Clonal Selection, Antibody Diversity & Recombination

• The adaptive immune response relies on clonal selection and expansion.
• Upon recognizing foreign antigen, an antigen-specific T cell or B cell clone is selected
• The clone then proliferates and differentiates into an effector cell: plasma B cells, T helper cells, and cytotoxic T cells.
• The human antibody repertoire has approximately 10^13 clones possible
• The human T cell receptor repertoire has approximately 10^18 clones possible
• The diversity of the human antibody and TCR repertoire is not encoded by individual genes.
• The diversity in the T cell and B cell receptor repertoire is generated through primary immunoglobulin gene rearrangement and T-cell receptor gene rearrangement.
• Structural variation occurs in immunoglobulin constant regions.

Primary Immunoglobulin Gene Rearrangement

• Hypervariable regions lie in discrete loops of the folded structure.
• Hypervariable regions are also called complementarity-determining regions (CDRs)
• Three CDRs in the heavy chain and three CDRs in the light chain variable domain contact the antigen
• Total of six CDRs in the heavy and light chains
• In a mature B cell, the variable domain of an antibody is encoded by a single V-region exon.
• Variable domains have the typical immunoglobulin fold composed of nine beta sheets
• The antibody-binding site is formed by three loops of amino acids known as CDR1, CDR2, and CDR3 (or hypervariable regions HV1, HV2, and HV3).
• The CDRs (red) are separated from each other by framework regions (yellow), which are mostly the same for all antibodies.
• The CDR3 (HV3) originates from two or three individual gene segments.
• The different variable regions (VH and VL) are not encoded in their final version in the germline.
• During development, B cells and T cells undergo rearrangement of the gene locus encoding their B cell and T cell receptor.
• The variable region exons are rearranged from 2 or 3 different gene segments: V, D and J segments.
• The V-segment encodes the CDR1 and CDR2.
• The J-segment encodes the CDR3 and the last framework region.
• The D-segment is only present in VH.
• The joint region (border) of the segments falls into the CDR3
• The CDR3 of the VH or VL chain is highly variable between different B cells.
• The light chain V-region genes are constructed from V and J segments.
• The heavy chain V-region genes are constructed from V, D and J segments.
• The light chain constant region is encoded in a separate exon.
• The heavy chain constant region is encoded in multiple separate exons
• Heavy and light chain C regions join to the V regions by splicing of the mRNA after transcription.
• Recombination of the heavy chain V-region occurs in two separate stages.
• First, a DH gene segment (D stands for diversity!) is joined to a JH gene segment.
• Then a VH gene segment rearranges to DJH to create a complete VH region exon.
• The border region of the V(D)J segments lies in the CDR3 loop.
• Random V, D, and J segment selection produces the high variability between V regions of immunoglobulins.
• Genes are organized into three clusters or genetic loci: the heavy chain locus and the K (kapa) and λ (lambda) light chain loci.
• The germline organization of the immunoglobulin heavy and light-chain loci in the human genome contains three sets of immunoglobulin chains: the heavy chain, and two equivalent types of light chains, the κ and λ chains
• Immunoglobulin gene segments that encode these chains are organized into three clusters or genetic loci: the κ, λ and heavy-chain loci
• Each locus can assemble a complete V-region sequence.
• The Heavy chain locus contains a series of C regions arrayed one after the other.
• Each C region corresponds to a different immunoglobulin isotype: IgM, IgD, IgG, IgE, IgA
• The isotype determines the effector functions of the antibody molecules.
• The first isotypes produced by B cells that leave the bone marrow are IgM and IgD.
• The expression of other isotypes (e.g. IgG) is regulated by DNA rearrangements that occur after the mature B cell gets activated later in life, in a secondary lymphoid organ called as "class switching"
• Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences
• DNA rearrangement is guided by conserved noncoding DNA sequences, i.e., conserved recombination signal sequences (RSSs) that flank the gene segments encoding the V, D, and J regions where the recombination takes place.
• Both heavy and light chain loci use a similar mechanism.
• Coding joint junctions are typically is imprecise, meaning nucleotides can be added or lost between joined segments during the rearrangement process, adding to the variability of the V-region sequence, called junctional diversity.

