Adaptive Immunity II – B-Cell Receptor and Antibody Diversity

Learning Outcomes

• Discuss generation of B-cell receptor (BCR) and antibody diversity, focusing on:

  • Genetic organisation of immunoglobulin (Ig) loci.

  • Molecular mechanisms that enlarge the antibody repertoire:

  • V-(D)-J recombination & junctional diversity.

  • Somatic hypermutation (SHM).

  • Class-switch recombination (CSR) and variable heavy (H)/light (L) chain pairing.

Antibody Repertoire & Quantitative Perspective

• Antibody repertoire = total number of distinct antigen‐binding specificities in one individual.

• Potential specificities ≈ 10^{8} (≈ 1\times10^{8} – 10^{11} quoted).

• Human protein-coding genes ≈ 2.5\times10^{4}; therefore diversity cannot arise from one gene per antibody → requires somatic DNA rearrangement.

Historical Landmark

• Susumu Tonegawa (1976) discovered somatic recombination in lymphocytes → 1987 Nobel Prize.

Mechanisms Generating Antibody Diversity (Overview)

1. Combinatorial diversity from gene-segment rearrangement ( V-(D)-J ).

2. Junctional diversity during those rearrangements (imprecise cutting/adding nucleotides).

3. Somatic hypermutation after antigen activation.

4. Variable combinations of independently generated H and L chains, including CSR.

Genetic Organisation of Immunoglobulin Loci

• Chromosomal localisation:

  • Heavy-chain (H) locus (isotypes \alpha,\,\delta,\,\varepsilon,\,\gamma,\,\mu) → chromosome 14.

  • Light-chain \lambda locus → chromosome 22.

  • Light-chain \kappa locus → chromosome 2.

• Germ-line arrangement (each locus contains):

  • V (variable), D (diversity – H only), J (joining), C (constant) gene segments.

• Order on chromosome (5′→3′): multiple V → multiple D (H only) → multiple J → series of C genes (isotypes).

V-(D)-J Recombination

Basic Rules

• Light chains ( \kappa,\,\lambda )

  • Require one recombination event: one random V joins one random J → \text{V}J.

• Heavy chains

  1. First: one random D joins one random J ( D\,J ).

  2. Second: one random V joins the pre-formed DJ → V\,DJ.

Quantitative Combinatorial Diversity (Human)

• \lambda-L: 30\,V \times 4\,J = 1.2\times10^{2} combinations.

• \kappa-L: 40\,V \times 5\,J = 2.0\times10^{2} combinations.

• Heavy: 40\,V \times 25\,D \times 6\,J = 6.0\times10^{3} combinations.

• Potential unique H+L pairings:
  \begin{aligned}
  6.0\times10^{3}\;H\times(2.0\times10^{2}+1.2\times10^{2})\;L &\approx 2.0\times10^{6}
  \end{aligned}
  (Only from 150 total germ-line segments → clearly insufficient vs 10^{8} repertoire → need further mechanisms.)

Recombination Signal Sequences (RSS) & 12/23 Rule

• RSS = conserved non-coding motifs flanking each V, D, J segment.

  • Structure: heptamer (7 bp) – spacer (12 bp or 23 bp) – nonamer (9 bp).

• 12/23 rule: segments flanked by a 12 bp spacer RSS can recombine only with segments flanked by 23 bp spacer RSS → ensures correct gene-segment order (e.g., prevents V joining directly to J in heavy locus).

Enzymatic Machinery (V-(D)-J Recombinase Complex)

• RAG-1 & RAG-2: recognise RSSs, introduce double-strand breaks.

• DNA-PK (DNA‐dependent protein kinase) + Artemis nuclease: open hairpins and create variability.

• TdT (terminal deoxynucleotidyl transferase): non-template N-nucleotide addition.

• DNA polymerase, exonucleases, DNA ligase: process & seal joints.

Molecular Steps

1. RAG-1/2 bind paired RSSs, cleave one DNA strand → free 3′-OH attacks opposite strand → hairpin at coding ends + blunt signal ends.

2. DNA-PK:Artemis randomly nicks hairpin → generates palindromic (P) nucleotides.

3. Exonuclease trimming may delete bases.

4. TdT adds up to 15 random N nucleotides.

5. DNA polymerase fills gaps; ligase forms coding joint (imprecise, highly diverse) and precise signal joint (excised circle).

Junctional Diversity

• Diversity concentrated in CDR3 (antigen-binding hotspot) due to:

  • Variable hairpin nick sites (P nucleotides).

  • Random N-nucleotide addition by TdT.

  • Exonuclease nucleotide deletions.

• Generates frame-shift/stop codons → many rearrangements non-productive (fails quality control).

Completion of Rearrangement & Early B-Cell Development

• Order of events in bone marrow (hematopoiesis):

  1. Stem cell → early pro-B: D!J heavy rearrangement begins.

  2. Late pro-B: V\,DJ rearrangement.

  3. Large pre-B: expression of μ heavy chain with surrogate light chain on surface (pre-B receptor) → signaling halts further H rearrangement (allelic exclusion).

