Antigens

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 Definitions
 Immunogen: is any agent capable of inducing a
specific immune response

 Antigen: is any agent reacts with the products of

a specific immune response

 Immunogenicity: is the ability of an antigen to

stimulate an immune response.

 Hapten :- A substance that is non-immunogenic but

which can react with the products of a specific
immune response.
• Are small molecules which could never induce an
immune response when administered by themselves
but which can when coupled to a carrier molecule 2
• All immunogens are antigens, but not all antigens are
immunogens.

• The structural portion of the antigen for binding antibodies

or TCR is called as an epitope or antigenic determinant.
• Antibody (Ab)- A specific protein which is produced in
response to an immunogen and which reacts with an
antigen

• The typical size of an epitope for Ab binding is about 5-7

amino acid residues.

• The region of the Ab for binding an epitope is termed

antigen-binding site.

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• The size of an epitope for binding the corresponding TCR
is generally larger of 10-15 amino acid residues.

• The aa residues of the antigenic peptide responsible for

binding MHC molecules are called the anchor residues.

• Antigen may have single epitope or may have multiple

epitopes of the same specificity (polysaccharides
polyvalency or multivalency); or combination of different
epitopes on the same molecule (protein).

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 Factors influencing immunogenicity
A. Contribution of the Immunogen

1. Foreignness

2. Size

3. Chemical Composition

4. Physical form

5. Degradability

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Factors related to the immunogen/antigen are:

1. Foreignness- The immune system normally discriminates

between self and non-self such that only foreign molecules
are immunogenic.

2. Size - There is not absolute size above which a substance

will be immunogenic. However, in general, the larger the
molecule the more immunogenic it is likely to be.

• Most potent immunogens have a molecular weight greater

than 50103 Daltons (Da)
• Few immunogens have molecular weight between 10103-
50103 Da
• A few immunogens are known to have molecular weight
less than 10,000 Da

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3. Chemical Composition - In general, the more complex the
substance is chemically the more immunogenic it will be.
Complex proteins are potent immunogens

4. Physical form - In general particulate antigens are more

immunogenic than soluble ones and denatured antigens
more immunogenic than the native form.
• Particulate > Soluble
• Denatured > Native
5. Degradability - Antigens that are easily phagocytized are
generally more immunogenic.
– This is because for most antigens (T-dependent antigens) the
development of an immune response requires that the antigen
be phagocytized, processed and presented to helper T cells by
an antigen presenting cell (APC). 7
B. Contribution of the Biological System
• Genetic Factors:
 The species or individuals may lack or have altered genes that
code for the receptors for antigen on B cells and T cells or they
may not have the appropriate genes needed for the APC to
present antigen to the helper T cells.
• Age: Age can also influence immunogenicity.
 Usually the very young and the very old have a diminished
ability to mount an immune response in response to an
immunogen.
C. Method of Administration
• Dose
• Route
• Adjuvants 8
Method of Administration
1. Dose - The dose of administration of an immunogen
can influence its immunogenicity. There is a dose of
antigen above or below which the immune response
will not be optimal.

2. Route - Generally the subcutaneous route is better

than the intravenous or intra gastric routes.
• The route of antigen administration can also alter
the nature of the response

3. Adjuvants - Substances that can enhance the

immune response to an immunogen are called
adjuvants.
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Chemical nature of immunogens
A. Proteins: The vast majority of immunogens are proteins.
These may be pure proteins or they may be glycoproteins
or lipoproteins. In general, proteins are usually very good
immunogens.
B. Polysaccharides: Pure polysaccharides and
lipopolysaccharides are good immunogens.
C. Nucleic Acids: Nucleic acids are usually poorly
immunogenic. However, they may become immunogenic
when single stranded or when complexed with proteins.
D. Lipids: In general lipids are non-immunogenic, although
they may be haptens.
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 Types of Antigens
A. T-independent Antigens –
• Directly stimulate the B cells
• Polysaccharides are T-independent antigens

 Properties of T-independent antigens

a. Polymeric structure – the same antigenic
determinant repeated many times

b. Polyclonal activation of B cells - Many of these antigens

can activate B cell clones specific for other antigens
(polyclonal activation)
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c. Resistance to degradation–
• more resistant to degradation and thus they persist
for longer periods of time and continue to stimulate
the immune system.
B. T-dependent Antigens
• Do not directly stimulate the production of antibody
without the help of T cells
• Proteins are T-dependent antigens
• Structurally these antigens are characterized by a
few copies of many different antigenic determinants

