This document discusses the generation of antibody diversity through various mechanisms in B cells. It covers topics like germline theories of diversity, somatic recombination and mutation of immunoglobulin genes, the roles of RAG proteins and AID enzyme, and the mechanisms of V(D)J recombination, somatic hypermutation, and class switching that generate a vast repertoire of antibodies from a relatively small number of gene segments. The regulation of immunoglobulin gene expression in B cells versus T cells is also summarized.
3. Generation of antibody diversity
How can all these diversity be generated?
- Germ-line theories
- Somatic-variation theories
Environment : 107 – 109 개 이상의 antigenic shapes (non-self)
Genome에 존재하는 antibody 이상의 antibody 생산이 요구됨
-Ehrlich side-chain theory
: antigen-induced selection
- Instructive hypothesis
: flexible antibody molecule
acted on by antigen to form
a complementary binding site
- Dreyer & Bennett : existence of a constant region
and a variable region
encoded by at least two different genes (??)
Tonegawa : somatic recombination
somatic mutation
4. Detection of Ig gene rearrangement by Southern blot hybridization
- Embryonic cell (nonlymphoid cell)의 Ig DNA
- Committed cells of B lymphocyte lineage (adult myeloma cells)
-- VJ rearrangement (pre-B cell)
: somatic recombination
Tonegawa’s bombshell-Immunoglobulin gene rearrange
9. - Gene segments are joined by recombinases
V(D)J recombinase
: recombination-activating genes
RAG-1 and RAG-2
Circular DNA isolated from thymocytes in which the DNA
encoding the chains of the TCR undergoes rearrangement
in a process like that involving the Ig genes
10. - Ig-gene rearrangements may be productive or nonproductive
- Allelic exclusion ensures a single antigenic specificity
- Allelic exclusion: A single specificity is maintained because only one of the two parental
alleles of Ig is expressed by every B cell clone from its earliest maturation stage
11. Generation of antibody diversity
- Multiple germ-line gene segments
- Combinatorial V-J and V-D-J joining generates diversity
12. - Junctional flexibility adds diversity - P-addition adds diversity
at palindromic sequences
- N-addition adds considerable diversity
by addition of nucleotides
13. Somatic hypermutation : Immunoglobulin heavy and light chain genes undergo
structural modifications after antigen stimulation
- Antigenic stimulation 후에 발생되는 structural alteration
- light chain 과 heavy chain 의 variable region 에서
single base change
- Germinal center- cells which have produced
a higher-affinity antibody are selected for survival
- Dependent on T cells and germinal center
- Athymic mice lack T cells and germinal center
- no affinity maturation
- Affinity maturation
- Somatic hypermutation adds diversity in already-rearranged gene
segments
15. Class switching among constant-region genes
- Switching regions : 2 – 3 kb upstream from each CH segment (except Cd)
Proposed mechanism for class switching induced by IL-4 in rearranged Ig heavy chain genes
16. - AID (activation-induced cytidine deaminase) mediates both somatic
hypermutation and class switching
- AID : RNA editing enzyme
: C U, repair G-C A-T
Experimental demonstration of the role of the enzyme AID
in class switching and hypermutation
17. Expression of Ig Genes
- Heavy-chain primary transcripts
undergo differential RNA processing
: Alternative RNA processing
20. Regulation of Ig-Gene transcription
- Promoter : AT-rich sequence (TATA box)
oct-2 (found only in B cells)
- Enhancer : in an orientation-independent manner
- Ig-gene expression is inhibited in T cells
: Ig-gene rearrangement (H, L chain –only in B cells
: TCR-gene rearrangement- T cells
why????, k-chain 3’ enhancer(3’kE) mutation Ig-genePU.1 binding site
21. Antibody genes
and antibody engineering
Human antibody from mice bearing a human artificial
chromosome (HAC) that includes entire human heavy-
and light-chain loci.
