Lecture 7: Genetics of Antigen Receptors

Basic Requirements of The Immune System

• Aim of the immune system is to recognise and remove all the dangers and mount a response to neutralise this all whilst avoiding self-harm through mistargeting/ collateral damage

Innate Immunity

• Broad recognition achieved via pattern recognition receptors – recognise PAMPs/MAMPs and DAMPs

• They are encoded in the Germline and by a single gene

  • They are Inherited – evolved to recognise molecular patterns typical of pathogens, microbes’ cells under stress, i.e. danger

• Has evolved over the years to recognise these patterns

Adaptive Immunity

• BCRs (antibodies) and TCRs recognise specific epitopes of pathogens and bacterium

  • There are over 20,000 Ab-specific genes, allowing for high specificity.

• They are somatically generated via rearrangement of gene segments in developing B and T cells.

• These receptor genes are inherited as partially finished segments (gene pools), which are reorganised during development. → somatic generation

• This enables a huge diversity of receptors, despite being encoded by relatively few genes.

Features and Implications of Diversity in the Adaptive Immune Receptor Repertoire?

• The system generates a highly diverse, random repertoire of receptor specificities.

• No prior exposure to pathogens is needed.

• Enables broad recognition and makes it hard for pathogens to evade detection.

• Risk: autoimmunity – potential development of self-reactive receptors.

• Clonal system – each B or T cell expresses only one type of receptor

Clonal Selection

• During development, progenitor cells give rise to large numbers of NAÏVE circulating lymphocytes, each with a different specificity

• When a lymphocyte recognises an epitope in a dangerous context, this useful clone is expanded to combat the threat.

• This also generates MEMORY cells, which will respond faster and better if the same threat is encountered again.

BCRs and TCRs

• They are multi-chain receptor complexes with chains that are specialised for recognition and signalling following recognition

B-Cell Receptor

• Fab region contains immunoglobulin folds and recognises native, unprocessed antigens.

• Upon activation, the receptor becomes a soluble antibody that binds the antigen.

• The Fc region mediates effector functions via interactions with Fc receptors (FcRs) on other immune cells.

• Different antibody isotypes (e.g., IgG, IgA) engage different effector mechanisms through the Fc region.

T-Cell Receptor

• Antigen must be processed, with peptides present in the context of MHC for recognition

• Fab region contains immunoglobulin folds

• Effector Mechanism: Mediated by cell-to-cell contact or local secretion of cytokines

• These receptors mediate their effects locally → don’t need to make secreted forms of the receptor

Structure of Typical Antibody (IgG)

• Composed of 2 identical heavy chains and 2 identical light chains.

• Light chains can be of two types: kappa or lambda, both functionally similar.

• N-terminal ends of both chains have variable regions (at first immunoglobulin domains), followed by constant regions.

• In heavy chains, the constant regions differ by isotype (e.g. IgG1).

• The antibody forms a Y-shaped structure with Fab regions (antigen binding) and an Fc region (effector functions).

How does the antibody variable region contribute to antigen binding?

• Kabat and Wu sequenced many antibody variable regions and analysed amino acid variability.

• They identified 3 hypervariable regions (CDRs) responsible for antigen binding.

• These regions are located in loops of the variable domain, allowing high variability without disrupting structure.

• Framework regions (shown in yellow) are less variable to maintain the structural immunoglobulin fold.

• The light chain immunoglobulin fold and heavy chain combine to form a beta-barrel, positioning the hypervariable CDRs to create the antigen-binding site.

Formation of B-Cell Receptor

• occurs via lineage-specific, developmentally regulated somatic recombination of gene segments.

• Only B cells rearrange immunoglobulin genes to form BCRs;
  TCR genes rearrange only in T cells.

• These gene rearrangements occur at defined developmental checkpoints during lymphocyte development and maturation.

Structure of a Typical Gene

• It is inherited as introns and exons in genomic DNA.

• It contains a promoter, followed by sequences transcribed until a stop signal, after which a poly-A tail is added.

• Splicing machinery removes introns, forming mature mRNA.

• The ribosome reads the single open reading frame in the mRNA to synthesise a protein.

Structure of Kappa Light Chain

• The constant region is encoded by the C-exon.

• The variable region is inherited as separate V and J gene segments (not full exons → incorrect ends).

• In genomic DNA, V, J, and C regions are separate.

