodies Peptide Dexamethasone EAE, NOD, RA Immunomodulator reduced adverse effects and increased effectiveness 207

Nanoemulsion Citrullinated self- Rapamycin RA Nanoemulsion accumulated in inflamed regions and suppressed disease 207
antigen activity

Mannosylated antigen Peptide EAE, R-EAE Amelioration of EAE, reduced CNS infiltration of immune cells 196

ADA, antidrug antibody; AIH, autoimmune hepatitis; APC, antigen-presenting cell; BAR, B cell-targeting antibody receptor; CAR, chimeric antigen receptor; CFA, complete Freunds adjuvant;
CNS, central nervous system; CTLA4, cytotoxic T lymphocyte associated protein 4; DC, dendritic cell; DSPG, 1,2-distearoyl-sn-glycero-3-phosphoglycerol; DTH, delayed-type hypersensitivity;
EAE, experimental autoimmune encephalitis; ICAM1, intercellular adhesion molecule 1; IFNγ, interferon-γ; GM-CSF, granulocyte-monocyte colony-stimulating factor; IDO, indoleamine-pyrrole
2,3-dioxygenase; LSECs, liver sinusoidal endothelial cells; moDC, monocyte-derived dendritic cell; NF-κB, nuclear factor-κB; NOD, non-obese diabetes; NP, nanoparticle; PBC, primary biliary
cholangitis; PD-L1, programmed cell death ligand 1; PEG, polyethylene glycol; PEI, polyethyleneimine; PEMA, poly(ethylene-maleic acid); PLA, poly(lactic acid); PLGA, poly(lactic-co-glycolic acid);

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
PSC, primary sclerosing cholangitis; PSL, phosphatidyl serine liposome; RA, rheumatoid arthritis; R-EAE, relapsing–remitting EAE; TGFβ, transforming growth factor-β.
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Table 2 ∣

Antigen-specific immunotherapy approaches in phase I or II clinical trials

Study type Vehicle or reagent Major findings Clinical trial ID

Kenison et al.

Cell-based approaches
Neuroinflammation
Tolerogenic fibroblasts Tolerogenic fibroblasts No significant safety concerns or side effects up to 16 weeks NCT05080270
after infusion

Tolerogenic DCs (TOLERVIT-MS) Vitamin D3-moDCs + IFNβ Interim report: no safety concerns NCT02903537

“Negative” DC vaccine (MS-tolDC) Vitamin D3-moDCs Interim report: no safety concerns NCT02618902

Regulatory DCs (TolDec-EM-NMO) Dexamethasone–GM-CSF–IL-4-moDCs with seven Safe; induction of TR1 cells and decreased CD8+ T cells, NK NCT02283671
peptides from MBP, MOG, PLP, AQP4 cells, CD14+CD56+ cells

Peptide-coupled PBMCs (ETIMS) Autologous PBMCs coupled with seven peptides No adverse effects, reduced antigen-specific T cell response, NCT01414634
no effects on immunoglobulins or recall antibody effect

Mesenchymal stem cells (MSCIMS) Mesenchymal stem cells Safe; neuroprotective; met some secondary visual end points NCT00395200

T cell vaccine Irradiated MBP-reactive T cells against nine epitopes Reduction in EDSS, walking time and relapses NCT01448252

T cell vaccine Irradiated MBP-reactive T cells Depletion of autoreactive T cells; smaller lesion volume 269

Rheumatoid arthritis
Autologous tolerogenic DCs (AutoDECRA) Dexamethasone–vitamin D-moDCs with synovia Safe, but no systemic effects detected NCT01352858

Autologous tolerogenic DCs (TolDCfoRA) Dexamethasone-moDCs Well tolerated NCT03337165

DC vaccine to suppress the immune NF-κB-inhibitor-moDCs Safe; fewer effector T cells and decreased pro-inflammatory ACTRN12610000373077
response to citrullinated antigen (BAY11-7082; cytokines
Rheumavax)
Autologous DCs (CreaVax-RA) moDCs pulsed with PAD4, citrullinated vimentin and Safe; reduction in autoantibody and IFNγ-producing T cells CRiSKCT0000035
fillagrin. HNRPA2/B1

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
Inflammatory bowel disease
Intralesional tolerogenic DCs (TolDecCDintra) Dexamethasone-moDCs Terminated (low recruitment) NCT02622763