  4. Small pre-B: light-chain VJ rearrangement.

  5. Immature B: complete IgM (μ heavy + genuine light) on surface.

  6. Mature naïve B: alternative RNA splicing co-expresses IgM (μ) and IgD (δ) BCRs.

• Allelic exclusion guarantees one antibody specificity per B cell.

Central & Peripheral Tolerance During Development

• Bone marrow central deletion: immature IgM+ B cells recognizing self undergo apoptosis (clonal deletion).

• Spleen (white pulp) final maturation:

  • Further deletion: IgM/IgD dual-expressing B cells tested again.

  • Survivors = mature naïve B cells entering circulation.

B-Cell Activation in Secondary Lymphoid Organs

• Antigen sources:

  • Tissue-derived antigens drain to lymph nodes/follicles.

  • Blood-borne antigens captured in spleen.

• Naïve B cell must:

  1. Recognise free or APC-presented antigen via BCR.

  2. Receive help from cognate CD4⁺ T helper (TH) cells.

• Outcomes after activation (germinal centre reaction):

  • Somatic hypermutation.

  • Class-switch recombination.

  • Massive clonal expansion → plasma cells or memory B cells.

  • Failure → apoptosis.

Somatic Hypermutation (SHM) & Affinity Maturation

• Timing/location: within 1 week of antigen exposure in rapidly dividing germinal-centre B cells.

• Enzyme: Activation-induced cytidine deaminase (AID).

  • Deaminates cytidine → uracil in DNA, triggering error-prone repair → point mutations.

• Target region: V-region exons (especially CDR1, CDR2, CDR3) in both H & L chains; constant regions spared.

• Consequences:

  • Creates variants with higher, equal, or lower affinity.

  • Positive selection by follicular dendritic cells & TH help retains high-affinity clones → affinity maturation (later antibodies bind stronger).

Class-Switch Recombination (CSR)

General Principles

• Initial antibody is always IgM (μ constant region) – sometimes co-expressed IgD by splicing.

• CSR replaces Cμ/Cδ with downstream constant region genes ( \gamma,\alpha,\varepsilon …) without altering antigen specificity (VDJ untouched).

• Requires B cell activation signals: CD40–CD40L plus cytokines from TH cells.

Molecular Mechanism

• Switch (S) regions (highly repetitive DNA) upstream of each constant gene.

• AID initiates double-strand breaks within the two selected S regions.

• Looping out and deletion of intervening DNA joins VDJ to new CH gene → irreversible; excised DNA lost as circle.

• Cytokines dictate target isotype:

  • IL-4 → IgG1, IgE.

  • TGF-β → IgA, IgG2b.

  • IFN-γ → IgG3, IgG2a (mouse isotypes; human parallels similar).

Functional Impact of Class Switching

• Preserves antigen specificity while altering effector functions:

  • IgG subclasses: opsonisation, placental transfer, complement activation.

  • IgA: mucosal immunity (dimeric form via J chain).

  • IgE: mast-cell binding, parasite & allergy responses.

  • IgM: pentameric (10 binding sites) → strong agglutination, complement activation.

Summary of Diversity Sources

• \textbf{1.} V(D)J combinational choice.

• \textbf{2.} Junctional diversification (P + N addition/deletion).

• \textbf{3.} Pairing of independently rearranged H & L chains.

• \textbf{4.} Somatic hypermutation post-activation.

• \textbf{5.} Class switching yields further functional variety (not new specificity).

Outcomes of B-Cell Differentiation

• Plasma cells: antibody factories; no surface BCR; most die in 1–2 weeks, some return to bone marrow as long-lived plasma cells (maintain serum antibody).

• Memory B cells: quiescent, circulate for years; rapid secondary response.

Tolerance, Autoimmunity & Implications

• Immune tolerance educates B/T cells to ignore self.

• Vast diversity means some BCRs intrinsically bind self (e.g., insulin) → central/peripheral deletion or anergy required.

• Failure leads to autoimmune disease (type 1 diabetes, SLE, etc.).

Advantages of Adaptive Immunity

• Antibody quality increases over time (affinity maturation).

• Clonal selection ensures self-tolerance and pathogen focus.

• Immunological memory provides rapid, heightened secondary responses.

Key Exam Point

• "Describe the genetic organisation of the heavy and light chain immunoglobulin loci and discuss the molecular mechanisms that lead to antibody diversity." Prepare to integrate points above: loci structure, RSS & enzymes, combinatorial + junctional diversity, SHM, CSR, functional outcomes.

Further Reading

• Kuby Immunology 8e – Ch 6.

• Janeway’s Immunobiology – Ch 3, 5, 8, 9.

• Parham – The Immune System 3e – Ch 3, 5, 8, 9.

• Roitt’s Essential Immunology (relevant chapters).