• During B-cell development, as a cell commits to the B-cell lineage, transcription machinery is activated, and epigenetic changes loosen the chromatin around the light chain locus.

• Somatic recombination joins a specific V segment (e.g., V36) to a J segment (e.g., J3) to form a functional V exon.

  • occurs at the level of the genomic DNA

• The primary transcript still includes the remaining J segments in an intron, which are removed during mRNA splicing.

• The resulting mature spliced mRNA is translated by ribosomes into a kappa light chain protein.

Gene Rearrangement

• Process where different B-cells choose different V and J segments through gene re-arrangement

• It results in clonal B-cells with light chains that have different antigen-binding properties

Generation of Complementary Determining Region (CDR)

• CDR1 and CDR2 are both encoded within the V (variable) kappa gene segment, and their variability depends on which V segment is selected for recombination.

• CDR3 spans the V-J junction:

  • The beginning of CDR3 comes from the V segment,

  • The end from the J segment,

  • The join introduces additional variability.

• This makes CDR3 the most variable of the CDRs.

Arrangement of Gene Segements at Human Immunoglobulin Gene Loci

• Light chains locus located on different chromosomes sit next to each other→ (κ and λ) involve rearrangement of V → J segments on separate chromosomes.

  • E.g., Vλ2 joins Jλ2, eliminating any DNA in between, forming the V exon and the C-terminus

• Heavy chains (IgH) contain an additional D (Diversity) segment → require two rearrangements:

  • D → J, then V → DJ.

• The final rearranged V exon is VDJ.

• CDR1 & CDR2 are encoded within the V segment.

• CDR3 is formed from the V, D, and J segments, with both joins contributing to its extreme variability.

Combinatorial Diversity in Immunoglobulin Gene Rearrangement

• It arises from the random recombination of V, D, and J segments:

  • Heavy chains (H): 40V × 23D × 6J = ~5520 combinations.

  • Light chains (κ + λ): 70V × 5J = ~350 combinations.

• Heavy + Light chain combinations:

  • H + L: 5520 × 350 = ~1.9 million combinations.

• Joining imprecision: During recombination, variability is introduced due to imprecision at the V-D, D-J, and V-J joins, increasing (junctional) diversity.

How do recombination signal sequences (RSS) and the 12-23 rule guide the V-D-J joining process?

• Each gene segment has a recombination signal sequence (RSS):

  • Heptamer, spacer (12 or 23 base pairs), and nonamer.

• The 12-23 rule ensures proper joining V-D-J joining order:

  • A segment with a 23-base pair spacer will join with a segment that has a 12-base pair spacer.

• Enzymes in the recombination machinery recognise these sequences and pull the ends of segments together, resulting in a cleavage near the heptamer, close to the V and J segment

• The intervening DNA sequence is removed bringing the segments together to form the V-D-J exon.

Molecular Mechanics of V(D)J Recombination

• V and J segments each have a recombination signal sequence (RSS) with 12 and 23 base pair spacers.

• As B-cells mature, they activate RAG1 and RAG2 genes, which, along with other proteins, recognise these sequences and pull the segments together.

• This causes a cleavage near the heptamer, forming hairpin loops at the coding ends.

• The coding ends are recognised as damaged DNA by DNA repair machinery, which nicks the ends to allow the V and J segments to join.

• The nicking process and subsequent end-resolution introduce some imprecision at the joining site.

• The enzyme TdT (a lymphoid-specific enzyme) adds random nucleotides to the junction, increasing diversity.

Generation of Junctional Diversity Through Imprecise Joining of Gene Segments

• RAG enzymes bind to D and J segments, pulling them together and cleaving the RSS heptamer, forming coding ends.

  • Coding ends are nicked and unfolded.

  • Palindromic P nucleotides are added as the DNA is unwound and is palindromic.

  • TdT adds N-nucleotides (random nucleotides) to the junction.

  • The strands pair and align until a good match is achieved.

  • Exonucleases trim and fill in the ends to form the coding joins.

• P-nucleotides are present at the ends, contributing variability to CDR3.

• Imprecision in the process, like random codon insertions, can lead to frameshifts or non-productive joins (e.g., stop codons, out-of-frame joins).

