Immunoglobulin Gene Recombination

Immunoglobulin Gene Recombination

Immunoglobulin (Ig) gene recombination is the process by which developing B cells assemble functional antibody genes from germline gene segments. This somatic DNA rearrangement generates the primary antibody repertoire—the initial diversity that allows the humoral immune system to recognize virtually any antigen before affinity maturation further refines the response.

Overview

Unlike most genes, which are inherited as complete units, immunoglobulin genes must be assembled from component parts during B cell development. The germline genome contains arrays of gene segments that are cut and rejoined to create complete variable regions. This combinatorial approach, combined with junctional diversity, generates an enormous repertoire from limited genetic material.

Key Features of Ig Recombination

The Immunoglobulin Loci

Humans have three immunoglobulin loci, each on a different chromosome, encoding the heavy chain and two types of light chains.

Heavy Chain Locus (IGH) — Chromosome 14q32

The largest and most complex immunoglobulin locus, spanning approximately 1.25 megabases.

Gene Segment Organization (5’ → 3’):

[VH segments]———[DH segments]———[JH segments]———[CH regions]
   ~45 genes       27 genes        6 genes      Cμ-Cδ-Cγ3-Cγ1-Cα1-Cγ2-Cγ4-Cε-Cα2

Segment Numbers:

Kappa Light Chain Locus (IGK) — Chromosome 2p12

Notable Feature: Contains the κ-deleting element (Kde), which rearranges to delete the Cκ region when κ chains are non-functional or autoreactive, allowing λ chain expression.

Lambda Light Chain Locus (IGL) — Chromosome 22q11

Organization: Unlike IGK, the lambda locus has multiple J-C pairs, each Jλ segment associated with its own Cλ gene.

κ:λ Ratio: In healthy humans, approximately 60% of B cells express κ light chains and 40% express λ. Deviation from this ratio can indicate clonality (malignancy) or selection pressure.

Recombination Signal Sequences and the 12/23 Rule

RSS Structure

Each V, D, and J segment is flanked by recombination signal sequences (RSS) that direct the RAG recombinase:

RSS Components:

[Coding segment]—CACAGTG—spacer (12 or 23 bp)—ACAAAAACC
                 ↑                              ↑
              Heptamer                        Nonamer

• Heptamer: Conserved 7-bp sequence; site of DNA cleavage
• Nonamer: Conserved 9-bp sequence; RAG binding
• Spacer: Either 12 bp or 23 bp (one or two turns of DNA helix)

The 12/23 Rule

Principle: Recombination occurs efficiently only between a 12-RSS and a 23-RSS.

This ensures proper segment ordering—V cannot join directly to V, only to D (which can then join to J).

Heavy Chain RSS Distribution:

This arrangement permits: VH(23)—(12)D(12)—(23)JH ✓ But prevents: VH(23)—(23)JH ✗

Light Chain RSS Distribution:

Note: κ and λ have opposite arrangements, but both follow the 12/23 rule for V-J joining.

The Recombination Machinery

The RAG Recombinase

RAG1 and RAG2 form the core recombinase complex:

Expression: Strictly limited to developing B cells in bone marrow (and T cells in thymus). Aberrant RAG expression is associated with lymphoid malignancies.

Step-by-Step Mechanism

Step 1: Synapsis

• RAG complex binds one RSS (preferentially 12-RSS first)
• Captures second RSS (23-RSS)
• Forms the synaptic complex with both segments
• 12/23 rule enforced at this stage

Step 2: Cleavage

The RAG complex introduces DNA double-strand breaks through two sequential reactions:

First: Nicking

• Single-strand nick exactly at heptamer/coding boundary
• Creates free 3’-OH group

Second: Hairpin Formation

• 3’-OH attacks opposite strand (transesterification)
• Creates hairpin-sealed coding end
• Creates blunt signal end

Result:
Coding end: [V segment with hairpin]
Signal end: [Blunt heptamer sequence]

Step 3: Hairpin Opening

Artemis (activated by DNA-PKcs) opens the hairpin:

Step 4: End Processing

Before joining, coding ends are processed:

P-nucleotides (Palindromic):

• Generated when asymmetric hairpin opening creates overhang
• Filled in by DNA polymerase
• Short palindromic sequences (0-4 nucleotides)

N-nucleotides (Non-templated):

• Added by Terminal deoxynucleotidyl transferase (TdT)
• Random sequence (template-independent)
• Can add 0-20+ nucleotides
• Primary source of heavy chain junctional diversity
• Note: TdT expression decreases after pro-B cell stage, so light chains have fewer N-nucleotides

Exonuclease Trimming:

• Germline nucleotides removed from segment ends
• Contributes to diversity but may cause frameshifts

Step 5: Joining

Non-Homologous End Joining (NHEJ) machinery ligates the ends:

Products:

• Coding joint: V(D)J junction → functional gene
• Signal joint: Joined signal ends → excision circle (KREC)

Order of Rearrangement

B cell development follows a strict rearrangement order with checkpoints ensuring productive rearrangements before proceeding.

