Immunology 146:474

1. Immunology 146:474 • Tu, Fri 1st period (8:40-10:00 AM) • Serc 118 • Dr. Lori Covey-Office hrs: 1-3 Thursday • Dept. of Cell Biology & Neuroscience Nelson Hall, B314 • covey@biology.rutgers.edu • Class web site: http://lifesci.rutgers.edu/~covey/Immuno/index.htm

2. VH region

3. Organization of the Light Chain Loci The Variable region of the Ig light chain is encoded by two Gene segments; The V segment 95-101 amino acids The J segment up to 13 amino acids

4. Figure 4-4

5. Figure 4-3 The heavy and light chain genes are located on autosomes so there are 2 copies of the heavy chain locus, 2 copies of the  light chain and  light chain loci Possibility of generating 1011 antigen specificities

6. First mechanism that generates diversity Combinatorial Diversity Any functional VH segment can recombine with any DH segment and any JH segment Any functional VL segment can recombine with any JL segment

7. Somatic recombination at the Ig heavy chain locus

8. D-->J rearrangements on both alleles VH DH JH C V-->DJ rearrangement VHDJH C

9. Figure 3-6 Framework and hypervariable regions define the Variable Regions of the Ig heavy and light chain polypeptides

10. 12/23 7-mer 9-mer Recombination Recognition Sequences (RSS) Only RSS elements that have different spacer lengths can recombine with each other

11. 12 or 23 7-mer 9-mer Recombination Recognition Sequences (RSS) Only RSS elements that have different spacer lengths can recombine with each other

12. Figure 4-6 part 1 of 2

13. Figure 4-6 part 2 of 2

14. Figure 4-7 Proteins required for carrying out recombination called “V(D)J recombinase RAG I and II enzymes required For variable region Somatic recombination Create hairpins Which are cleaved. Terminal deoxynucleotidyl transferase (TdT) are added, nt can be subtracted as well by nucleases

15. Figure 4-7 part 3 of 3 2nd mechanism for creating diversity: Junctional Diversity Enzymatic processes creates Diversity in the joint between gene segments

16. Figure 4-8 part 1 of 3 Creates tremendous diversity at the joins of the three segments

17. Figure 4-8 part 2 of 3

18. Figure 4-8 part 3 of 3

19. Figure 4-7 part 3 of 3 3rd mechanism for creating diversity: Combinatorial- at the heavy and light chain level Pairing of individual heavy chains with different light chains

20. 4th mechanism for creating diversity: Somatic hypermutation Occurs after stimulation with antigen in the antigen-dependent stage of B cell differentiation Introduces point mutations into the V regions of the rearranged heavy- and light-chain genes at a very high rate Requires a signal from CD4+ T cells

21. Figure 4-13

22. T Cell Receptor (TCR) Comprised of two distinct chains,  chain and  chain, linked by S-S bond Each chain has a V region and a C region Carbohydrate-moeties Hydrophobic trans-membrane region

23. Figure 4-11 T cell receptor gene rearrangement Arranged in a similar pattern to Ig gene segments and are rearranged by the same enzymes

24. Figure 4-13

25. Figure 4-14 Colored Loops are the TCR CDR loops

26. Figure 4-15 part 1 of 2

27. Somatic recombination occurs at the TCR

28. Classes or “Isotypes” of immunoglobulins • Antibody (Ab) = immunoglobulin (Ig) • Each B cell expresses a unique Ig • The “C” part of the antibody molecule is conserved among classes of antibodies

29. Antibody Isotypes, cont’d • An isotype refers to the class of heavy chain polypeptides • isotypes, IgM, IgD, IgG, IgA and IgE. Heavy chain polypeptide is designated: VHCH Mature, naïve or resting B cells express only IgM and IgD on their surfaces

30. Heavy chain isotypes (classes) are distinguished by the structure of their constant regions • 5 different isotypes-IgM, IgD, IgG, IgA and IgE. • 4 subclasses of IgG, 2 subclasses of IgA • Critical to maintain the antigen-binding specificity of the antibody molecule (CDRs) in all isotypes

31. Effector functions of the antibody isotypes

32. Differences in Isotype Structure • # of interchain disulfide bonds • # of oligosaccharide moieties • # of C domains, IgM and IgE extra domain • Length of hinge region

33. Figure 4-18

34. IgM and IgD are expressed on mature naïve B cells • IgM is first immunoglobulin expressed during development • IgM and IgD expressed on surface together in mature, resting (naïve) B cells • Very little IgD secreted • IgM and IgD are produced by differential cleavage of a common mRNA transcript

35. Figure 4-20 part 1 of 2

36. Figure 4-20 part 2 of 2

37. IgM and IgD, cont’d • 2 cleavage and polyadenylation sites, pA1 and pA2 • When cell is activated, ceases to make both IgM and IgD only produced IgM

38. Organization of the heavy chain locus VDJ

39. Question-How do you make antibodies that have identical antigen binding recognition (F(ab)2) but different effector functions (Fc regions)?

40. Isotype switch recombination • Assembled VDJ variable region to be associated with different constant regions • Irreversible DNA recombination • Regulated by cytokines expressed by T cells and signals produced by pathogens

41. Isotype switch cont’d • Occurs only after B cells have been stimulated by antigen • Involves regions of repetitive DNA called “switch” or S regions • Non-homologous recombination event • REQUIRES TRANSCRIPTION THROUGH THE REGION

42. Isotype switch recombination

43. Figure 4-21 part 2 of 2

44. Membrane and secreted form • Each heavy chain gene has a membrane-coding (MC) and a secretion-coding (SC) exon. • Each mRNA transcript has 2 cleavage sites for polyadenylation (pAm and pAs) • Membrane form-25 amino acid hydrophobic tail • Secreted form- has a hydrophilic COOH end

46. All isotypes are expressed as a membrane and secreted form Membrane form-specific cleavage and polyadenylation

47. Membrane and secreted form, cont’d • Resting, naïve B cells express membrane IgM and IgD. • In late-stage plasma cells-almost all the expression is secreted form

48. Somatic hypermutations create diversity • Occurs later after B cells are in secondary lymphoid organs • Introduces single nt mutations into germline DNA • Results in increased binding affinity of antibody for antigen

49. Somatic hypermutations create diversity

50. Figure 4-9 Important enzyme involved in this process called “activation induced cytidine deaminase” or AID Requires transcription