NATURE OF ANTIGENS
Antigens
 Immunogens
 Materials that trigger immune response of
lymphocytes
 Macromolecules capable of eliciting the formation of
immunoglobulins (antibodies) or sensitized cells in an
immunocompetent host
 Specifically react with the antibodies or sensitized
cells that have been induced
 Antigens
 Term used to refer to a substance that reacts with
antibody or sensitized cells but may or may not be
able to evoke an immune response in the first place
 Conclusion
“All Immunogens are antigens”
How and why we respond to particular immunogens?
 Response is caused by a combination of factors:
1. The nature of the immunogen
2. Genetic coding of molecules (MHC molecules) that
must combine with an immunogen before T cells are able to
respond
3. Immunogen processing and presentation
Factors Influencing the Immune Response
1. Age
 Older individuals are more likely to have a decreased
response to antigenic stimulation
 Neonates do not fully respond to immunogens because
their immune systems are not completely develope
2. Dose
 the larger the dose of an immunogen one is exposed to,
the greater the immune response is
 very large doses can result in T and B cell toleranc
3. Route of inoculation
4. Overall health
 Individuals who are malnourished, fatigued or stressed
are less likely to mount a successful immune response
5. Genetic capacity
 There is a genetic predisposition that allows individual
to respond to particular immunogens, linked to the
MHC and to the receptors generated during T and B
lymphocyte development
Traits of Immunogens
 In general, the ability of an immunogen to stimulate a
host response depends on the following
characteristics:
1. Molecular size
- must have a molecular weight of 100,000 (with
exceptions)
- for the most part, the rule of the thumb is that the
greater the molecular weigh, the more potent the
molecule is as an immunogen
2. Chemical composition and molecular complexity
 proteins
 polysaccharides
PROTEINS
 good immunogens because they are made of variety of
amino acids
 The particular sequential arrangement of amino acids
(primary structure) determines the secondary structure
(relative orientation of amino acids within the chain),
the tertiary structure, which embodies the spatial or
three-dimensional orientation of the entire molecule,
and the quaternary structure, which based on the
associations of two or more chains into a single
polymeric unit
CARBOHYDRATES
 Less immunogenic than protein because the units of
sugars are more limited than the number of amino acids
in protein
 Often occur in the forms of glycolipids or glycoproteins
Ex. blood group antigens
Glycolipids – A, B, and H blood group
Glycoprotein – Rh and Lewis
PURE NUCLEIC ACIDS AND LIPID
 not immunogenic by themselves
 response can be generated when they occur in
conjunction with other substance
Ex. DNA-protein complex in LE that stimulates
autoantibodies to DNA
3. Foreignness
 immune system is able to distinguish self from nonself
 nonself are immunogenic
 typically, the more distant taxonomically the source of the
immunogen is from the host, the better it is as a stimulus
4. Ability to be processed and presented with MHC molecules
 for a substance to be elicit an immune response, it must be
subject to enzymatic digestion to create small peptides
or pieces that can be complexed to MHC molecules to
present to responsive lymphocytes => immune response
 The particular MHC molecules produced determine genetic
responsiveness to individual antigens
 T cells are only able to respond to peptides when MHC
molecules present them
Nature of Epitopes
Epitopes
 Also known as the determinant site
 It is the key portion of the immunogen
 They are molecular shapes or configurations that are
recognized by antibody or by T cells
 For proteins, an epitope may consist of as few as 6to 15 AAs
(B-cells)
 Large molecules may have numerous epitopes, and each one
may be capable of triggering specific antibody production or
a T cell response
 These epitopes may be repeating copies, or they may
have differing specificities
 They may be:
1. Sequential or linear
– amino acids following one another on a single
chain
2. Conformational
– results from the folding of one chain or
multiple chains, bringing certain amino acids
from the different segments of a liner sequence
or sequences into close proximity with each