Heavy Chain First

Stage 1: D-J Rearrangement (Early Pro-B)

• Occurs on both IGH alleles
• D segment joins to J segment
• Intervening DNA deleted
• Does not require surface expression

Stage 2: V-DJ Rearrangement (Late Pro-B)

• V segment joins to existing DJ
• First attempt on one allele
• If non-productive (frameshift, stop codon):
  • Attempts second allele
  • If both fail: cell death

Stage 3: Heavy Chain Checkpoint

Productive VDJ rearrangement leads to:

1. μ heavy chain expression (in cytoplasm)
2. Pre-BCR formation: μ chain + surrogate light chain (VpreB + λ5)
3. Pre-BCR signaling triggers:
   • Survival (rescue from default apoptosis)
   • Proliferation (4-6 cell divisions)
   • Allelic exclusion (stops further heavy chain rearrangement)
   • Light chain locus opening

Light Chain Second

Stage 4: Light Chain Rearrangement (Pre-B)

• V-J joining only (no D segments in light chains)
• κ locus typically rearranges first
• Sequential attempts:
  1. First κ allele
  2. Second κ allele
  3. κ-deletion (Kde rearrangement)
  4. First λ allele
  5. Second λ allele

If light chain is autoreactive: Receptor editing (new V-J rearrangement) before trying next allele

Stage 5: Immature B Cell Checkpoint

Successful light chain rearrangement produces:

• Complete IgM BCR on surface
• Testing against self-antigens in bone marrow
• Central tolerance checkpoints (see Immune Tolerance)

Sources of Antibody Diversity

1. Combinatorial Diversity

Random selection from gene segment pools:

Heavy Chain:

45 VH × 27 DH × 6 JH = 7,290 combinations

Light Chain:

Kappa: 40 Vκ × 5 Jκ = 200 combinations
Lambda: 30 Vλ × 5 Jλ = 150 combinations
Total light chain: ~350 combinations

Heavy × Light:

7,290 × 350 ≈ 2.5 × 10⁶ combinations

2. Junctional Diversity

The imprecise joining process dramatically amplifies diversity:

Combined junctional diversity: ~10^7 additional variants at heavy chain junction

3. D Segment Flexibility

The D segment adds unique diversity:

• Can be read in all three reading frames
• Can be used in inverted orientation
• Different reading frames produce completely different amino acids
• Central portion may be deleted entirely
• Results in enormous HCDR3 variability

4. Total Primary Repertoire Diversity

Before somatic hypermutation:

Primary diversity = Combinatorial × Junctional × D-frame
                  ≈ 10^11 to 10^13 unique BCRs

Note: Actual repertoire limited by B cell numbers and selection

The Heavy Chain CDR3 (HCDR3)

The HCDR3 is the most diverse and most functionally important region of the antibody:

HCDR3 Composition

[End of VH]—[N-nts]—[D segment]—[N-nts]—[Start of JH]
    FR3      N₁        (any)      N₂       CDR3-JH
                       frame

HCDR3 Features

HCDR3 Length Distribution

Different antigens favor different HCDR3 lengths:

Regulation of Recombination

Chromatin Accessibility

V(D)J recombination requires accessible chromatin:

Developmental Control

Accessibility is strictly developmentally regulated:

Allelic Exclusion

Each B cell expresses only one heavy chain and one light chain allele:

Mechanism:

1. Productive rearrangement triggers surface expression
2. Pre-BCR (heavy) or BCR (light) signaling
3. RAG expression suppressed
4. Chromatin at remaining allele becomes inaccessible

Purpose: Ensures each B cell produces antibodies with a single specificity (monospecificity)

Receptor Editing

A tolerance mechanism that can rescue autoreactive B cells:

Process

1. Autoreactive immature B cell recognized self-antigen
2. RAG genes re-expressed (or maintained)
3. Secondary V-J rearrangement:
   • Upstream V joins to downstream J
   • Replaces original light chain junction
4. If new BCR non-autoreactive → survival
5. If still autoreactive → further editing or deletion

Evidence of Editing

• ~25-50% of mature B cells show molecular signs of editing
• Multiple Vκ-Jκ rearrangements on same allele
• κ-deleting element (Kde) rearrangements
• Skewed Vκ-Jκ combinations (upstream V, downstream J favored)

Editing vs. TCR Editing

Clinical Significance

Primary Immunodeficiencies

B Cell Malignancies

Recombination signatures help classify lymphomas:

Clonality Testing:

• Monoclonal IGH/IGK rearrangement → suggests malignancy
• Polyclonal pattern → reactive process
• PCR or sequencing-based detection

Minimal Residual Disease (MRD):

• Track specific VDJ sequence after treatment
• Deep sequencing detects 1 in 10⁵-10⁶ cells
• Guides treatment decisions

Aberrant Recombination and Translocations

RAG occasionally targets cryptic RSS in non-Ig loci:

These result from RAG-mediated breaks at RSS-like sequences with subsequent aberrant joining.

Key Concepts

1. V(D)J recombination assembles Ig genes from germline segments using the RAG recombinase

2. The 12/23 rule ensures proper segment joining through RSS spacer length requirements

3. Junctional diversity (P- and N-nucleotides) is the primary source of CDR3 variability

4. Ordered rearrangement proceeds heavy chain → light chain, with checkpoints at each stage

5. Allelic exclusion ensures one antibody specificity per B cell

6. Receptor editing rescues autoreactive B cells by replacing light chains

7. HCDR3 is the most diverse region, central to antigen recognition and clonotype identification

Related Articles

• V(D)J Recombination — General mechanism shared with TCR
• B Cell Development — Developmental context
• Somatic Hypermutation — Secondary diversification
• Germinal Centers — Site of affinity maturation
• Immune Tolerance — Central B cell tolerance

References

1. Alt FW, et al. (2013). Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell, 152:417-429.

2. Tonegawa S. (1983). Somatic generation of antibody diversity. Nature, 302:575-581.

3. Nemazee D. (2017). Mechanisms of central tolerance for B cells. Nature Reviews Immunology, 17:281-294.

4. Wardemann H, Nussenzweig MC. (2007). B-cell self-tolerance in humans. Advances in Immunology, 95:83-110.