Antigen-specific Treg cell therapy Treg cells with ovalbumin No adverse effects; symptom reduction 2006-004712-44

Autologous tolerogenic DCs Dexamethasone–vitamin D-moDCs Mixed clinical response 2007-003469-42

Type 1 diabetes
Immunotherapy vaccine (PIpepTolDC) Autologous tolerogenic DCs loaded with proinsulin Ongoing NCT04590872
peptides

Autologous tolerogenic DCs Autologous moDCs primed with peptides Ongoing NCT05207995

AVT001 Autologous moDCs Ongoing NCT03895996

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Study type Vehicle or reagent Major findings Clinical trial ID

Tolerogenic DCs (D-Sense) Vitamin D3-moDCs with proinsulin Not published NTR5542

Polyclonal Treg cells + IL-2 (TILT) Polyclonal Treg cells No adverse effects but poor Treg cell survival NCT02772679

Tolerogenic DCs Dexamethasone-vitamin D3-moDCs pulsed with islet Long-lasting CD4+ T cell tolerance and temporary bystander 2013-005476-18
antigen
Kenison et al.

tolerance

Autologous immunoregulatory DCs Anti-CD40/CD80/CD8-moDCs with six ODNs Not published NCT02354911

CD4+CD127lowCD25+ polyclonal Treg cells Polyclonal Treg cells + IL-2 Treg cell survival but also expansion of cytotoxic T cells NCT01210664

Autologous DCs Antisense CD40, CD80, CD86 ODNs-moDCs Safe; induction of B220+CD11c+ B cells NCT00445913

Treg cells Autologous ex vivo-expanded Treg cells No adverse effects; elevated C-peptide levels and lower ISRCTN06128462
insulin dependence after 1 year

Systemic lupus erythematosus

Autologous polyclonal Treg cells Treg cells Increased Treg cells in inflamed tissue NCT02428309

Graft rejection after kidney transplantation

DCreg Donor-derived tolerogenic DCs Ongoing NCT03164265

Donor alloantigen reactive Treg cells Donor alloantigen reactive Treg cells No adverse effects NCT02474199
(ARTEMIS)

Autologous tolerogenic DCs (ONEatDC) GM-CSF-moDCs No adverse effects, fewer infections NCT02252055

Regulatory macrophages (ONEmreg12) Donor-derived regulatory macrophages induced with No adverse effects, fewer infections NCT02085629
GM-CSF + IFNγ

Donor alloantigen-reactive Treg cells (The ONE Donor-alloantigen-reactive Treg cells with donor antigen No adverse effects, fewer infections NCT02244801
Study) (DART)
Natural Treg cells (ONEnTreg13) CD4+CD25+FOXP3+ Treg cells No adverse effects, fewer infections NCT02371434

Treg cells (ONETreg1) CD4+CD25+FOXP3+ Treg cells No adverse effects, fewer infections NCT02129881

Treg cells (The ONE Study) Treg cells induced with belatacept ex vivo with donor No adverse effects, fewer infections NCT02091232

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
antigen

Graft-versus-host disease
Ex vivo expanded donor Treg cells Donor Treg cells cultured with recipient DCs Not published NCT01795573

T cell-depleted graft with simultaneous infusion Conventional T cells and Treg cells in predefined ratio Safe, no adverse effects NCT01660607
T cells

Treg cells Induced Treg cells No adverse effects NCT01634217

Treg cells Induced Treg cells Safe, low risk of acute GVHD NCT00602693

Free antigen approaches

Multiple sclerosis
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Study type Vehicle or reagent Major findings Clinical trial ID

ATX-MS-1467 Synthetic peptides of four epitopes of MBP Study 1 showed temporary lesion reduction; study 2 showed NCT01973491
lesion reduction up to 48 weeks with higher doses

Myelin peptides Transdermal peptide mix Reduction of gadolinium-enhancing lesions 270

Myelin peptides Transdermal peptide mix 271

Kenison et al.

