University of Balamand

Faculty of Medicine and

Medical Sciences

IMUN402
Lecture#4
Genetics of Antibodies and Lymphocyte Receptors /
Antibody responses/Isotype switching

Instructor: Samer Bazzi, PhD

- Diversity (repertoire) is the total sum of all Ab specificities that an organism is capable of expressing
- Theories:
• Germ line theory: different V region genes for each Ab
• Somatic mutation theory: few V region genes are present, diversity generated by somatic mutation.
How big is the Ig repertoire?
Answer: the book says > 1011. It is so big that an individual may not be able to express the whole repertoire at
one time. In reality, the expressed repertoire may not exceed 10-20 million different specificities.

How is it possible to generate immunoglobulins (Igs) and many millions of B or T cell receptors
with few hundred genes?
Answer: gene rearrangements (somatic DNA recombination) for B and T cells, and somatic
hypermutations (only in B cells)

Immunoglobulin Genetics: History Antigen Binding Specificity: Ig/B-Cell Receptor

- Same C region gene could associate

with many V region genes (IgG Abs
with different specificities)

- Same V region gene could associate

with many C region genes (same
idiotype on IgG and IgM Abs)

- V and C regions are coded for by

separate genes in both H and L chains
 Primary Immunoglobulin Gene Rearrangements and Expression
Three genetic loci encode immunoglobulin molecules:
- 1 locus (IGH) encoding the heavy chain (human chromosome 14)
- 2 loci (IGK and IGL) encoding the light chains:
• Kappa (κ) locus (human chromosome 2)
• Lambda (λ) locus (human chromosome 22)
- The 3 loci are located on different chromosomes and have a unique structure composed of "gene segments"

Heavy Chain Locus

- Has multiple V (variable) segments, multiple D (diversity) segments, multiple J (joining)
segments and multiple C (constant) segments.
- During maturation, one of each V, D, J and C segment is randomly “chosen” and used to encode the final
antibody molecule.

Light Chain Locus

- Has multiple V (variable) segments, multiple J (joining) segments and multiple C (constant) segments
- A kappa chain is encoded by one V segment, one J segment and one C segment.
Heavy and Light Chain Gene Segments
- In heavy chains, the V, D and J segments encode the variable domain while the C segment
encodes the constant domain.

- In light chains, the V and J segments encode the variable domain while the C segment encodes
the constant domain.

Heavy Chain Light Chain

 Production of Heavy Chain of Ig

- B cells undergo a strictly programmed series of gene

rearrangements in the bone marrow.

- In the B-cell stages of development, gene

rearrangements proceed to assemble the Ig molecule.

- Rearrangement starts with the D and J segments.

- VDJ rearrangements in DNA produce the diversity

of heavy chain variable domains
 Production of Light Chain of Ig

- VJ rearrangements in DNA produce the

diversity of light chain variable domains

- K or λ constant domains are added to

complete the light chain
Expression of IgM and IgD Differential Splicing for µ and δ H Chains
- Whether a cell expresses mu (µ)
and/or delta (δ) heavy chain
depends on RNA processing
- Same primary transcript, but
different processed transcripts.
- Both IgM and IgD (of identical
specificity) are co-expressed on most
mature naive B cells. Immature B
cells make mostly mu transcripts
Mechanism of DNA Rearrangements
- Rearrangement takes place at the correct location relative to coding region and is guided by DNA
sequences called recombination signal sequence (RSS) that flank each gene segment.

- These sequences are recognition sites for the joining process.

- Recombinase enzyme complexes RAG1 and RAG2 (recombination activating genes 1 and 2)
recognize the RSS and catalyzes the joining process.

- Deficiency in RAG1 or RAG2 can produce nonfunctional B cells.

 Order of Ig Gene Expression
- A cell always starts by rearrangement of H chain

Random use of
paternal or
maternal genes

- Only when H chain rearrangement stops, L chain rearrangement can start

 Order of Ig Gene Expression
- For light chain, a cell always starts with Kappa genes
 Parameters contributing to Ab diversity

- Having multiple V, D, and J segments

- Rearrangements of the V, D, and J segments

- Junctional diversity: while heavy chain gene segments are undergoing recombination, the
enzyme terminal deoxyribonucleotidyl transferase (Tdt) randomly inserts bases at the junctions of
V, D, and J segments (N-nucleotide addition). The random addition of the nucleotide generates
junctional diversity

- Combinatorial diversity: diversity is created by the random pairing of the heavy and light
chains.

- Somatic hypermutation: point mutations occur with repeated antigen stimulation (from primary
to secondary responses). This increases affinity to antigen and creates additional diversity to the
antibody. This process occurs in peripheral lymphoid organs.

• Mutations that alter aa in C region disrupt the basic structure of Ab and are selected against

- Ig diversity is ~ 1011
 Germline Organization of Human TCR Loci
- T-cell receptor gene rearrangement takes place in the thymus

- The T-cell receptor β-chain is comparable to the heavy chain Ig genes and they rearrange first

- The T-cell receptor α-chain gene is comparable to the Ig κ and λ light-chain Ig genes in it does does
not have D gene segments and is rearranged only after its partner receptor β-chain has been expressed.
As with the immunoglobulin light-chain genes, repeated attempts at rearrangement are possible.
 TCR Gene Rearrangements
- The same RSS and the same enzymes are used to rearrange both the TCR genes and the Ig genes.
- Nucleotides are added at the junctions between rearranged segments.
- The C region of TCR has no effector function, but serves as part of membrane-bound Ag receptor
linking the V region to a receptor-associated signaling complex.
 Mechanisms of Generating TCR Diversity
1- Combinatorial V(D)J joining

2- Alternative joining of D gene segments

3- Junctional flexibility

4- N-region nucleotide addition

* Somatic hypermutation does not appear to be important

- Potential diversity generated is ~ 1016 α,β TCRs and ~1018 γ,δ TCRs
Phases of Humoral Immune Responses
 Fate of the Immunogen
Clearance after 1o Exposure
- Equilibrium phase: Ag equilibrates between
vascular and extra vascular compartments by
diffusion. This is rapid process but does not
apply to particulate Ag.