Combinatorial Diveristy

• Source of diversity in the primary antibody repertoire

• Multiple gene segments

• any H with any L

Junctional Diveristy

• Source of diversity in the primary antibody repertoire

• P-nucleotide addition

• N-nucleotide addition

• exonuclease activity – removal of things out

• most of this diversity is localised at the CDR3 joins

T-Cell Development

• Cells develop in the bone marrow with the progenitors moving to the thymus to make alpha beta and gamma delta t-cells

• alpha/beta (ab) T-cells:

  • most abundant type of T-cell (~95%)

• gamma/delta (g/d) T-cells:

  • play roles in immune responses at epithelial surfaces. Innate?

How are the TCR alpha and beta chains encoded in the germline, and how do they compare to antibody chains?

• TCR gene loci consist of multiple gene segments.

• The TCR alpha chain is similar to the immunoglobulin light chain:

  • Formed from V and J segments that together make the V-exon.

• The TCR beta chain is similar to the immunoglobulin heavy chain:

  • Includes D, J, and V segments.

  • Rearrangement occurs in two steps: D + J, then V → DJ to form the functional beta chain

TCR Gene Segment Recombination

• TCR gene segments are flanked by Recombination Signal Sequences (RSSs).

• These RSSs are recognised by the same lymphoid-specific RAG1 and RAG2 proteins used in B cells.

• T and B cells share a similar gene organisation and recombination mechanism.

• Epigenetic changes loosen chromatin around the TCR α and β loci to allow recombination machinery access.

• B cell receptor genes recombine in B cells during development in the bone marrow.

• T cell receptor genes recombine in T cells during development in the thymus.

Comparison of Diversity Sources in Immunoglobulins vs. αβ TCR

• TCRs exhibit greater junctional diversity than immunoglobulins.

• Both immunoglobulins and TCRs incorporate P-nucleotides at coding joins (due to hairpin resolution).

• TdT (Terminal deoxynucleotidyl Transferase) adds N-nucleotides:

Terminal deoxynucleotidyl Transferase (TdT)

• It is active during heavy chain rearrangement, contributing to greater variability in heavy chain joins

• Its activity decreases during light chain rearrangement, leading to reduced variability in light chain joins

  • Turned on during both α and β chain rearrangements in T cells → adds N-nucleotide diversity in both chains.

Difference in Reading Frame flexibility in D segments between TCR β chains and immunoglobulin heavy chains

• TCR β chains: D segments can be read in 3 frames, and with 2 recombination joins (V-D & D-J), there's more flexibility to stay in-frame → higher chance of producing a functional receptor.

• Ig heavy chains: Also have 3 potential reading frames, but out-of-frame joins often lead to stop codons, resulting in non-productive rearrangements.

• Overall: TCR β chains allow for more diversity without loss of function.

Sources and Distributions of Diversity between Immunoglobulins and T Cell Receptors

• Both use somatic recombination of gene segments → similar level of combinatorial diversity.

• TCRs have greater junctional diversity due to:

  • D segments read in all 3 reading frames.

  • TdT activity at all junctions.

• More J segments in TCRs.

  • TCR diversity is concentrated in CDR3, which has the main contact with the peptide presented by MHC.

• Greater CDR3 diversity in TCRs → directly interacts with the antigenic peptide in the MHC groove.

Clonality in B-Cells

• Each B cell inherits:

  • 2 heavy chain alleles

  • 2 light chain kappa alleles

  • 2 light chain lambda alleles

• Clonality requires expression of only one heavy and one light chain allele from the parents.

• This is enforced by checkpoints through:

  • Allelic exclusion (prevents both alleles of a gene from being expressed)

  • Isotypic exclusion (only one type of light chain—kappa or lambda—is used)

Mature Activated B-Cell

• Only 1 productively rearranged heavy chain allele is active

• Only 1 productively rearranged light chain allele is active

• All other alleles are excluded from expression

Gene Rearrangements in B Cell Development

• Stepwise, regulated process:

  1. Heavy chain rearrangement starts first:
     • D joins to J segment
     • Then V joins to DJ to form the V exon

  2. If a productive heavy chain is formed (passes the checkpoint):
     • Rearrangement of the other heavy chain allele is stopped
     • Light chain allele rearrangement begins

  3. If the first heavy chain rearrangement is non-productive:
     • Rearrangement occurs on the second chromosome
     • If still non-productive, the cell is eliminated

