Download Isotype switching Diversification of Igs after B cells encounter antigens

Antigens and Antibody Structure
Lecture 2
General Concepts
 An antigen is any agent capable of binding specifically to components
of the immune system, such as the B cell receptor (BCR) and
antibodies, or the T cell receptor (TCR).
 Typically elicit an immune response.
 Can be foreign or they can be self (autoantigen).
 Sources of antigens include bacteria, viruses, parasites, pollen, tissue
and organ transplants, blood transfusion etc.
Antigen vs Immunogen
 An antigen is any agent capable of binding specifically to components of
the immune system such as the BCR or TCR
 An immunogen is any agent capable of inducing an immune response and
is therefore immunogenic
 Antigenicity is the ability of an antigen to combine with the final products
that arise from an immune response such as antibodies and cell surface
receptors
 Immunogenicity is an antigens ability to elicit a humoral (antibody mediated)
and or cell mediated immune response
 All immunogens are antigens but not all antigens are immunogens
e.g. haptens
Major classes of antigens
 Complex carbohydrates - components of bacterial cell walls, antigens
determining ABO blood groups
 Lipids- immunogenic when coupled to large carrier proteins
 Nucleic acids - poor immunogens by themselves. Can be immunogenic
when conjugated to protein carriers
 Proteins - Excellent immunogens
 Haptens - small antigenic substances. Immunogenic only when coupled to
large carrier molecules
Various properties of antigens that influence their
immunogenicity
 Foreignness - the more foreign the antigen is to the body the more
immunogenic
 High molecular weight and complexity - proteins of 100kD or more are
potent immunogens, while 5kD or less are usually poor immunogens
 Susceptibility to processing and presentation - if a macromolecule
cannot be ingested, processed and presented by APCs, it is a poor
immunogen
 Chemical nature - proteins and polysaccharides make potent
immunogens
Further requirements for Immunogenicity
 Immunogen dosage:
a. Each immunogen has a certain dose that may be required to elicit
an immune response.
b. Higher doses do not necessarily induce a stronger response.
c. Repeated doses e.g. vaccine boosters may be needed to
induce a sufficient immune response
Further requirements for Immunogenicity
 Routes of administration - different routes carry antigens to different
organs of the immune system
Subcutaneous – Generally elicit the strongest immune responses.
Due to their uptake, processing, and presentation to
effector Langerhans cells present in the skin, which
are among the most potent APCs.
Responses take place in draining lymph nodes
Intravenous – Antigens are carried first to the spleen
Gastrointestinal – Antigens elicit local antibody responses within the
intestine.
Adjuvants
An adjuvant is a substance that when mixed with an immunogen,
enhances the immune response of the immunogen
Examples:
 Aluminium hydroxide
 Aluminium phosphate (Alum)
 Freund’s complete adjuvant: (Immunogen + killed mycobacteria, oil and
water)
Adjuvant mechanisms include:
Increasing the biological or immunological ½ life of vaccine antigens
 Increasing the production of local inflammatory cytokines
 Improving antigen delivery, processing and presentation by APCs
Epitopes
 Epitope (antigenic determinant) –the part of an antigen to which an
antibody binds or gives rise to the MHC-binding peptide that is
recognized by a TCR.
 Epitopes for antibodies are exposed on the surface of antigens while
T cell epitopes are usually located in the internal regions of the
protein
 T cells do not recognize soluble native antigen but rather recognize
antigen that has been processed into antigenic peptides and
presented by MHC molecules of APCs.
 Paratope are small regions of antibodies which bind to epitopes
Epitopes
Antibody
epitope
T cell
epitope
B cell epitopes
 B cell epitope - can bind to an antigen-binding site on an antibody
molecule
 Linear epitopes – the antibody binds parts of the molecule that
are adjacent in the linear sequence (For example a string of amino
acids that are in linear sequence in a protein).
 Conformational or discontinuous epitopes – an epitope that is
formed by parts of a protein brought together in the folded protein,
but which are separated in the amino acid sequence.