- Catabolic decay phase: host cell enzymes,

mostly in Macrophages, catabolize Ag and the
duration of this phase is Ag- and host-
dependent.

- Immune elimination phase: Ag combines with

Ab and the complex is phagocytosed and
degraded. Ab titer in serum increases after this
phase

Clearance after 2o exposure

- More rapid onset of immune elimination phase
because of presence of Ab in serum or tissues
 Kinetics of the Ab Response
T-Dependent Ag; 1o Response
- Lag phase (induction): Ag is recognized as foreign and cells begin to proliferate and
differentiate. It takes 5-7 days
- Log phase: exponential phase where Ab [C] increases exponentially as B cells differentiate into
plasma cells.
- Plateau phase: steady state, Ab synthesis is balanced by Ab decay.
- Decline phase: rate of Ab synthesis is lower than rate of Ab decay and level of Ab keeps falling
down to reach baseline.
 Kinetics of the Ab Response
T-Dependent Ag; 2o Response
- Lag phase: shorter than 1° response due to presence of memory cells
- Log phase: more rapid and of higher magnitude
- Plateau phase
- Decline phase: slower than 1° response with Abs persisting for months-years

Specificity: 2° responses are induced against determinants recognized in the 1° response.

However, closely related determinants may produce secondary responses on rare occasions.
 Qualitative Ab Changes during1o and 2o Responses
Class variation
- 1o Response: IgM
- 2o Response: IgG, IgA or IgE
with IgG persisting during in 2o
response
Affinity Maturation
Due to somatic mutation after class
switching; the strength of binding of
IgG increases upon secondary
challenge, specially to low Ag dose,
because high affinity clones are
mainly triggered to proliferate (clonal
selection).
Cellular Events in 1o Response to T-Dependent Ags
- Lag: Clonal selection: clones of naïve T and B cells with appropriate Ag receptors will bind to
Ag, become activated and proliferate. Few expanded B cells become plasma cells

- Log: Completed differentiation. Ab produced is IgM and B cells switch to produce other classes
with higher affinities.

- Stationary: with depletion of Ag, T and B cells are no longer activated. Additional regulatory
mechanisms (such as anti-idiotypic Abs) start to operate and plasma cells start to die

- Decline: no new Ab is made, Ag is not there.

- Memory: some stimulated B and T cells do not die.

Cellular Events in 2o Response to T-Dependent Ags
- Lag phase
• Virgin cells
• Memory cells

- Log phase
• Pool size
• IgG, IgA or IgE

- Stationary

- Decline
• Sustained production
Features of Primary (1°) and Secondary (2°) Ab responses
 Kinetics of Ab Response to T-Independent Ags
- 4 Phases (Lag, Log, Plateau, Decline)

- IgM Ab; No or little isotype switching

- No or little affinity maturation

- No secondary response; no memory cells (rarely seen with few Ags due to memory B, but
not T cells)
 Membrane versus Secreted Ig molecule
-Each constant domain-encoding
region ends by a hydrophilic Differential Splicing to Generate Secreted and
secretory tail (S) followed by Membrane Forms of µ H Chains
hydrophobic membrane domain made
of 2 exons, one encoding a
transmembrane region
(M1) and the other a
cytoplasmic Tail (M2).

- Differential RNA splicing and

polyadenylation can
either lead to a secreted
or to a membrane-bound protein.

- Immature B cells express mostly

IgM whereas mature ones express
both IgM & IgD
 Isotype/Class Switching
- In order to change the function of the Ig molecule and keep the specificity, one needs to
conserve the light chain and the heavy chain variable region, but to replace the heavy chain
constant region.
- IgM to other Igs → the constant region of the heavy chain (C) changes the μ (IgM) segment to
γ (IgG), ε (IgE), or α (IgA).
- This process is driven by antigenic stimulation, occurs in peripheral lymphoid tissues, is not
random and requires additional stimuli (i.e. Th-cytokines and CD40L). Certain cytokines can
direct selective switching: IL-4→IgE or IgG4; IFN-γ→IgG1 or IgG3; TGFβ/IL-5→IgA.
- The process involves a DNA rearrangement at switch sites, present in the introns of the
constant region of H chains, and implicates enzymes that are different from those used in VDJ
rearrangement at RSS.
Organization of the Heavy Chain C regions

 = pseudogene

~200,000 bp
Note: Cm is always first and Cd is always second
- To make isotypes other than µ and δ requires gene splicing (i.e., switching*)
- Switch regions mark the sites for isotype switching.
- All switch regions consist of short (~20bp) DNA sequences that are repeated about 150 times.
- Switch regions are in the introns (not next to exons like the RSS) upstream of all C regions
genes except Cd. Thus no cell can switch if only producing IgD

- Since switch sequences are in the

introns, there are no frame shift
mutants and all switches are
productive
*Usually, one refers to VJ and VDJ
splicing as “rearrangements” and
isotype splicing as “switching”
Role of the Complement Protein C3d in B cell Activation
Antigen Receptor-Mediated Signal Transduction in B Lymphocytes
Functional Consequences of Ig-Mediated B Cell Activation