Ch. 5. How can the immune system recognize so many different epitopes?

1. Ch. 5. How can the immune system recognize so many different epitopes? Antibody H and L chains are composed of gene segments Many unique variable segments are inherited A limited variety of constant region sequences are used They must be rearranged into functional genes before they can be transcribed Ch. 5

2. p. 112 Ch. 5

3. Organization of Ig genes Germline DNA- gene segments surrounded by noncoding regions These are rearranged to form functional genes by a “cut and paste” method Light chains- V domain is composed of V and J segments C domain is composed of C segment Ch. 5

4. p. 114 Ch. 5

5. In Heavy chains- - V domain is composed of V, D, and J segments - C domain is composed of one C segment - Segments in V domains rearrange first - A single V domain can join to one C, then rearrange subsequently to join to another C domain Ch. 5

6. Multigene families Two types of L chains:  or  In humans: 40 V, 5 J, 1 C Similar number of  genes in humans Heavy-chain gene families are similar but more complex (also have D segment) CH regions formed from exons Ch. 5

7. p. 117 Ch. 5

8. Heavy chain DNA D-J and V-DJ rearrangements occur separately On a mature B cell, both mIgM and mIgD are expressed on the cell surface On any one cell, mIgM and mIgD have same V domains, but different C domains Ch. 5

9. How does rearrangement occur? Each V, D and J is flanked by RSS’s (Recombination Signal Sequences) Mechanism is controlled by RAG-1 and RAG-2 (recombination-activating genes) proteins and an enzyme, TdT (terminal deoxynucleotidyl transferase) If any of these proteins is defective, no mature B cells can form; nor T cells Ch. 5

10. p. 118 Ch. 5

11. p. 121 Ch. 5

12. B cells are diploid and contain chromosomes from both parents. However, heavy chain genes are rearranged on only one chromosome, as are light chain genes (on another chromosome). Therefore, any one B cell will contain one VH and one VL ( antigen specificity) How? Allelic exclusion – one allele gets turned off Ch. 5

13. p. 122 Ch. 5

14. Generation of antibody diversity (why are there so many possible antigen combining sites?) Many reasons… Ch. 5

15. 1. Multiple germline gene segments are inherited In human germline: 51 VH, 27 D, 6 JH 40 V, 5 J  30 V , 4 J Ch. 5

16. 2. Combinatorial V-J and V-D-J joining 57 V X 27 D X 6 J= 8262 possible combinations for VDJ joining (H chain) 40 V X 5J = 200 possible V and 120 possible V (L chain) 8262 X (200+120) = 2.64 X 106 possible combinations (random combination of H and L chains) Without taking into account other sources of diversity Ch. 5

17. 3. Junctional flexibility in V-J or V-D-J junction 4. Additional nucleotides added at junctions if a single-stranded region is created during the joining process * * * * * * * * * * * * * * 5. Somatic hypermutation (AFTER Ag stimulation) mutations occur AFTER rearrangement tend to occur in CDR regions affects antigen affinity (tends to increase): called “affinity maturation” (late in IR) occurs in B cells but not T cells Ch. 5

18. Class switching After antigen stimulation, heavy-chain DNA can rearrange so VDJ can join to another isotype Cytokines help determine the isotype IgG2a or IgG3 (mice): IFN- IgM: IL-2, IL-4, IL-5 IgE: IL-4 Ch. 5

19. p. 129 Ch. 5

20. Mature B cells express both mIgM and mIgD “Alternative RNA splicing” to give IgM and IgD - The VDJCC contains 4 polyadenylation sites - mIgM or mIgD can be generated depending on which polyadenylation site is used “Alternative RNA splicing” to give membrane-bound and secreted Igs Synthesis, assembly, & secretion of Igs then occurs. Ch. 5

21. Regulatory elements of transcription Promoters: upstream of initiation site, promote init. of RNA transcription in a specific direction Enhancers: activate transcription, not in a specific direction Gene silencers: down-regulate transcription Gene rearrangement brings enhancers close to the promoter they influence Ch. 5

22. p. 131 Ch. 5

23. Ch. 5

24. p. 134 Ch. 5

25. Ch. 5

26. What is a monoclonal antibody? Derived from a single clone and specific for a single epitope 1975- Kohler and Milstein developed the hybridoma technique for developing monoclonal antibodies Ch. 5

27. Antibody genes and genetic engineering Mouse mAb’s generate HAMA (Human Anti-Mouse Ab) Cleared quickly; Allergic reactions Now we have mouse CDR’s in human constant regions (“humanized Ab’s”) Cattle and mice producing human Ab’s (due to HAC) Bacteriophage libraries of Ab combining sites Ch. 5

28. p. 137 Ch. 5

29. p. 138 Ch. 5

30. Ch. 5

31. Ch. 5

32. p. 139 Ch. 5

33. p. 139 Ch. 5

34. Ch. 5

35. Ch. 5

36. Ch. 5

37. p. 141 Ch. 5

38. Behavior of monoclonal vs polyclonal antibodies Monoclonal antibodies tend to have high affinity Polyclonal antiserum will have mixture of low and high affinity antibodies Avidity vs. affinity Antibodies can be cross-reactive (source of some autoimmune disorders) Ch. 5

39. Genetically-engineered monoclonal antibodies are widely produced Advantages over hybridoma technology can choose isotype as well as specificity Can be expressed in a variety of host cells non-lymphoid mammalian cells bacteria (antibody fragments), phage plants, yeast mice, cattle Mutations of interest can be introduced Ch. 5

40. Therapeutic applications Cancer treatment Imaging Immunotoxins Catalytic antibodies (Abzymes)? Research applications Structure-function analysis Recombinant antibodies “Humanized” antibodies Ch. 5