B cell epitopes
Multivalent antigen – any antigen that contains more than one
epitope or more than one copy of an epitope.
Antigens can bind in pockets, grooves, or extended
surfaces in the binding sites of antibodies.
Antigens that stimulate B cells can be T cell dependent
or T cell independent
T-dependent antigens requires the presence of helper T cells to stimulate
antibody production by B cells.
T-independent antigens are able to trigger B cells to produce antibodies
without the presence of T cells.
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T cell dependent
Require T cell help
Complex proteins
Very common
Induce IgG and all other isotypes
Much stronger immune response
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T cell independent
Do not require T cell help
Simple polysaccharides
Less common
Induce IgM
T cell dependent antigens
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Require T cell help
Complex proteins
Very common
Induce IgG and all other isotypes
Much stronger immune response
Antibodies
 Soluble proteins that recognize antigens (foreign or self) with high
specificity.
 Because of their globular shape, they are called immunoglobulins
 Expressed as secreted or membrane bound forms.
 They are highly specific for an antigen
General characteristics
 The function of an immunoglobulin is to:
 Bind pathogens
 Make the pathogen a target for cells and molecules of the
immune system.
General characteristics
 The variable (V) region (varies greatly from one antibody to the
next) and is responsible for binding pathogens.
 The constant (C) region contains binding sites for receptors on
phagocytes and complement proteins and is responsible for
recruiting cells and molecules for pathogen destruction.
General characteristics
 Igs are composed of 4 polypeptide chains.
 2 identical H chains (heavy polypeptide chains) and
 2 identical L chains (light polypeptide chains)
 Linked by disulfide bond(s)
 Two types of light chains found in Igs:
 Either 2 Lambda (λ) L chains or
 2 kappa (κ) L chains
Basic Antibody Structure
 Hinge region– flexible region of the
antibody where all of the arms of the
Y come together, allows the Ig to be
flexible for binding to antigen and
proteins that mediate immune
responses.
 Fab - contains the antigen binding
site. Obtained by papain digestion
 F(ab’)2 - obtained by pepsin digestion
 FC region–the portion of the constant
region that forms the "stem" of the Y
shaped Ig molecule.
Hypervariable Regions
 Hypervariable regions (paratope)
– 3 regions of high variability within
the V domain, designated HV1, HV2,
HV3.
 Framework regions – the regions of
the V domain between the
hypervariable domains which are
relatively invariant.
 HV1, HV2, and HV3 are sometimes
called complementarity
determining regions (CDR1,
CDR2, CDR3) because they form
the antigen binding site.
General characteristics
 There are 5 types (isotypes) of heavy chains
1) Mu (µ)- IgM
2) Delta (δ)- IgD
3) Gamma (γ)- IgG………(IgG1, IgG2, IgG3 and IgG4)
4) Alpha (α)- IgA ...............(IgA1 and IgA2)
5) Epsilon (ε)- IgE
 Antibodies of different isotypes have different functions
Antibody classes or Isotypes
 The constant region of the H chain determines the Ig’s class or
isotype.
 There are 5 main isotypes of Igs
– IgA
IgD
IgE
IgG IgM
 Some Ig classes are subdivided into subclasses that are encoded
by different constant region genes that are similar in sequence.
 Subclasses are designated by a number after the isotype that
identifies the relative abundance of the subclass within the main
isotype, i.e. IgG1 is more abundant than IgG2 which is more
abundant than IgG3 in serum.
 In humans there are two subclasses of IgA and 4 subclasses of
IgG.
Immunoglobulin Variants
Isotype- Large differences in C regions that distinguish each Ig class and
subclass.
 IgM, IgG1, IgG2 etc.
Allotype- Small differences in C regions that are encoded by different
alleles of isotypic genes (Allelic difference)
 IgG1a versus IgG1b
 These differences occur in some but not all members of a species
Idiotype- Small differences in V regions within an individual
Isotype polymers
 IgM and IgA can form polymers.
