Chapter 7 Organization and Expression of Immunoglobulin Genes

1. Chapter 7 Organization and Expression of Immunoglobulin Genes

2. 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?

3. 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

5. 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

6. DNA rearrangement: produces variable region • Happens before the B cell encounters antigen • Later mRNA splicing: produces constant region • Happens after that particular B cell encounters antigen it’s specific for • Now the B cell can switch from making IgM to IgD to IgG, etc • All with the same variable region

8. 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 • J • C

10. 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

12. Heavy Chain Rearrangement • The B cell receptor is made up of two kinds of proteins, the heavy chain (Hc) and the light chain (Lc)., Each of these proteins is encoded by genes that are assembled from gene segments. • Each B cell has 2 chromosome 14s (Mom + Dad)…but a B cell makes only one kind of Ab. So the segments on one chromosome have to be “silenced”.

13. This works like a card game with 2 players. It is “winner takes all”..each player tries to rearrange its card (gene segments) until it finds a arrangement that works. The first player to do this wins. • The players in the card game first choose one each of the possible D and J segments, and these are joined deleting the DNA sequences in between.

15. Then one of the many V segments is chosen, and this “card” is joined to the DJ segment, again by deleting the DNA in between. • Next to the rearranged J segment is a strong of gene segments (CM, CD, etc) that code for the various constant regions. • By default, the constant regions for IgM and IgD are used to make the BCR, simply because they are first in line.

17. Next, the rearranged gene segments are tested. (if the gene segments are not lined up right, the protein translation machinery will encounter a stop codon and terminate protein assembly. • If the segment passes the test, that chromosome is used to construct the winning Hc protein. This heavy chain protein is then transported to the cell surface, where it signals to the losing chromosome that the game is over.

18. If the heavy chain rearrangement is productive, the baby B cell proliferates for a bit, and then the light chain players step up to the table. The rules of the game are similar to those of the heavy chain game, but there is a second test…the completed heavy chain and light chain proteins must fit together properly to make a complete antibody. If this does not occur, the B cell commits suicide.

19. To produce antibody the B cell has to be activated. “Naïve” or “virgin” B cells have never encountered their antigen. • Activation of a naïve B cell requires 2 signals: the first is the clustering of the B cell’s receptors and their associated signaling molecules. A second signal is required (co-stimulatory signal) (Note: in T cell-dependent activation, this second signal is supplied by a helper T-cell). • In response to certain antigens, naïve B cells can also be activated with little or no T cell help (T-cell independent)

20. Once B cells have been activated, and have proliferated to build up their numbers, they are ready for maturation. Maturation occurs in 3 steps: • class switching (where a B cell can change the class of antibody it produces) • somatic hypermutation (rearranged genes for the BCR can undergo mutation and selection that can increase the affinity of the BCR for the antigen • career decision (B cell decides whether to become a plasma or memory cell)

21. Virgin B cells first produce IgM (default). As the B cell matures, it has the opportunity to change the class of Ab to either IgG, IgE or IgA. • The gene segments that code for the constant region for IgM are next to the constant regions for IgG, IgE or IgA, switching is easy: • Cut off the IgM constant region DNA and paste on one of the other constant regions.

22. Somatic Hypermutation • Normal overall mutation rate of DNA is extremely low (~ 1 mutated base /100 million bases). However, the chromosome area where B cell are encoded is highly restricted, which means that an extremely high rate of mutation can occur (~1 mutated base per 1000 cases). • This high rate of mutation is called somatic hypermutation. It occurs after the V,D, and J segments have been selected, and usually after class switching.

23. Somatic hypermutation changes the part of the rearranged Ab gene that encodes the antigen binding region of the Ab. Depending on the mutation, there are three possible outcomes. • The affinity of the Ab for the Ag may remain unchanged, my increase or may decrease • For maturing B-cells to continue to proliferate, they must be continually re-stimulated by binding to their Ag. Therefore, because those B cells whose BCRs have mutated to a higher affinity are stimulated more easily, they proliferate more frequently.

24. Because they proliferate more frequently, the result is that you end up with many more B cells whose BCRs have high affinity for their Ag.

25. BUT, hypermutation in TCRs is not beneficial (remember you want them to recognize self---but not over react>>autoimmune problems)

27. 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

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

30. 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: • http://www.youtube.com/watch?v=AxIMmNByqtM • 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

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

35. 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

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

37. 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

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

39. 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

40. 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

41. μ→δ→γ→ε→α IgM→IgD→IgG→IgE→IgA

42. 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

45. 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

46. 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

50. 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