other so they can be recognized together
Epitopes recognized by B cells differ from those recognized by T
cells
 Surface antibody to B cells may react with both linear and
conformational epitopes on the surface of an immunogen
 Any that is capable of cross-linking surface immunoglobulin
molecules is able to trigger B cell activation (no need for
degradation)
 T cell on the other hand, only recognize an epitope as a part
of a complex formed with MHC proteins on the surface of an
antigen-presenting cell
 T cell epitopes are linear but may be molecules found  It is released slowly from the injection site
anywhere in the cell, rather than strictly surface molecules  Enhance immune response by:
1. Prolonging the existence of immunogen in the
Haptens area
 Substances that are too small to be recognized by themselves, 2. Increasing the effective size of the immunogen
but if they are complexed to larger molecules, they are then 3. Increasing the number of macrophages involved
able to stimulate a response in antigen processing
 Nonimmunogenic materials that, when combined with a
carrier, create new antigenic determinants MAJOR HISTOCOMPATIBILITY COMPLEX
 Once antibody production is initiated, the hapten is capable HLA/MHC
of reaction with antibody even when the hapten is not  Accounts for differences in how individuals respond to
complexed to a carrier molecule; however, precipitation or particular immunogens
agglutination reactions will not occur  HLA (Human Leukocyte Antigens)
 May be complexed artificially in the lab or naturally within a  Jean Dausset (French scientist) 1958
host  were first identified on circulating WBCs
Example: allergic reaction to poison ivy and penicillin  Aka MHC molecules
 Most famous study of haptens was conducted by Karl  determine whether transplanted tissue is
Landsteiner histocompatible and accepted, or recognized as
 German scientist who discovered ABO blood groups foreign and rejected (tissue rejection)
 The Specificity of Serological Reactions (1917)  MHC molecules
 Found not just on WBCs but on all nucleated cells in
Relationship of Antigens to the Host the body
 Antigens can be placed in broad categories according to their  Play a pivotal role in the development of both humoral
relationship to the host and cellular immunity
1. Autoantigens  Main function is to bring antigen to the cell surface for
 Antigens that belong to the host recognition by T cells => antigen presentation and
 Do not evoke an immune response under normal recognition (because only when antigen is combined
circumstances with MHC molecules does T cell activation occur)
2. Alloantigens  Clinical relevance:
 Are from other members of the host’s species,  transfusion reactions
 Capable of eliciting an immune response  graft rejection, and
 autoimmune diseases
 Important to consider in tissue transplantation and in
 Genes controlling expression of these molecules are the
blood transfusions
system of genes known as the major
3. Heteroantigens
histocompatibility complex (MHC)
 Are from other species
Genes Coding for MHC molecules (HLA Antigens)
 Animals, plants, or microorganisms MHC system
4. Heterophile antigen
 Most polymorphic system found in humans
 A particular type of heteroantigen
 Genes coding for the MHC molecules in humans are found
 Antigens that exist in unrelated plants or animals but on the short arm of chromosome 6
which are either identical or closely related in structure
 divided into 3 categories or classes
so that antibody to one will crossreact with antigen of
1. Class I
the other
 coded for at 3 different locations or
- Human blood group antigen, which is
loci (A, B, and C)
related to pneumococcal
2. Class II
polysaccharide type XIV antigen
found in pneumococcal bacteria  genes are situated in the D region
- Human blood group B antigen reacts  Different loci: DR, DQ, and DP
with antibody to Escherichia coli  There is a gene that codes for the
-Heart muscles and M protein of alpha chain and one or more genes
streptococcus=> rheumatic fever (cross- that code for the beta chain
reaction)
* Class I and II gene products are involved in antigen recognition
ADJUVANTS and influence repertoire of antigens to which T cells can respond
 A substance administered with an immunogen that increases 3. Class III
the immune response  Located on chromosome 6 situated