Enzymatic steps in RAG-dependent V(D)J rearrangement

• The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase
• The lymphoid-specific components of the recombinase, called RAG-1 and RAG-2, are encoded by two recombination-activating genes, RAG1 and RAG2.
• RAG1 and RAG2 are essential for V(D)J recombination.
• RAG-1/2 cleaves RSSs and covalently closed DNA hairpin ends
• This is only active during lymphocyte development in order to assemble antigen receptors.
• CDR1 and CDR2 are encoded within the V gene segment
• In Vl the CDR3 comprises the border of V and J gene segments
• In Vh the CDR 3 is formed by V, D and J gene segments
• The diversity of CDR3 is increased by the addition and deletion of nucleotides during the formation of the junctions between gene segments.
• RAG proteins generate DNA hairpins at the coding ends of V, D, or J segments, after which Artemis catalyzes single-stranded cleavage at a random point within the coding sequence - but near the hairpin that was first formed.
• P-nucleotides make up palindromic sequences that are added to the ends of the gene segments.
• Random N-nucleotides are added by the enzyme terminal deoxynucleotidyl transferase (TdT)
• Diversity within the immunoglobulin repertoire is generated by four processes: multiple versions of V(D)J segments that leads to different combinations, junctional diversity through the addition and subtraction of nucleotides, combinations of VH and VL, and somatic hypermutation after the B cells are activated in secondary lymphoid organs.
• The first three mechanisms contribute to approximately 10^11 different receptors.

Summary of Take Home Messages:

• The antigen receptors of lymphocytes are diverse
• The same basic mechanism accounts for diversity in T and B cells during their development.
• Immunoglobulin and T-cell receptor chains assembled by somatic recombination from sets of separate gene segments: V, (D,) J segments
• Diversity is generated by V(D) J joining, imprecise joining at the junctions, and the association of different chains
• Key enzymes mediating recombination RAG-1/RAG-2, along with Artemis, TdT, and DNA ligase.

T-Cell Receptor Gene Rearrangement

• TCR α and β chains consist of a variable (V) amino-terminal region and a constant (C) region.
• Variable domains (Vα and Vβ) can be superimposed with an antibody's VH and VL.
• The TCR gene segments are arranged similarly to immunoglobulin gene segments, and they use the same enzymes
• The a-chain locus includes V (70-80) and J (61) segments.
• The β-chain locus includes V (52), D (2) and J (7) segments.
• The T-cell receptor gene rearrangement takes place in the thymus
• The mechanics of gene rearrangement are similar for B and T cells
• T-cell receptor gene segments flanked by analogous 12 and 23 base pair recombination signal sequences (RSS)
• Recombination occurs in T-Cell receptor locus (TCRα and TCRβ)
• The most variable parts of the T-cell receptor (TCR) interact with the peptide of a peptide:MHC complex and the TCR also binds with the MHC molecule itself.
• The less variable CDR1 and CDR2 loops mainly contact the relatively less variable MHC component of the ligand
• The highly variable CDR3 regions mainly contact the unique peptide component

TCR Summary:

• T cell receptors (TCRs) of alpha:beta T cells are structurally similar to immunoglobulins due to homologous genes and the ability to assemble via somatic recombination using V(D)J segments analogous to Ig.
• The greatest amount of T cell diversity lies in the junction of the V(D)J segments, which are a part of the CDR3
• CDR3 makes contact with the peptide:MHC complex.
• TCR’s are less diverse in the Constant region

Structural Variation in the Immunoglobulin Constant Regions

• Immunoglobulin C regions can function as both a trans-membrane receptor and a secreted antibody.
• Heavy chain locus encodes C regions that determine the antibody isotype, depending of the C region used by the heavy chain.
• The light-chain C regions (CL) provide only structural attachment for V regions
• There seem to be no functional differences between lambda and kappa light chains
• The TCR C regions support the V regions, anchor the receptor into the membrane, and do not vary after assembly of a complete receptor gene IgMs are the first antibody secreted after B-cell activation and have a high molecular weight.
• Since IgMs exist as pentamers and are present in the bloodstream instead of tissues (too big to extravasate), the low number of IgMs can be compensated.
• Despite the low-affinity in the bloodstream, IgM is an excellent activator
• IgDs are co-expressed with IgM on the surface of almost all mature B cells, but secreted rarely.
• IgG (immunoglobulin G is the most abundant immunoglobulin, which contain four subclasses that are named by an order of abundance. In addition, the subclasses vary in their effector function
• Most therapeutic antibodies are IgGs, have long half lives of 2-4 weeks, and transfer across the placenta
• IgA can be found in the bloodstream and mucosal surfaces.
• This immunoglobulin is secreated to defend the gut and respiratory tract, as well as in mothers milk as monomers.
• IgE, is contained in an extra constant heavy chain domain. In addition, IgE induces most cell degranulation or activation or basophiles, which is very effective from multicellular disease to allergic diseases.
• Immunoglubulins assemble as multimers with a J chain and constant segments downstream from V(D)J segments
• V-region initially pairs with µ & d (CH) genes, which are co-expressed via alternative splicing of exons
• The antibody that a B cell secretes upon activation recognizes the antigen that initially activated the B cell – but the constant parts may vary (isotype switching).

Explore clonal selection and expansion in adaptive immunity, detailing antigen recognition and effector cell differentiation. Learn about antibody variable domain structure, CDRs, and V(D)J recombination for antibody diversity.