1. Lecture 5 Antibody genes I Problem of the generation of diversity V(D)J recombination Surface immunoglobulin B lymphocyte development Heavy chain class switch

2. ~10 different constant regions Millions of different variable regions

3. Antibody structure (IgG) Figure 2.2

4. Theories of the Generation of Diversity (G.O.D.) Pauling 1940- Antigen serves as template around which antibody is folded. Germline model- one gene for each antibody. Problems: need lots of genes, allotypes (i.e. constant region polymorphisms behave like simple Mendelian genes). Somatic mutation model- one gene that gets independently mutated in individual cells. Problem: mutator must spare C-region Recombination between C and V at the level of Protein RNA DNA- Dryer and Bennett

5. VHVH D J // 200 134 CHCH J // VV 754 J V 2 C V J C 2 CC C Mouse antibody genes

6. Human antibody genes

9. Combinations 40 X 530 X 4 65 X 27 X 6 200 + 12010,530 320 10,530X= 3,369,600 V(D)J: H/L: Number of gene segments  40+5=45  30+4=34 H 65+27+6=98 total gene segments 177

10. Concepts Antibodies genes are assembled by DNA recombination. A relatively small number of gene segments can be combined in different ways to generate extensive diversity.

11. Human Ig gene loci 65 40 30

12. Mouse Ig gene loci 200 100

13. “transposon” Germline configuration DNA Configuration in B cell DNA mRNA V(D)J recombination makes a permanent change to the genome (RNA splicing does not) hnRNA DNA recombination intron

14. Figure 2-18 V(D)J recombination is initiated by the Recombinase Activator Gene Products, RAG1 and RAG2 (found only in developing lymphocytes)

15. Fugmann et al, 2000 Lieber, M Recombination signal is a conserved 7mer-spacer-9mer. One coding element with a 12 bp spacer joins with another carrying a 23 bp spacer OH V(D)J recombination initiated by RAGs completed by general non- homologous end joining (NHEJ) DNA repair enzymes (present in all cells) “coding” and signal ends are processed differently!! 12 or 23bp spacer 7-mer9-mer

16. Concepts V(D)J recombination involves dsDNA breaks and ligation. Recombination is initiated by the lymphoid specific RAG1/2 complex and resolved by components of the non-homologous end joining machinery (which is important for DNA repair in other contexts as well, such as radiation damage, etc. Antibody V, D, J gene segments carry adjacent recombination signals composed of a conserved heptamer/nonamer motif that is recognized by the RAGs. V(D)J recombination is imprecise at the coding junctions by design.

17. Figure 2-19 part 1 of 3 Imprecision of coding joins promotes generation of diversity

18. Figure 2-19 part 2 of 3 TdT= Terminal deoxynucleotidyl transferase (Lymphocyte specific) can add nucleotides randomly to the single stranded ends exposed by nicking of the hairpin Untemplated!

19. Figure 2-19 part 3 of 3

20. Junctional Diversity

21. Nucleotides can be added to the junction by terminal deoxynucleotidyl-transferase (Tdt)

22. Mechanisms of diversity Naïve B cells (pre-antigen selection): Large number of germline genes Combinatorial Joining (V x [D] x J) Combinatorial Association (H x L) Junctional Diversity (3rd CDR) Loss of nucleotides N-region nucleotides added by Tdt P-region nucleotides added by repair Antigen-experienced B cells (post-antigen stimulation) Somatic mutation

23. Risks and benefits of DNA manipulation by the immune system Generation of diversity -RAG1/2 DNA breaks, hairpins -Terminal nucleotidyl transferase untemplated nucleotide additions Drawback: These processes probably contribute to cancer Many (lymphoid) tumors involve somatic mutation and translocation associated with antigen receptor genes

24. The potential diversity generated by V(D)J recombination is virtually infinite. >Total lymphocytes in a human ~10 12 >Genome size ~3X10 9 bp

25. Figure 2-31

26. B cell maturation

27. B cell receptor is membrane bound form of antibody B cell Differential RNA splicing regulates the expression of these forms

28. Membrane-bound vs. Secreted Figure 2.23

29. Figure 2-23 Ig-  is a critical signal transducer for all surface immunoglobulin (sIg). It is required both for expression on the membrane and for transmitting signals to the cell.

30. Reminder about gene structure and the central dogma of molecular biology Regular gene: (eukaryote) DNA Exon 1Exon 2 Exon 3 promoter RNA AAAA RNA splicing AAA mRNA AAA Many genes generate alternative splicing isoforms Translation on ribosomes to protein Translation Protein product Alternative protein product

31. Early stages of B cell development up to the point where B cells start to express sIgM occurs in the bone marrow. Mature IgM/IgD+ cells populate the secondary lymphoid organs (spleen, lymph nodes, Peyer's patches

32. Bone marrow B cell development occurs in stages

33. Figure 4-4

34. Figure 4-5 B cell development is regulated by stromal cells and cytokines

35. Quality control problems caused by generation of diversity must avoid expression of more than one specificity -allelic exclusion -isotype exclusion (  vs L-chain) non-functional genes -out of frame rearrangements common (2/3) self-reactivity Lymphocyte learning and quality control

36. Quality control of B cells This is used to make sure that cells make in-frame antibody genes and only one H and L chain 1/3 2/3 2/9 4/9

37. Figure 4-8

38. Figure 4-14

39. Figure 4-9

40. Figure 4-10

41. Figure 4-21

42. IgM vs. IgD In a mature, naïve B cell, sIgM and sIgD are coexpressed and carry the identical V region. This occurs by generating 2 different splicing products from the same rearrangement.

43. Concepts V(D)J recombination creates enormous diversity. Recombination is ordered during B cell development in the bone marrow, starting with H-chain gene D-to-J recombination, followed by Vh-to-DJ, then recombination of L-chain genes. Recombination is essentially random, and error-prone leading to out-of-frame rearrangements, and receptors that are autoreactive. A quality control process ensures that the vast majority of B cells carry just one H/L pair, and that the receptor is non- autoreactive. Quality control involves preB and B cell receptor signaling through the Ig-  transducers. Final maturation of pre-immune B cells involves migration to the spleen and lymph nodes, and coexpression of IgM and IgD antigen receptors. P.S., this is Not the last slide

44. Antibody gene DNA One exon is assembled from separate pieces by DNA rearrangement in immature lymphocytes DNA V Naïve B cell IgM, IgD DNA On the antibody H chain, other exons are swapped in by a DNA rearrangement process distinct from V(D)J recombination C heavy Antigen stimulated B cell *** ** Point mutations are introduced Next time