Immunology

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

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 • B cell is committed to antigen specificity • B cell DNA looks different from germ line DNA • 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

13. 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

15. Mechanism of Variable-Region DNA rearrangements • Catalyzed by enzymes • V(D)J recombinase • Proteins mediate V-(D)-J joining • RAG-1 and RAG-2 • Proteins that cleave the DNA at the RSSs and then aid in the joining of fragments

16. 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 videos: • http://garlandscience.com/garlandscience_resources/resource_detail.jsf?landing=student&resource_id=9780815342434_CH05_QTM01

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

21. 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

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

23. 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

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

25. 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

26. Class Switching • Isotype switching • After antigenic stimulation of B cell • VHDHJH until combines with different CH gene segment • Activation-induced cytidine deaminase (AID) • IL-4 also involved • Watch this video on class switching: • https://www.youtube.com/watch?v=bk_RJogk_o0

27. Class switching order: μ→δ→γ→ε→α IgM→IgD→IgG→IgE→IgA

28. 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

31. 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

33. Videos on monoclonal antibodies and phage display: • https://www.youtube.com/watch?v=QfShgQ_x_vA • https://www.youtube.com/watch?v=AqQDZxoCGqE • https://www.youtube.com/watch?v=ybda36T-NXo

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

39. 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