T lymphocyte development and pathophysiology of SCID.pptx

T cell development# Understand SCID# Understand pathophysiology of Combined Immunodeficeincy# Understand the flowcytometric classification of SCID

1. T cell development Dr. Gayathri DM Resident Pediatric Immunology/ Rheumatology PGIMER

2. To know the immunobasis of SCID To understand newer disease/new defects For approaching targeted therapy Why understanding T cell development is important ?

3. Overview of lymphocyte development

4. How are they formed ?

5. How are they formed ?

6. How are they formed ? In utero life

7. Where do they get trained ?

8. What do they become ? CD4+ and CD8+ αβ cells γδ T cells Natural Killer T cells (NKT) Mucosa associated invariant T cells

9. How do they get trained ?

10. Commitment to T cell lineage TCR- γδ cells arise from fetal liver hematopoietic stem cells (10%) TCR- αβ cells arise from bone marrow derived hematopoietic stem cells (90%)

11. What is Commitment? Commitment of the common lymphoid progenitor to the T cell lineage It depends on transcription regulators that drive the development towards T cells

12. Notch 1 and GATA 3 are transcription factors that commit developing lymphocytes to the T cell lineage. They cause the expression of number of genes that are required for the development of T cells eg. Components of pre-TCR receptor, RAG-1/RAG-2 proteins.

13. Checkpoints in T cell development

14. Proliferation Committed progenitors proliferate first in response to cytokines The second proliferation happens in response to signal generated by a pre-antigen receptor (Occurs after select cells have rearranged the first set of antigen receptor genes successfully)

15. Proliferation IL-7 drives the proliferation of T cell progenitors Produced by stromal cells in the bone marrow, and the thymus

16. Receptor of IL-7 (and also of IL-2, IL-15) have a common gamma chain – mutation occurs in X-linked SCID (CD-132) IL-7 needed for T lymphocytes IL-15 needed for NK cells B cells remain apparently unaffected

17. What is the clinical significance ?

18. Steps of maturation in thymus

19. Rearrangement of Antigen Receptor Genes in T lymphocytes The genes that encode diverse antigen receptors of individual T lymphocytes are generated by the recombination of different Variable region gene segments (V) Diversity gene segments (D) Joining gene segments (J)

20. Variable region Constant region C β 1-Four exons along with exons in C β 2 coding the extracellular constant region, short hinge region, transmembrane segment and cytoplasmic tail

21. Also has non-germline junctional sequences that are added (by addition or removal of nucleotides) between the V, D, and J regions

22. Why 12/ 23 ? Is it random ? What is the significance ? QUESTION

23. Recombination Signal Sequences (RSS)

24. Process of VDJ recombination Synapsis : two distinct selected coding segments ad their adjacent RSSs have acquired specific histone marks and are brought together by a chromosomal looping event and held in position Cleavage : RAG-1: cleaves, RAG-2: activates RAG-1 Hair pin opening and processing : ARTEMIS opens the hair pins at the coding ends (type of endonuclease), TdT – terminal deoxynucleotidyl transferase: adds nucleotides to broken DNA ends Non homologous end joining : ubiquitous proteins CLINICAL SIGNIFICANCE?

25. Clinical significance ?

26. Generation of Diversity in T cells Combinatorial diversity -Different combinations of gene segments united by the V(D)J recombination – generate different binding sites It is further enhanced by the juxtaposition of two different randomly generated- alpha and beta chain in TCR molecules LARGEST contribution however is by the junctional diversity

27. However, the actual number of antigen receptors on B or T cells expressed in everyone at any one point in time is probably 10 7 OR 10 8 only – this reflects the finite number of lymphocytes that an individual can accommodate at any time.

28. Double-negative Thymocyte development Rearrangement of TCR-Beta CD4 and CD8 expressed also

29. Contributes to the largest proliferative expansion during the T cell development Here beta chain allelic exclusion also occurs. Clinical significance? Lack of any component in the pre-TCR complex results in a block in maturation of T cells at the double negative stage

30. Role of thymus in T cell maturation T lymphocytes originate from precursors that arise in the fetal liver and adult bone marrow and seed the thymus First detected around week 7 or 8 of human gestation Thymic environment provides stimuli that are needed for proliferation and maturation of thymocytes

31. Role of thymus in T cell maturation The movement of cells into and through the thymus is driven by chemokines. The progenitors of thymocytes express the chemokine receptor CCR9. Entry of these precursors into the thymus is dependent on CCR9 binding the chemokine ligand CCL25, which is produced in the thymic cortex. Chemokines such as CCL21 and CCL19 , which bind to the CCR7 chemokine receptor on thymocytes , direct the movement of developing T cells from the cortex to the medulla. Eventually, newly formed T lymphocytes, which express the sphingosine 1-phosphate receptor , exit the thymic medulla following a gradient of sphingosine-1 phosphate into the blood stream .

32. Role of thymus in T cell maturation Thymic stromal cells, including epithelial cells, secrete IL-7, which is a critical lymphopoietic growth factor. The rates of cell proliferation and apoptotic death are extremely high in cortical thymocytes. A single precursor gives rise to many progeny, and 95% of these cells die by apoptosis before reaching the medulla.

33. Largest proliferative expansion during the T cell development

34. Proliferation The second proliferation happens in response to signal generated by a pre-antigen receptor (Occurs after select cells have rearranged the first set of antigen receptor genes successfully) It is needed to ensure a large enough pool of progenitors to produce a highly diverse repertoire of mature, antigen-specific lymphocytes

35. Proliferation The greatest proliferative expansion of lymphocyte precursors occurs after successful rearrangement of the genes encoding the TCR-beta pre-antigen receptor. The signals produced by the pre-antigen receptors are responsible for far greater proliferation of developing lymphocytes than cytokine stimulation .

36. Why? If a cell succeeds in productively arranging its TCR-Gamma as well as TCR-Delta before a successful arrangement of TCR-Beta – it gets selected into TCR Gamma-Delta lineage This happens in 10% of developing double negative T cells These cells serve as an early defense against a limited number of commonly encountered microbes

37. Variable region Constant region

38. Thymic cortical epithelial cell

39. Stochastic model vs Instructive model ? (CD4=GATA3, CD8=RUNX3)

40. Therefore – Negative selection occurs twice Double positive stage – at cortex – mediated by the cortical thymic epithelial cells Single positive stage – at medulla – mediated by bone marrow derived dendritic cells, macrophages and medullary thymic epithelial cells (these express – AIRE (autoimmune regulator gene)

41. Theory: When there is intermediate level of affinity of interaction (not clear) – the self recognizing T cells develop into T regulatory cells

42. Role of thymus in T cell maturation The cell death is due to a combination of factors: F ailure to productively rearrange the TCR β chain gene F ailure to pass the pre-TCR/ β selection checkpoint, F ailure to be positively selected by self MHC molecules in the thymus Self antigen–induced negative selection (CENTRAL TOLERANCE)

43. Thank you