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Antigenic Determinants Recognized by B cells
and Ab
• Composition
– Proteins, polysaccharides, nucleic acids
– Sequence (linear) determinants
– Conformational determinants
• Size
– 4-8 residues
• Number
– Limited (immunodominant epitopes)
– Located on the external surfaces of the Ag

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 Antigenic Determinant Recognized by T cells
• Composition
– Proteins (some lipids)
– Sequence determinants
• Processed
• MHC presentation (lipid presentation by MHC-like
CD1)
• Size
– 8 -15 residues
• Number
– Limited to those that can bind to MHC

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 Superantigens
• Conventional T-dependent antigen, only a small fraction
(1 in 104 -105) of the T cell population is able to recognize the
antigen and become activated (monoclonal/oligoclonal
response).

• However, there are some antigens which polyclonally

activate a large fraction of the T cells (up to 25%)-
Superantigens

• The intact protein binds to class II MHC molecules and to one or

more Vβ regions of the TCR. The antigen is not bound to the
peptide binding groove of the MHC molecule or to the antigen
binding site of the TCR.
o Thus, any T cell that uses a particular Vβ in its TCR will be
activated by a superantigen, resulting in the activation of a large
numbers of T cells. 15
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• Examples of superantigens include:
• Staphylococcal enterotoxins (food poisoning),
• Staphylococcal toxic shock toxin (toxic shock syndrome),
• Staphylococcal exfoliating toxins (scalded skin
syndrome)
• Streptococcal pyrogenic exotoxins (shock).

• Although the bacterial superantigens are the best studied

there are superantigens associated with viruses and other
microorganisms as well.
• The diseases associated with exposure to superantigens are,
in part, due to hyper activation of the immune system and
subsequent release of biologically active cytokines by
activated T cells.
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Antigen processing and presentation

• Antigen Processing – is a metabolic process that digests

proteins into peptides, which can then be displayed on the
cell membrane together with MHC class I or II molecules

• Endogenous antigens- peptides derived from antigens that

have been processed within the cytoplasm of the cell

• Exogenous antigens- Peptides derived from antigens that

are Internalized by phagocytosis /endocytosis and
processed within the endocytic pathway

• T cells recognize Ag in the form of short peptides in the

context of either MHC class-I or MHC class-II molecules

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• The two types of T cells (CD4+ and CD8+) recognize Ag
bound and presented by two distinct MHC classes:
• CD4+ T cells recognize Ag in the context of MHC class-II
while CD8+ T cells recognize Ag in the context of MHC
class-I molecules

• This dichotomy is imposed by the fact that the CD4 and CD8
molecules ligate (bind to) the MHC class-II and the MHC
class-I molecules, respectively.
• The CD4 and CD8 molecules are called co-receptors.
• The CD4 molecule binds to the outer surface of the β2
domain of the MHC class-II molecule.
• The CD8 molecule makes contact with the α3

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• All cells expressing MHC class I or class II molecules can
present peptides.
• However, only cells presenting peptides associated with
MHC class II molecules to CD4 T cells are called antigen-
presenting cells (APC).

• Cells presenting peptides with MHC class I molecules are

called target cells.

There are two types of APC:

A. Professional APC
- Dendritic cells: Are the most effective APC. They
express constitutively high levels of MHC class II
molecules and co-stimulatory molecules.

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- Macrophages: Need to be activated by phagocytosis of
microorganisms before they express MHC class II
molecules or co-stimulatory molecules.

- B cells: Express MHC class II molecules constitutively

but need to be activated before they express co-
stimulatory molecules.

B. Nonprofessional APC
• These are cells that can serve as APC for short periods of
time during inflammation.
Eg. epithelial cells and endothelial cells.

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• The APCs process Ag along two distinct pathways
depending to how the Ag/pathogen has gained access and
where it is localized within the cell:

1. The cytosolic (Endogenous) pathway

2. The vesicular pathway (Endocytic) pathway

• Pathogens replicating in the cytosol are processed and

presented to CD8+ T cells in the context of MHC class-I
molecules.

