1. Chapter 7
Organization and Expression of Immunoglobulin Genes
Dr. Capers
Immunology

2. Kindt • Goldsby • Osborne
Kuby IMMUNOLOGY
Sixth Edition
Chapter 5
Organization and Expression of Immunoglobulin Genes
Copyright © 2007 by W. H. Freeman and Company

3. How does antibody diversity arise?
What causes the difference in amino acid sequences?
How can different heavy chain constant regions be associated with the same variable regions?

4. In germ-line DNA, multiple gene segments code portions of single immunoglobulin heavy or light chain
During B cell maturation and stimulation, gene segments are shuffled leaving coding sequence for only 1 functional heavy chain and light chain
Chromosomal DNA in mature B cells is not the same as germ-line DNA

6. Greater complexity was revealed later
Dreyer and Bennett – 1965
2 separate genes encode single immunoglobulin heavy or light chain
1 for the variable region
Proposed there are hundreds or thousands of these
1 for the constant region
Proposed that there are only single copies of limited classes
Greater complexity was revealed later
Light chains and heavy chains (separate multi-gene families) are located on different chromosomes

7. DNA rearrangement: produces variable region
Later mRNA splicing: produces constant region

9. Kappa (κ) and lamda (λ) light chain segments:
L – leader peptide, guides through ER
V VJ segment codes for variable region J
C – constant region
Heavy chain L
V VDJ segment codes for variable region D C

11. Variable-region gene rearrangements
Variable-region gene rearrangements occur during B-cell maturation in bone marrow
Heavy-chain variable region genes rearrange first
Then light-chain variable region
In the end, B cell contains single functional variable-region DNA sequence
Heavy chain rearrangement (“class switching”) happens after stimulation of B cell

14. Mechanism of Variable-Region DNA rearrangements
Recombination signal sequences (RSSs)
Between V, D, and J segments
Signal for recombination
2 kinds
12 base pairs (bp) – 1 turn of DNA
23 bp – 2 turns of DNA
12 can only join to 23 and vice versa

16. Mechanism of Variable-Region DNA rearrangements
Catalyzed by enzymes
V(D)J recombinase
Proteins mediate V-(D)-J joining
RAG-1 and RAG-2

17. Gene arrangements may be nonproductive
Imprecise joining can occur so that reading frame is not complete
Estimated that less than 1/9 of early pre-B cells progress to maturity
Gene rearrangement video:
Look at Figure 7-8 – VDJ recombination
1. Recognition of RSS by RAG1/RAG2 enzyme complex
2. One-strand cleavage at junction of coding and signal sequences
3. Formation of V and J hairpins and blunt signal end
4. ligation of blunt signal end to form signal joint
2 triangles on each end (RSS) are joined
5. Hairpin cleavage of V and J regions
6. P nucleotide addition (palindromic nucleotide addition – same if read 5’ to 3’ on one strand or the other
7. Ligation of light V and J regions (joining)
8. Exonuclease trimming (in heavy chain)
Trims edges of V region DNA joints
9. N nucleotide addition (non-templated nucloetides)
10. Ligation and repair

20. Allelic Exclusion
Ensures that the rearranged heavy and light chain genes from only 1 chromosome are expressed

23. Generation of Antibody Diversity
Multiple germ-line gene segments
Combinatorial V-(D)-J joining
Junctional flexibility
P-region nucleotide addition
N-region nucleotide addition
Somatic hypermutation
Combinatorial association of light and heavy chains
This is mainly in mice and humans – other studied species differ in development of diversification

24. Ab diversity – Multiple gene-line segments AND combination of those segments

25. Ab diveristy – junctional flexibility
Random joining of V-(D)-J segments
Imprecise joining can result in nonproductive rearrangements
However, imprecise joining can result in new functional rearrangements

26. Ab diversity – P-addition and N-addition

27. Ab diversity – somatic hypermutation
Mutation occurs with much higher frequency in these genes than in other genes
Normally happens in germinal centers in lymphoid tissue

28. Class Switching Isotype switching
After antigenic stimulation of B cell
VHDHJH until combines with CH gene segment
Activation-induced cytidine deaminase (AID)
Somatic hypermutation
Gene conversion
CLASS-SWITCH recombination
IL-4 also involved

29. μ→δ→γ→ε→α
IgM→IgD→IgG→IgE→IgA

30. Ig Gene Transcripts
Processing of immunoglobulin heavy chain primary transcript can yield several different mRNAs
Explains how single B cell can have secreted and membrane bound Ab

33. Regulation of Ig-Gene Transcription
2 major classes of cis regulatory sequences in DNA regulate
Promoters – promote RNA transcription in specific direction
Enhancers – help activate transcription
Gene rearrangement brings the promoter and enhancer closer together, accelerating transcription

34. Antibody Engineering
Monoclonal Abs used for many clinical reasons (anti- tumor Ab, for instance)
If developed in mice, might produce immune response when injected
Can be cleared in which they will not be efficient
Can create allergic response
Creating chimeric Abs or humanized Abs are beneficial

39. Rearrangement of TCR genes
Similar to that of Ig
Rearrangement of α and γ chains
V, J, and C segments
Rearrangement of β and δ chains
V, D, J, and C segments

42. Generation of TCR diversity (a lot like Ig)
Multiple germ-line gene segments
Combinatorial V-(D)-J joining
Junctional flexibility
P-region nucleotide addition
N-region nucleotide addition
Combinatorial association of light and heavy chains
However, there is no somatic mutation with TCR
May be to ensure that after thymic selection, the TCR doesn’t change to cause self-reactive T cell