1. Aluminum salts between the class I and class III
 The only ones approved for clinical use in the regions
U.S.  Genes code for complement
 Are used to complex with an immunogen to proteins and cytokines (tumor
increase its size and to prevent a rapid escape necrosis factors alpha and
from the tissues beta)
2. Freund’s complete adjuvant  Class III proteins are secreted
proteins that have an immune
 Consists of mineral oil, emulsifier, and killed
function,
mycobacteria (0.5 mg/ml)
 they are not expressed on cell
 Produces granuloma, or large areas of scar
surfaces
tissue (not used in humans)

 Antigen is mixed with adjuvant and then injected


Alleles
 Different forms of a gene that code for slightly different
varieties of the same product
 Examples: MHC (polymorphic)
HLA-A – 580 different alleles (>2,013)
HLA-B – 921 alleles (>2,065)
HLA-C – 312 alleles (>1,551)
 The probability that any two individuals will express the
same MHC molecules is very low
 An individual inherits two copies of chromosome 6, and thus
there is a possibility of two different alleles for each gene on
the chromosome, unless the individual is homozygous (same
alleles) at a given location (genes are described as
codominant)
 Haplotype
 inherited chromosomal region
 consists of a package of genes for A, B, C, DR, DP, and
DQ
 Full genotype
 consist of two of each gene at a particular allele
 Because there are numerous alleles or variant forms at each
locus, an individual’s MHC type is about as unique as a
fingerprint
HLA Nomenclature
 Traditionally, HLA nomenclature has been defined
serologically through the use of a battery of antibodies
 Currently, advances in DNA analysis have made
identification of actual genes possible but the nomenclature
has become correspondingly more complex
 Example: HLA DRB1*1301
- Indicates the actual gene involved in coding for an
HLA DR1 antigen, with the B standing for the beta chain,
which is part of the antigen, and the 1301 indicating a
specific allele
 HLA antigens
 The uniqueness of the HLA antigens creates a major
problem in matching of organ donors because these
antigens are highly immunogenic
 However, in cases of disputed paternity,
polymorphisms can be used as a helpful identification
tool
 HLA testing can show that a man is not a
child’s father if the child has an HLA allele that
is not present in either the mother or the
alleged father or if the child does not possess
either of the two alleles that are present in the
alleged father at a given locus
 If the alleged father does in fact possess all the
alleles present in the child that are not from the
mother, he is likely a candidate, but paternity
cannot be absolutely proven
Example:
Mother: HLA A2, A3 / B8, B12 / Cw1, Cw3
Child: HLA A3, A9 / B8, B16 / Cw2, Cw1
Alleged Father: HLA A1, A9 / B7, B14 / Cw2, Cw7
 Is the man the father of the child?
 Alleles not found in the mother are A9, B16,
Cw2
 Father has A9 and Cw2, but not B16
 Result: The man is NOT the child’s father
Structure of Class I Molecules
 Each of the MHC gene codes for a protein product
that appears on cell surfaces
 All of the proteins of a particular class share structural
similarities and are found on the same types of cells
 Class I MHC molecules are expressed on all
nucleated cells, although they differ in the level of
expression
MHC Class I Molecules
 Higher on lymphocytes
 Lowest on liver hepatocytes, neural cells, and muscle
cells (explains why HLA matching is not done in liver
transplant)
 HLA-C antigens
 are expressed at a lower level than HLA-A and HLA-
B antigens
 2 most important to match for transplantation
 Each class I antigen is a glycoprotein dimer, made up
of two noncovalently linked polypeptide chains
 Alpha chain
 has as molecular weight of 45,000 (44,000)
 ß2 – microglobulin
 the lighter chain
 molecular weight of 12,000
 encoded by a single gene on chromosome 15
 does not penetrate the membrane
 Folded into three domains: α1, α2, α3; is
inserted into the cell membrane via
hydrophobic regions
 The three external domains consist of about 90 amino
acids each
 The transmembrane domain has about 25
hydrophobic amino acids along with a shorter stretch
of about 5 hydrophilic amino acids, and an anchor of
30 amino acids
 processing
 The main role of class I and class II antigens is to bind
peptides within cells and transport them to the plasma