• While intravesicular pathogens are processed and presented

to CD4+ T cells in the context of MHC class-II molecules

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 Degradation and transport of antigens that bind major histocompatibility complex
(MHC) class II molecules.
(a) In an antigen-presenting cell (APC), newly synthesized MHC class II molecules
bind the invariant chain (IC), which prevents binding of peptides that are present
in the endoplasmic reticulum (ER).
(b) The IC allows transport of MHC class II molecules from the ER into the Golgi
apparatus to acidified endosomes.
(c) Endosomes contain peptides that are derived from either resident pathogens
(e.g. bacteria) or
(d) Engulfed extracellular proteins (or pathogens)
(e) In the phagosomes
(f) Proteases within the endosome degrade proteins into peptides.
(g) The endosome fuses with the Golgi to form the trans-Golgi.
(h) Here, the IC is cleaved and released from the MHC class II molecule. This
allows the binding of peptides within the endosome to the peptide-binding cleft
of the MHC molecules. A class II-like molecule (HLA-DM) binds to MHC class II
molecules to facilitate the release of the IC (not shown).
(i) The MHC class II-peptide complex is then transported to the cell surface of the
APC for
(j) Recognition by the T-cell receptor (TCR) of (CD4 + , CD3 + ) T helper (Th) cells
and
(k) Intracellular signalling for activation.
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Degradation and transport of antigens that bind major histocompatibility complex
(MHC) class I molecules.
(a) In an antigen-presenting cell (APC), newly MHC synthesized C class I molecules
bind to calnexin (Cx), which retains them in a partially folded state in the
endoplasmic reticulum (ER).
(b) Binding of MHC class I molecules to β 2 microglobulin (β 2 m) displaces Cx and
allows binding of chaperonin proteins (calreticulin and tapasin; not shown).
(c) The MHC class I-β 2 m complex binds to the TAP complex (TAP1-TAP2), which
awaits the delivery of peptides.
(d) Peptides (e.g. from antigens) are formed from the degradation of cytosolic
proteins ('self'-, pathogen-and tumour-derived proteins in the cytoplasm).
(e) These are degraded by proteasomes into
(f) short peptides.
(g) Peptides are transported into the ER by the TAPs, where they meet the MHC
class I-β 2 m complex
(h) This peptide binding in the antigenic groove of the MHC stabilizes the structure of
the MHC class I molecule and
(i) releases the transporter associated with antigen processing (TAP) complex.
(j) The fully folded MHC class I molecule with its peptide is transported to the cell
surface via the Golgi apparatus.
(k) Recognition of the MHC class I-peptide complex by the T-cell receptor (TCR) of
an antigen-specific (CD8 + , CD3 + ) cytotoxic T lymphocyte (CTL) takes place
and (l) a signal transduction event activates effector functions in the MHC-class-I-
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restricted T cell;
• Group 1 CD1 molecules mainly present lipid antigens to
clonally diverse T cells that mediate adaptive immunity to
the vast range of microbial lipid antigens.

• By contrast, CD1d (group 2) molecules present lipid

antigens to natural killer T (NKT) cells

• CD1 proteins survey the endocytic pathway to intersect

and bind lipid antigens

• Saposins mediate the loading of lipids onto CD1

molecules in lysosomes

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The Major Histocompatibility
Complex (MHC)

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• Major Histocompatibility Complex Cluster of genes found
in all mammals

• Its products play role in discriminating self/non-self

participant in both humoral and cell-mediated immunity

• MHC act as antigen presenting structures

• In Human MHC is found on Chromosome 6 referred to

as HLA complex

• In Mice MHC is found on Chromosome 17 referred to as

H-2 complex

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 The MHC Genes
• Genes Of MHC Organized In 3 Classes
– Class I MHC genes
• Glycoproteins expressed on all nucleated cells
• Major function to present processed Ags to TC
– Class II MHC genes
• Glycoproteins expressed on M, B-cells, DCs
• Major function to present processed Ags to TH
– Class III MHC genes
• Products that include secreted proteins that have
immune functions. Ex. Complement system,
inflammatory molecules
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• Class I MHC Genes Found In Regions A, B and C In Humans
(K and D In Mice)

• Class II MHC Genes Found In Regions DR, DP and DQ (IA

and IE In Mice)
• Class I and Class II MHC Share Structural Features

– Both involved in APC

• Class III MHC Have No Structural Similarity To Class I and

II
– Ex. TNF, heat shock proteins, complement components

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 The MHC is Polygenic and Polymorphic

• MHC is polygenic because it has several genes

for each class (3 for class-I, 3 for class-II).

• MHC is polymorphic due to the relatively

large number of alleles within each MHC
class.

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MHC Polymorphism

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 Consequences of MHC polymorphism

• The combined effect of polymorphism and

polygeny is the generation of a highly diverse
repertoire of MHC molecules capable of
presenting a large variety of Ag peptides to T
cells.