 Constant regions of IgM and IgA antibodies contain a region essential for
polymerization.
 J chain–small peptide that links the polymerization region of Ig molecules to
form a polymer.
 J chains allow IgA to form dimers and IgM to form pentamers.
Neutralization and Opsonization functions of
Immunoglobulins
 Neutralization: Neutralizing antibodies directly
inactivate a pathogen or toxin by binding tightly to it and
preventing it from interacting with human cells to cause
harm.
 Opsonization: Some antibodies are opsonins,
they function in opsonization – the coating of a
pathogen with an immune system protein.
Opsonization can eliminate pathogens in two
ways:
 By inducing phagocytosis –phagocytes
have receptors that bind complement and
the Fc (constant ) region of some Igs .
 By complement mediated cell lysis–
antibodies bound to cell surfaces can also
lead to direct lysis of the cell by
complement.
Complement and Antibodies working together
IgM
1. When bound to B cells it forms the B cell receptor
2. A soluble form (pentamer) is produced in a primary immune response
3. Eliminates pathogens in the early stages of B cell mediated immunity
before there is sufficient IgG
4. Can form a pentamer therefore has high agglutination ability
5. Excellent complement fixing or complement activating antibody
Ig G
1. 80% of total Ig, therefore provides the majority of antibody related
immunity against pathogens
2. Has a longer half life (23 days) than other antibodies
3. Distributed intravascularly and extravascularly
4. Can pass through the placenta
5. Possesses both anti-viral and antibacterial activity
IgA
1. Monomeric IgA functions within fluids and tissues
2.Dimeric form is most common.
3. Dimeric form, protects mucosal surfaces (GI, respiratory and urogenital)
Predominant antibody in mucus secretions, saliva, tears, sweat and breast
milk.
Antibody classes or Isotypes
IgD
They are present on B lymphocyte surfaces
with little known activity
IgE
1. Present on mast cells and basophils
2. Binds to allergens and triggers histamine release
from mast cells and basophils (involved in allergy)
3. Protects against worms
4. It has the shortest half life of 2 days
Antibody classes or Isotypes
Elevated levels of IgM usually indicates a recent infection
The number of different antibodies that can be
produced seems limitless

Ig heavy (H) and light (L) chain genes are inherited in germline
configuration–families of fragmented gene segments that
must be rearranged before the Ig can be expressed.

Gene segments: Portions of a gene
The DNA sequence encoding a variable region is
assembled from two or three gene segments.
Light Chain
 L chain V domains are encoded by 2 gene segments:
 VL gene segment –makes up most of the V domain
 JL gene segment –joining gene segment
The DNA sequence encoding a variable region is
assembled from two or three gene segments.
Heavy Chain
 Heavy chain V domains (VH) are encoded by 3 gene segments:
 VH gene segment
 DH gene segment–diversity gene segment
 JH gene segment
 There are multiple copies of each type
of gene segment in the germline DNA.
Generation of Ig diversity in B cells before
encounter with antigen
 Somatic recombination – random recombination of different V,J gene segments in
variable region of light chains and V,D, J gene segments in variable region of heavy
chains.
 Primarily responsible for diversity of antibodies
Generation of Ig diversity in B cells before
encounter with antigen
Somatic recombination–the Ig gene rearrangement process where V,D,
and J gene segments are cut and spliced.
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Involves one rearrangement event (V-J) in the L chain gene and 2
rearrangement events (V-D-J) in the H chain gene.

Occurs only between gene segments on the same chromosome.

Involves enzymes that specifically recognize, bring together, cut
and then rejoin DNA.
V(D)J recombinase– the set of enzymes needed to recombine V,D and J segments
Naive B cells use alternate mRNA splicing to make IgM and
IgD.
 After Ig gene rearrangement the heavy chain locus can be transcribed and
functional mRNA is produced by removing introns from the primary
transcripts.