membrane where they can be recognized by T cells
 Evolved to deal with 2 types of infectious agents
 Class I molecules present peptides that have been
synthesized within the cell to CD8 T cells
 Class II molecules contribute to antigen recognition by
CD4 T cells
 They mainly bind exogenous proteins, those taken into
the cell from the outside and degraded
 Class I molecules are the watchdogs of viral, tumor,
and certain parasitic antigens within the cell
 Class II molecules alert the CD4 T cells to the presence
of foreign proteins found outside the cell
 For a T cell response to be triggered
1. Peptides must be available in adequate supply for
MHC molecules to bind
 α1 and α2 domains each form an alpha helix and that
these serve as the walls of a deep groove that function
as the peptide-binding site in antigen recognition
(peptide-binding cleft/groove)
 Binding site is able to hold peptides that are
between 8 to 10 amino acids long only
 Most of the polymorphism (variation) resides
here
 α3 and β2 are similar to the constant regions found in
immunoglobulin molecules

Nonclassical class I antigens

 designated E, F, and G
 not expressed on cell surface except for G
 Do not function in antigen recognition but may play
other roles in the immune response
G antigens
 are expressed on trophoblast cells during the
first trimester of pregnancy
2. They must be able to be bound effectively
 thought to help ensure tolerance for the fetus
3. They must be recognized by the T cell receptor
 The difference in functioning of the two molecules is tied to
Structure of Class II Molecules
the mechanisms by which processed antigen is transported
 Occurrence is much more restricted because they are found
to the surface
primarily on antigen presenting cells (B lymphocytes,
 Both types, however, must be capable of presenting an
monocytes, macrophages, dendritic cells, and endothelium
enormous array of different antigenic peptides to T cells
 Major class II molecules are DP, DQ, and DR
 The chemistry of the MHC antigens controls what sorts of
 Consist of two noncovalently bound
peptides fit in the binding pockets
polypeptides that are both encoded by genes in
the MHC complex
Role of Class I Molecules
 DL is expressed at the lowest levels  Both class I and class II molecules are synthesized in the
 DR is expressed at the highest levels rough ER
 DR3- most polymorphic (close to 2,000)  Class I molecules bind peptides while still in the ER
 Both the α chain (mol.wt. 33,000/ 34,000) and the β  Binding helps to stabilize the association of the
chain (mol.wt. 27,000/ 29,000) are anchored to the α chain with the β2- microglobulin
cell membrane  Before binding with antigen occurs, newly
 Each has 2 domains synthesized α chains freely bind calnexin
 Α1 and β1 domains come together to form the (keeps the α chain in a partially folded state
peptide-binding site, similar to the one found in class while it awaits binding to β2- microglobulin
I molecules, however both ends of the binding cleft from
are open, and this allows for capture of longer chromosome
peptides than is the case of class I molecules (can 15)
accommodate 13-18 amino acids chains+ side chains)
 When β2-
Nonclassical class II genes:
microglobulin
DM, DN, and DO
binds,
- Products of these genes play a regulatory role in antigen
calnexin is
released, and
calreticulin
and tapasin
(+ ERP57
protein) are
 associated with the complex (help stabilize
complex for protein binding)
 Peptides that associate with the class I molecules are
approximately 9 AAs in length and derived from
partial proteolytic digestion of proteins previously
synthesized in the cytoplasm
 Digestion appears to be carried out by proteases in
large cylindrical cytoplasmic complexes
(proteosomes)
Proteosomes
 Consist of approximately 16 to 20 components that degrade
proteins in the cytosol
 Once cleaved, the peptides are then pumped into the
lumen of ER in an ATP-dependent process by
specialized transporter proteins
Transporters Associated with Antigen Processing (TAP)
 Responsible for the ATP-dependent transport from the
cytoplasm to the lumen of ER of short peptides
 TAP1 and TAP2 may function as “molecular rulers” to
measure the distance between the amino and carboxyl termini
of peptides so that only peptides of the correct size are
transported
 Most efficient at transporting peptides that are 12 residues or