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 MHC Classification and structure

• The two types of polymorphic genes, namely the

class I and II encodes two groups of structurally
distinct but homologous proteins.

• MHC class-I is made up of two polypeptide chains:

One large called  and one small called 2
microglobulin.
• The  chain has 3 extracellular domains: 1, 2
and 3, a transmembrane and a cytoplasmic
segment.

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 MHC Classification and structure
•The 1 and 2 domains form a cleft where the Ag
fragment binds.
•The 2 microglobulin has only one extracellular
domain
• No transmembrane portion
• No cytoplasmic tail
• Binds to the 3 domain non-
covalently
•Immunoglobulin-like region – highly conserved α3
domain - site to which CD8 on T cell binds 38
 MHC Class-I
α
1 2
NH
NH2
Alloantigenic
sites β
NH2
2
α COO
CH
O H
2
α3 Disulfide
bridge
Papain
cleavage

Plasma membrane

O P
H Cytoplasm
COO
H
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 MHC Class-II
• Composed of two polypeptide chains  and .
• Each chain has two extracellular domains 1 2
and 1 and 2
• A transmembrane segment
• A cytoplasmic tail
• The  1 and  1 domains form the cleft for
the Ag fragment
• Immunoglobulin-like region – conserved α2
and β2 domains – β2 is site to which CD4 on
T cell binds

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 The MHC class-II
NH2
NH2
CH
O
α β CH
O
1 1

CH α β
O
2 2

Plasma membrane

Cytoplasm
COO COO
H H
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Class I or II MHC Molecules and Interaction between Peptides
• MHC molecules show a broad specificity
• Each MHC molecule has a single peptide binding cleft

• The peptide sequences that bind to MHC molecules are

distinct from those recognized by T cells

• The affinity of peptide-MHC interactions is much lower

than that of antigen-antibody binding

• Peptide-MHC complexes persist long due to low rate of

dissociation

• MHC molecules don’t discriminate between self and non Self

antigens 42
• The binding of peptides to MHC molecules is
o A non-covalent interaction mediated by residues both in
the peptides and in the clefts of the MHC molecules.

• Amino acids from both the antigenic peptide and the

MHC molecules contribute to T cell antigen recognition,

o With the peptide being responsible for the fine specificity

and
o The MHC residues accounting for the MHC restriction of
the T cells

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 Genomic Organization of MHC I and II
• In Humans, the MHC is located on the short arm of
chromosome 6 and  Microglobulin is encoded by a gene
on chromosome 15
• Human MHC occupies 3500kb

• Class I genes are in the most telomeric region and class II

are in the most centromeric region of HLA locus
• Other genes in the class II locus
-TAP 1 and 2
- subunits of a cytosolic protease complex
(Proteasome)
- HLA-DM

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• MHC class III = Between class I and II gene
• Complement factors C2, C4,& Factor B
• Genes encoding in this MHC III do not play any
role in antigen presentation

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 Expression of MHC complex Molecules
• Class I expressed on all nucleated cells
• Class II expressed on APCs (Macrophages, B cells
and Dendritic cells
• Expression of MHC molecules is increased by
cytokines produced during innate and adaptive
immune responses
- Interferon   and 
- Tumor necrosis factor and lymphotoxin
• increases expression of class I molecules
- Interferon  stimulates expression of class II
molecules
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 Peptide-binding grooves for class I and class II
MHC are structurally similar

• Both have a peptide-binding groove with a wall of two α

helices and a floor of eight β-pleated sheets

• Close-ended groove for class I MHC requires an 8- 10

amino acid-length peptide to bind; open-ended groove for
Class II MHC lets it bind a peptide 13-25 amino acids long

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 Aspects of MHC
1. MHC molecules are membrane-bound. Recognition by
T cells requires cell-cell contact.

2. Peptide from cytosol associates with class I MHC and is

recognized by Tc cells. Peptide from vesicles associates with
class II MHC and is recognized by Th cells.

3. Mature T cells must have a T cell receptor that recognizes

the peptide associated with MHC.

4. Each MHC molecule has only one binding site. The different
peptides a given MHC molecule can bind all to the same
site, but only one at a time.
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6. MHC polymorphism is determined only in the germ
line. There are no recombinational mechanisms for
generating diversity.

7. Because each MHC molecule can bind many different

peptides, binding is termed degenerate.

8. Cytokines (especially interferon-γ) increase level of

expression of MHC.

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Reading Assignment
• The cytosolic (Endogenous) pathway

• The vesicular pathway (Endocytic) pathway

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