 The same rearranged V region gene can be used with any of the C region
genes.
 Naive B cells– B cells that have not encountered specific antigen – express IgM
and IgD.
Each B Cell Produces Ig of a Single Antigen
Specificity.
 Allelic exclusion–a process of using genes from only one parental
chromosome which assures that each B cell expresses only
one type of heavy chain and one type of light chain and therefore
antibody of a single specificity.
or
Ig is first made in a membrane-bound form that is
present on the B cell surface.
 B cell receptor (BCR)–the cell surface
complex of an immunoglobulin and 2 other
proteins (Igα and Igβ).
 Igα and Igβ proteins–two invariant
transmembrane proteins with long cytoplasmic
tails that interact with intracellular signaling
molecules when antigen is bound by the BCR.
 Igs with a transmembrane domain must be
associated with Igα and Igβ proteins to be
transported to the surface of the B cell.
Diversification of Igs after B cells encounter
antigens
 After an encounter with antigen, IgM and IgD isotypes can be produced
as secreted antibodies
 IgM antibodies are produced in large amounts and are important in
protective immunity
 IgD antibodies are produced only in small amounts and have no
known effector function
 All the isotypes of Ig can be made in two forms:
1) membrane bound BCR, or
2) secreted antibody
Secreted Igs (antibodies) are produced by an alternate pattern of heavy
chain mRNA processing.
 The membrane anchor at the carboxy terminus of membrane bound Ig is
encoded by one exon.
 The carboxy terminus of secreted Ig is encoded by an alternate exon.
 During differentiation to a plasma cell mRNA is alternately processed and from
that time secreted Igs are produced.
Diversification of Igs after B cells encounter
antigens
 Somatic hypermutation – mutations that occur in the V genes of heavy
and/or light chains of a B cell.
 Competes mot effectively for binding to antigen
 Preferentially selected to mature into antibody producing plasma cells
 Leads to affinity maturation (progressively higher affinity for antigen)
 Isotype switching – an individual B cell switches to make an antibody of a
different class such as IgG, IgA or IgE.
 Antigen specificity is the same
 Effector functions are different
Diversification of Igs after B cells encounter
antigens
Somatic hypermutation
 Somatic hypermutation – mutations that occur in the V genes of heavy
and/or light chains of a B cell.
 Affects V regions only, not C regions or any other B cell genes.
 Occurs at a rate of 1 mutation per cell division –1 million times the mutation rate
normally seen in genes.
Diversification of Igs after B cells encounter
antigens
Somatic hypermutation
 Binding of antigen stimulates B cells to divide rapidly and differentiate into
plasma cells.
 Somatic hypermutation gives rise to B cells with mutant Ig on their surfaces.
Some of these have a higher affinity for antigen. They will be more likely to bind
antigen and generate plasma cells.
 Somatic hypermutation results in affinity maturation–the production of
antibodies with progressively higher affinities during the course of an immune
response. Seen particularly after secondary and subsequent exposure to antigen
(example: after booster vaccinations).
Diversification of Igs after B cells encounter
antigens
Isotype switching
 Isotype switching produces Igs with different C regions but identical antigen
specificities.
 IgM is the first Ig produced in an antibody response but has limited mechanisms for
fighting pathogens.
 Antibodies with other effector functions can be generated by isotype switching – a
further DNA recombination event that allows the rearranged V region gene to
associate with other heavy chain C region genes.
Diversification of Igs after B cells encounter
antigens
Isotype switching
 Isotype switching is stimulated by cytokines released by T cells during the
course of an immune response after antigen stimulation.
 Switch regions – stretches of repetitive DNA which guide recombination that
results in isotype switching. Switch regions lie in introns upstream of all of the
heavy chain constant regions except the one that gives rise to IgD.
Diversification of Igs after B cells encounter
antigens
Isotype switching
 Once one isotype switching event has occurred a portion of DNA has been
removed and the switch is not reversible, however a subsequent event may
occur involving a downstream C region gene.