Microarray analysis indicates that different subsets of B cells express

1. Microarray analysis indicates that different subsets of B cells express specific “gene signatures.”

2. Adaptive Immunity • Central objective: • Protect against foreign invaders • Create memory of invasion to prevent recurrent infection • Response must be highly specific to the invader

3. Ig Isotypes: Classes and Subclasses • Ig isotypes are defined by the C-terminal portion of the molecule • There are 5 classes of Ig and there are 4 sub-classes of IgG. • Isotype class determines effector function.

4. Ig Isotypes

5. Ig Isotypes Localize to Different Anatomical Sites in the Body

6. Ig Isotypes Localize to Different Anatomical Sites in the Body

7. Ig Isotypes Have Different Effector Functions

8. When Do Isotypes Matter? • The primary -> secondary • immune response: • Increase Ig titer • Switch from IgM to IgG • Increase Ag binding affinity

9. When Do Isotypes Matter? Primary ->Secondary Immune Response Transition Slide 2

10. Primary/Secondary Immune Response • Events associated with an immune response over time: • Increase of IgM titer • Increase of IgM titer is specific to the immunizing Ag Slide 2

11. Isotypes Switch: IgM->IgX • Three Types of Switch: • RNA level • IgM->IgD • Membrane->Secreted • DNA level • Class Switch Recombination

12. IgM and IgDAre Coexpressed in Mature B Cells Differentiation to the mature B cell stage is marked by the co-expression of IgM and IgD on the plasma membrane

13. A Shift of IgM to IgM/IgD Expression in Mature B cells Mechanism: Alternative RNA polyadenylation and splicing

14. In Mature B Cells Ig is Located on the Mb and Functions as the BCR Location of Ig on the cell surface is transmb and requires the presence of the mb exon

15. In Plasma Cells Ig is Secreted Secretion of Ig from the cell requires the presence of the secretion exon

16. Secreted Ig Promotes Critical Functions • In mature B cells Ig is located on the plasma mb. • Ig is secreted from terminally differentiated plasma cells

17. Secreted IgM and IgA are Multimerized by J Chain • In mature B cells Ig is located on the plasma mb. • Ig is secreted from terminally differentiated plasma cells

18. Isotypes Switch: IgM->IgX • Three Types: • RNA level • IgM->IgD • Membrane->Secreted • DNA level • Class Switch Recombination

19. C Regions Are Arrayed in the IgH Locus

20. Looping Out and Deletion Model of SR

21. Germline Transcripts are Required for SR

22. When Do Isotypes Matter? • The primary -> secondary • immune response: • Increase Ig titer • Switch from IgM to IgG • Increase Ag binding affinity

23. Affinity Maturation and Ig Isotype Switch • In a primary immune response, affinity of Ig for its Ag is usually not high enough to immediately clear the pathogen from the system. However, affinity is increased by somatic hypermutation (SHM). • SHM alters V genes at the DNA level

24. Primary/Secondary Immune Response Protein level Slide 2 Acquisition of mutations in the V region of the VH and VL proteins: nonrandom distribution

25. Primary/Secondary Immune Response Protein level The position of the newly acquired mutations is in the antigen binding pocket. Slide 2

26. Somatic Hypermutation and Affinity Maturation • In SHM, individual nucleotides in VJ or VDJ units are mutated, thus adjusting the specificity of and potentially increasing the affinity for Ag.

27. Somatic Hypermutation and Affinity Maturation cont... • Mutations translate into a change in the Ag binding pocket of the Ab. Some mutations are deleterious while others are neutral or advantageous for Ab:Ag fit. Some deleterious mutations generate autoreactive Ig. Question: are mutations random or directed at the level of the genes?

28. Somatic Hypermutation and Affinity Maturation DNA level

29. Somatic Hypermutation and Affinity Maturation cont... • Selection of high affinity B cell clones leads to affinity maturation of the immune response. • B cells expressing high affinity BCR are favored by clonal selection. This occurswhen Ab:Ag complexes are highly compatible and possess a “good fit”. The high affinity Ab:Ag interaction transduces a strong signal to the B cell to proliferate.

30. Clonal Selection: Type I Clonal selection occurs following VDJ in the BM to delete B cells expressing autoreactive BCR.

31. Clonal Selection: Type II Mutations which increase affinity of Ab for Ag will create a BCR which promotes strong proliferation. Selection also occurs in the periphery following SHM on the GC to delete newly autoreactive BCR and to preclude the escape of self-reactive B cells.

32. SHM Occurs in the Germinal Center B Cells

33. The Surprising Link between CSR, SHM and Gene Conversion • Activation induced deaminase (AID) is responsible for CSR and SHM. • It is the only B cell specific gene product required for both these genetic alterations.

35. The Surprising Link Between CSR, SHM and Gene Conversion How does AID work? • AID has homology to the APOBEC family of proteins which are involved in RNA editing • AID is also a cytidine deaminase and converts dC ->uracil • Uracil is mutagenic and triggers the BER pathway

36. AID Deaminates dC and Triggers the BER Pathway

37. What happens when AID is mutated? What happens when UNG is mutated? What happens when DSB repair proteins are mutated?

38. Hyper-IgM Syndrome: No Secondary Isotypes • Four Types: Cause???? • Mutations in: • CD40 (T cell); CD40L (B cell) • AID • UNG • Subgroup with unknown cause

39. CSR Requires DNA Repair Proteins DNA Repair Proteins Involved in CSR: DNA-PKcs, Ku70, Ku80, H2AX, NBSl, ATM MMR (Msh2, Msh6, Pms2, Mlhl)

40. The End

41. End of part II

42. U=T During Replication

43. BCR and TCR Break the Rules of Mendelian Genetics Monospecific Ag receptors: Only one H chain and one L chain is expressed per B/T cell whereas each cell has 2 alleles for H and L chains