  4. Light chain rearrangement:
     • V joins to J
     • If unsuccessful on the first allele, attempts rearrangement on the second allele
     • Must pair with heavy chain to produce a functional antibody on the surface

Heavy Chain Rearrangement in B-Cell Development

• D to J, then V-D to J; first checkpoint occurs

• If a productive heavy chain is formed, it combines with a surrogate light chain and reaches the surface

• Crosslinking with the surrogate light chain signals the cell to produce more heavy chains

• RAG genes are active during this phase of recombination

RAG Gene Activity During B-Cell Development

• RAG genes are active during the recombination steps

• RAG genes temporarily turn off during the proliferation phase

• This results in the production of multiple B cells with the same heavy chain but potentially different light chains

• Once proliferation finishes, heavy chain genes become inaccessible

Light Chain Rearrangement in B Cell Development

• After proliferation, light chain genes become accessible

• RAG genes are reactivated and V to J rearrangement occurs

• If this doesn't work, rearrangement proceeds on the lambda chain

• A functional light chain that can combine with the heavy chain and reaches the surfaces, signals to turn the RAG genes off

TCR Alpha and Beta Chain Rearrangement

• The beta chain of the TCR binds with a surrogate alpha chain

• TdT is active throughout the rearrangement of the alpha and beta chains

• The rearrangement of the alpha chain contributes to the diversity in CBR3

Fate of BCRs Following B-Cell Activation

• BCRs are made soluble and become an antibody → recruit effector function by binding with the antigen through the Fc receptor

Membrane Bound BCRs

• The C-terminal region of the membrane-bound BCR contains cytoplasmic and transmembrane domains, which are encoded by the ‘yellow’ exons.

• A splice site between the ‘blue’ exons (coding for the antibody region) and the ‘yellow’ exons allows for alternative splicing to generate the membrane-bound form of the BCR.

Secreted BCR

• Generated by alternative RNA processing.

• The C-terminus o is encoded by the blue exon.

• Upon B cell activation, an alternative polyadenylation (poly A) site is used, which determines where the polyadenylation signal is added.

• The yellow exons (which encode the transmembrane domain) are no longer spliced in.

• Instead, the transcript runs through the blue exons to the next stop codon, giving rise to a splice site for the secreted form.

Role of C-Region Exons in Class Switching

• Class switching is organised within the heavy chain gene locus.

• C-region exons for different isotypes (IgM, IgD, IgG, IgE, IgA) are located 3’ of the JH gene segments.

• These C exons are lined up along the genome, allowing a B cell to take its recombined V segment and join it to different C-region exons.

• This enables a single B cell clone to switch the antibody isotype it produces, while retaining antigen specificity.

• Although each B cell has the potential to express all isotypes, it only expresses one at a time (except for IgM and IgD, which can be co-expressed before class switching).

Co-Expression of IgM and IgD

• Occurs early on B-cell development → regulated by alternative RNA processing

• The only time that two isotypes are expressed at the same time

  •  If it stops at the polyadenylation site = IgM.

  • If it uses the other 2^nd polyadenylation site=IgD

Mechanism Behind Isotype/ Class Switching in B-Cells

• Isotype switching occurs via recombination between switch (S) regions upstream of constant (C) region exons.

• For example, to switch from IgM to IgE, recombination loops out the DNA between the Sμ (IgM) and Sε (IgE) regions.

• This process is irreversible, as the intervening DNA is excised and lost.

• It allows the same VDJ exon to be expressed with a different C-region, changing the antibody isotype without altering antigen specificity

Process Following Activation of Antibody Clones from the Primary Repertoire

• Affinity maturation occurs in germinal centres, introducing mutations in the V regions (somatic hypermutation).

• Primary repertoire is formed in the bone marrow; a secondary wave of diversification happens in germinal centres.

• Mutations in framework regions can disrupt antibody structure → these cells are not selected.

• B cells producing higher-affinity antibodies are preferentially selected and expanded.

Somatic Hypermutation → Antibody Diversity

• Occurs in germinal centres during immune response.

• Introduces point mutations into the heavy chain V-region, especially in CDRs (CDR1 & CDR2 )

• Leads to affinity maturation— improves antibody response (higher affinity) overtime via cycles of mutation and selection

• (Note): Would be harmful in T cells due to risk of generating self-reactive TCRs—T cells must remain tolerant.