Induction of TR1 cells but not FOXP3+ Treg cells

MBP8298 (MAESTRO-03) MBP8298 No difference to placebo NCT00468611

BHT-3009 MOG-DNA Fewer lesions and reduction of a spectrum of antibodies NCT00382629

Glatiramer acetate Random peptides resembling MBP Reduced number of lesions 272

Type 1 diabetes
TOL-3021 in new onset disease (DAWN) DNA-plasmid encoding proinsulin Ongoing NCT03794973

TOL-3021 in established disease (DAY) DNA-plasmid encoding proinsulin Ongoing NCT03794960

Proinsulin peptide (MonoPepT1De) Proinsulin No adverse effects, reduced insulin use increase, FOXP3 and NCT01536431
IL-10 induction

BHT-3021 DNA-plasmid encoding proinsulin No adverse effects, C-peptide level increased but no effect on NCT00453375
insulin requirement

DPT-1 Insulin, GAD65 No effect on prevention of familial disease development

Rheumatoid arthritis
Chicken type II collagen Chicken type II collagen Improvement of symptoms, albeit less than methotrexate- ChiCTR-TRC-00000093
treated control group

Altered peptide ligand approaches

Multiple sclerosis
RTL1000 TCR ligand No adverse effects at <100 mg; no worsening of disease NCT00411723

NBI-5788 Altered peptide ligand Suspended after detection of increased brain lesions in some NCT00079495
patients

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
CGP77116 Altered peptide ligand Aborted owing to exacerbation of disease in three patients NCT00001781

NBI-5788 Altered peptide ligand Hypersensitivity reactions, reduced lesion load 200

Type 1 diabetes
NBI-6024 Altered peptide ligand No effect compared with placebo NCT00873561

Antigen and adjuvant approaches

Multiple sclerosis
NeuroVax TCR peptides in IFA Ongoing NCT02057159

Type 1 diabetes
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Study type Vehicle or reagent Major findings Clinical trial ID

Diamyd (DIAGNODE-3) rhGAD65–Alum+vitamin D HLA-DR3-DQ2+ Ongoing NCT05018585
haplotype
Diamyd booster Booster Diamyd+vitamin D3 Ongoing NCT05351879

MER3101: MAS-1 adjuvanted (MER3101) Ongoing NCT03624062

Kenison et al.

β-chain + MER3101 administered into lymph nodes

Diamyd (DIAGNODE-2) rhGAD65–alum+vitamin D3 administered into lymph No significant improvement in overall groups but increased NCT03345004
nodes glycaemic control in HLA-DR3-DQ+ subjects

Diamyd (DIAGNODE-1) rhGAD65–alum+vitamin D No adverse effects; TH2 cell induction; TH1 cell reduction; NCT02352974
C-peptide baseline increased

Islet β-chain IFA–β-chain emulsion Insulin-specific antibody and T cell induction; induction of NCT00057499
long-lasting antigen-specific Treg cells, no HBA1C or insulin
use changes

Diamyd in newly diagnosed disease rhGAD65–alum No change in C-peptide levels NCT00751842

(DIAPREVENT)

Diamyd in newly diagnosed disease GAD–alum No adverse effects; no difference in insulin secretion NCT00529399
(DIAPREVENT)

Nanoparticle-based approaches
Multiple sclerosis
Xemys CD206-targeted liposomes with three MBP peptides Cytokine normalization #930 [FASEMS-01/01]

Rheumatoid arthritis
DEN-181 Liposomes with collagen peptide + NF-κB inhibitor Reduced effector T cells, increased Treg cells 176

AG4263 Iron particles coated with peptide–MHC class II-bound No long-lasting effects 273
antigen

Coeliac disease
TIMP-GLIA PLGA nanoparticles with gliadin TAK-101 Reduction of IFNγ-producing cells on challenges NCT03738475

Nat Rev Immunol. Author manuscript; available in PMC 2024 June 03.
AQP4, aquaporin 4; DC, dendritic cell; EDSS, expanded disability status scale; GAD, glutamic acid decarboxylase; GVHD, graft-versus-host disease; GM-CSF, granulocyte-monocyte colony-stimulating
factor; IFA, incomplete Freunds adjuvant; IFNβ, interferon-β; IFNγ, interferon-γ; MBP, myelin basic protein; moDC, monocyte-derived dendritic cell; MOG, myelin oligodendrocyte glycoprotein; NF-κB,
nuclear factor-κB; ODNs, oligodeoxynucleotides; PAD4, peptidylarginine deiminase 4; PLGA, poly(lactic-co-glycolic acid); PLP, proteolipid protein; TCR, T cell receptor; Treg cell, regulatory T cell.
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