less in length
 Allelic difference in TAP proteins also influence the
immune response because they help determine the types
of peptides that are best transported
Tapasin
 May help to bring TAP transporters into close proximity to
the newly formed MHC molecules so that numerous peptides
are available to them
 May also help in the loading of peptides into the class I
molecules
 Once α chain has bound the peptide, the complex is rapidly
transported to the cell surface
 Of the thousands of peptides that may be processed in this
manner, only a small fractions of them (approximately 1 in
2000) actually induce a response in association with class I
molecules (Immunodominant)
Immunodominant peptides (1 in every 2,000)
 Peptides that bind with a strong affinity to class I molecules
and that produce enough complexes to find CD8+ T cells
with a complementary receptor
 Binding is based on the interaction of only 2 or 3 amino acid
residues with the class I groove
 Different class I molecules will have slightly different
binding affinities, and it is these small differences that
determine to which particular antigen one individual will
respond
 It is estimated that a single cell may express about 105
copies of each class I molecules, so many different peptides
can be captured and expressed in this manner
 In healthy cells, self peptides are ignored by the T cells
 In diseased cells, peptides are derived from viral proteins or
proteins associated with cancerous states
 Display of hundreds of class I molecules complexed to
antigen allows CD8+ T cells to continuously check cell
surfaces for the presence of other than self-antigen
Role of Class II Molecules
 Class II molecules must be transported from the ER to
an endosomal compartment before they can bind
peptides
 While still in the ER, class II molecules associate with a
protein (Invariant chain), which may cause steric
obstruction and prevent interaction of the binding site
with any endogenous peptides in the ER
Invariant chain
 A 31- kd protein that is made in excess so that enough is
available to bind with class II molecules shortly after they are
synthesized
 May be responsible for helping to bring α and β chains
together in the ER lumen then moving them out through
Golgi complex to the endocytic vesicles
 Also serves to protect the binding site of class II molecules
 Once bound to the invariant chain, class II molecule is
transported to an endosomal compartment where it
encounters peptides derived from endocytosed, exogenous
proteins
 Antigen processing may help to unfold molecules and
uncover functional sites that are buried deep within the
native protein structure
 Invariant chain is degraded by a protease, leaving a small
fragment (corticotropin-like intermediate lobe peptide/
CLIP) attached to the peptide-binding cleft
 CLIP is then exchanged for exogenous peptides
 Selective binding of peptides may be promoted by:
 low pH of the endosomal compartment
 HLA-DM molecules help to mediate the reaction
 Generally, peptides of approximately 13 to 18 amino acid
residues can bind
 Conserved amino acids are distributed all along the actual
binding site
 Allows hydrogen binding to take place along the
length of the captured peptide
 There are also several pockets in the class II proteins that
easily accommodate amino acid side chains
 This gives class II proteins more flexibility in the
types of peptides that can be bound
 Once binding has occurred, class II protein-peptide complex
is transported to the cell surface
 On the cell surface, class II molecules are responsible for
formation of a trimolecular complex that occurs between
antigen, class II molecule, and an appropriate T cell receptor
 If binding occurs with a T cell receptor on a CD4+ T cell,
the T helper cell recruits and triggers a B cell response,
resulting to antibody formation
Clinical Significance of MHC
 Testing for MHC antigens has typically been done because
both class I and class II molecules are capable of inducing a
response that leads to
 graft rejection
 Transplantation
 Play a role in development of autoimmune
diseases
 Evidence that both class I and class II molecules play a major
role in antigen presentation has more far-reaching
consequences
 They essentially determine the types of
peptides to which an individual can mount an
immune response (Ex. hepatitis B vaccine)