Immunology 2nd test Chapter 14

What is the antibody repertoire?

The antibody repertoire is the total collection of different antigen specificities that a person's B cells can recognize.

Approximately how large is antibody repertoire in humans?

In humans, naive B cells can express up to ~10¹¹ (100 billion) different B-cell receptors.

Why do we need such enormous B-cell receptor diversity?

Because there is enormous antigen diversity in nature .The immune system must be prepared to recognize virtually any foreign molecule it might encounter, including pathogens that have never existed before.

List the 5 mechanisms by which antibody diversity is generated.

1) Combinatorial shuffling of heavy and light chains.

2) V, D, J gene segment recombination (somatic recombination).

3) Hairpin nicking by Artemis at coding joints.

4) Junctional diversity — P-nucleotides and N-nucleotides added by TdT/NHEJ.

5) Allelic exclusion.

On which chromosomes are the three immunoglobulin gene loci located?

• Heavy chain → Chromosome 14

• κ (kappa) light chain → Chromosome 2

• λ (lambda) light chain → Chromosome 22

What gene segments encode the V region of a light chain? A heavy chain?

• Light chain V region (V_L ) = 1 V segment + 1 J segment.

• Heavy chain V region (V_H) = 1 V segment + 1 D segment + 1 J segment.

• The C region is always encoded by a separate C gene segment.

What is somatic recombination and why is it called 'somatic'?

DNA cutting-and-rejoining process in developing B cells that assembles V, D, and J gene segments into a single V-region exon. It is called somatic because it occurs in somatic cells (B-cell precursors), not in germ cells, so the rearrangement is NOT heritable.

What is the order of recombination for the heavy chain?

• Two sequential recombinations

• First - D joins to J (forming DJ)

• Second - V joins to the pre-formed DJ (forming VDJ). V cannot join directly to J because of the 12/23 rule.

What are Recombination Signal Sequences (RSS)? What

• conserved DNA sequences flanking each gene segment that guide the RAG enzymes to the correct cleavage sites.

• Each RSS consists of a heptamer (CACAGTG) and a nonamer (ACAAAAACC)

• 2 types: 12-bp or 23-bp spacer.

Where are RSS located relative to each gene segment?

V segments are flanked on their 3' side by a 23-RSS. D segments are flanked on BOTH sides by 12-RSS. J segments are flanked on their 5' side by a 23-RSS.

 Explain the 12/23 rule of recombination.

The 12/23 rule states that recombination can only join segments when one RSS has a 12-bp spacer and the other has a 23-bp spacer. Since V and J segments are both flanked by 23-RSS, they cannot join directly. The D segment (12-RSS) must bridge them — enforcing the order D→J then V→DJ.

What are the components and 3 steps of the V(D)J recombinase?

• Components: RAG1 and RAG2 proteins.

• Step 1: RAG binds one RSS and recruits the partner RSS (12+23) into a synaptic complex.

• Step 2: RAG endonuclease cuts the DNA at both heptamers, forming blunt RSS ends and hairpin coding ends.

• Step 3: RSS ends are ligated (signal joint); coding hairpins are opened by Artemis, modified, and rejoined by NHEJ (coding joint).

What is junctional diversity?

additional sequence variability introduced at the V-J and V-D-J coding joints during recombination. It results from: (1) palindromic P-nucleotides from Artemis hairpin opening at variable positions, (2) trimming by exonucleases, and (3) random addition of non-templated N-nucleotides by TdT. It is not encoded in germline DNA.

What enzyme adds N-nucleotides during junctional diversity, and where are these added?

TdT (terminal deoxynucleotidyl transferase) adds random N-nucleotides to the coding ends after hairpin opening. These are added at the VJ junction in light chains and at both the DJ and VDJ junctions in heavy chains.

Which CDR is most affected by junctional diversity, and why?

CDR3 is most affected

• In light chains, CDR3 is at the VJ junction

• In heavy chains, CDR3 spans the entire VDJ junction (two junctions), making it the most variable loop and the primary determinant of antigen-binding specificity.

 How many combinatorial antibody variants are possible from recombination alone?

• κ chain: 175 combos

• λ chain: 120 combos → 295 total light chain variants.

• Heavy chain: 40V × 23D × 6J = 5,520 variants.

• Random pairing of light + heavy chains: ~1.6 million antibodies. Junctional diversity then amplifies this to ~10¹¹.