Lymphocyte Development & Generation of Lymphocyte Antigen Receptors

1. Lymphocyte Development & Generation of Lymphocyte Antigen Receptors • Pin Ling (凌 斌), Ph.D. ext 5632; lingpin@mail.ncku.edu.tw • References: 1. Abbas, A, K. et.al, Cellular and Molecular Immunology (6th ed., 2007), Chapter 8

2. Questions What is the Bare Lymphocyte Syndrome? • An immunodeficiency disease • Lacking Class-II MHC expression • Mutations in transcriptional factors regulating Class II expression, e.g. CIITA What is the advantage of MHC Polymorphism? Is that good if MHC is as diverse as Ig or TCR? • MHC polymorphism may provide a broader protective immunity in a population during pathogen infection .

3. Outline • Overview of Lymphocyte Development • Generation of Diversity in Lymphocyte Ag receptors • B & T Lymphocyte Development • Summary & Question

4. Key Concepts in lymphocyte development 1. Lymphocyte development-A process of differentiation of lymphoid progenitor cells into mature lymphocytes (T & B). 2. Rearrangement and expression of Ag receptor genes are associated with lymphocyte development. 3. Selection events are involved in preserving cells w/ correct Ag receptors and eliminating dangerous cells w/ self-recognition Ag receptor. 4. Proliferation in the early lymphocyte development is stimulated by IL-7.

5. Stages of Lymphocyte Development-I

6. Checkpoints in Lymphocyte Development

7. Selections in Lymphocyte Development

8. Pluripotent HSCs =>Lymphocyte Lineages

9. Outline • Overview of Lymphocyte Development • Generation of Diversity in Lymphocyte Ag receptors • B & T Lymphocyte Development • Summary & Question

10. Key Concepts in Diversity of lymphocyte Ag receptors 1. The germline organizations of Ig & TCR are similar. a. Multiple (V)ariable, (D)iversity, & (J)oining gene segments. b. These gene segments are spatially separated on the chromosomes 2. During lymphocyte development, gene rearrangement of Ag receptor genes occurs: a. Combinatorial diversity - Non-homologous DNA recombination (VDJ recombination) - Lymphoid-specific enzymes- RAG1 & RAG2 Other factors- DNA-PK, TdT, Ku, DNA ligase IV… b. Junctional diversity - Addition or removal of nucleotides among V (D)J joints - The largest contribution to diversity of Ag receptors

11. In 1970s, Scientists thought • 105 genes in human body => Antibodies over 1010 ? • => Limited gene segments • Hybrid-DNA nature (Recombination) • Diverse Ab products

12. Germline organization of Human Ig loci D segment in H chain

13. Germline organization of Human TCR loci D segment in b chain

14. Domains of Ig & TCR proteins HV3 or CDR3

15. Generation of Diversity in Ag receptor genes

16. V(D)J Recombination-I 1. Recombination Signal Sequence (RSS): Heptamer & Nonamer => separated by 12- or 23- spacers => Recognized by Recombinase 2. Deletion-VJ exons have the same orientation 3. Inversion – VJ have the different orientation

17. V(D)J Recombination-II

18. V(D)J Recombination III –Junctional Diversity Junctional diversity=> the greatest variability at HV3 (CDR3)

19. Generation of Diversity in Ig or TCR

20. Generation of Diversity in Ig or TCR

21. Overview of Ig gene rearrangement

22. Outline • Overview of Lymphocyte Development • Generation of Diversity in Lymphocyte Ag receptors • B & T Lymphocyte Development • Summary & Question

23. Pluripotent HSCs => lymphocyte lineages II 1. Regulated by transcriptional factors 2. Notch-1 & GATA3 => T EBF, E2A & Pax-5 => B

24. Features of B lymphocyte development 1. Maturation of B Lymphocytes development - Rearrangement & expression of Ig gene in a precise order - Selection & proliferation of pre-B cells via pre-Ag receptor 2. Selection of the mature B cell repertoire - Self Ag => Affect the strength of the BCR signal - Immature B cells => self Ag/high avidity => Receptor editing => Additional L chain recombination => Not Self-reactive Fail to receptor editing => Apoptosis 3. During this maturation, B cell lineage cells go through distinct stages => A specific Ig gene expression => Distinct surface markers 4. At Pre-B cell stage, H chain recombination occurs first and associates w/ Surrogate light chains (l5 & VpreB). - l5 & VpreB are similar to k & l light chains but invariant - form pre-B cell receptor => Development

25. Stages of B lymphocyte development-I

26. Stages of B lymphocyte development-II

27. Pre-B cell & Pre-T cell receptors

28. Stages of B lymphocyte development-III

29. Receptor Editing–2nd Chance

30. Features of T lymphocyte development 1. Maturation of T Lymphocytes development - Sequential Rearrangement & expression of TCR genes - Selection & proliferation of T cell repertoire 2. Selection of the mature T cell repertoire occurs in Thymus - Positive selection => Self MHC-restricted - Negative Selection =>Self Ag-MHC/high avidity=> Apoptosis => Central Tolerance 3. CD4 & CD8 are surface markers for differentiation of Thymocytes (immature T cells).

31. Stages of T lymphocyte development-I

32. Maturation of T lymphocytes in Thymus

33. CD4 & CD8 expression on Thymocytes

34. Selection of T lymphocytes in Thymus

35. Specificity & Avidity are different in T-cell Selection

36. TCR transgenic mouse model => T cell selection

37. Positive selection-Bone marrow chimera mice

38. Outline • Overview of Lymphocyte Development • Generation of Diversity in Lymphocyte Ag receptors • B & T Lymphocyte Development • Summary & Question

39. SUMMARY 1. Rearrangement and expression of Ag receptor genes regulate B & T lymphocyte development. 2. Selection events are involved in preserving cells w/ correct Ag receptors and eliminating dangerous cells w/ self-recognition Ag receptor 3. Gene rearrangement of Ag receptor genes occurs during lymphocyte development. - The basic mechanism is common to both B & T cells - Generate the Combinatorial diversity by randomly combining different V(D)J gene segments - Generate the Junctional diversity by filling the gap betweenjointed gene segments

40. Question What is happened to the immune system if RAG1 & RAG2 are mutated?