Mechanisms of lymphocyte-mediated cytotoxicity Dr. Ronald Smeltz Medical Sciences Building Room 325 rbsmeltz@vcu.edu

1. Mechanisms of lymphocyte-mediated cytotoxicityDr. Ronald SmeltzMedical Sciences BuildingRoom 325rbsmeltz@vcu.edu

2. Outline • Identify the effector cells of cytotoxicity • NK cell recognition of target cells • CTL differentiation into cytotoxic T cells • Mechanisms of cytotoxicity • Granules • Receptor-mediated • Signaling cytotoxicity • Transition of cytotoxic T cells into memory cells

3. Examples • Pathogens • Tumors • Transplantation • Homeostasis • Tolerance • Elimination of antigen-bearing dendritic cells

4. Cells that mediate cytotoxicity • Natural killer (NK) cells • CD8+ cytotoxic T cells (CTL)

5. NK cell recognition of target cells

6. NK cell recognition

7. The “Missing Self” Hypothesis • States that altered expression/down-regulation of MHC Class I on target cells leads to spontaneous NK-mediated destruction of the target cell • Down-regulation of MHC Class I OR over-expression of NK cell activating molecules leads to NK cell-mediated killing of target cell

8. The “Altered Self” hypothesis Activation of NK cells is the net effect of inhibitory and activating signals

9. NK cell recognition molecules NK cell receptorsTarget cell ligands • Ly49 (mouse) H-2K, H-2D • KIR (human) HLA-A, HLA-B, HLA-C • CD94/NKG2 Qa-1b HLA-E • NKG2D Rae-1 MIC-A,MIC-B • NKp ?? Genetic polymorphism

10. Ly49 Family (mouse) • Most Ly49 members are inhibitory receptors, some are activating receptors • Bind to Class I • Inhibitory receptors bind Class I with high affinity • Example: Ly49A • Activating receptors bind Class I with low affinity, but bind additional ligands with high affinity • Examples: Ly49D, Ly49H

11. KIR family (humans) • KIR (Killer cell Ig-like receptors): • Immunoglobulin (Ig)-like domains • Two types of KIR • Long: “L”, inhibitory • 1-2 ITIM motifs • Short: “S”, activating • No ITIMS, no cytoplasmic domains • Similar to Ly49 family, inhibitory KIR molecules bind Class I with high affinity

13. Similarities between Ly49/KIR • Expressed on NK cells, activated CD8+ T cells • Bind to determinants of MHC Class I expressed by target cell • Inhibitory receptors have cytoplasmic ITIMs • Activating receptors bind to ITAM-bearing DAP12 adaptor proteins • Asp- in transmembrane domain of DAP; Lys+ in transmembrane domain of activating receptor • Different structures, very similar functions!

14. CD94/NKG2 • Inhibitory and activating receptors • CD94/NKG2A heterodimer: Inhibitory • NKG2A has a cytoplasmic ITIM • CD94/NKG2C heterodimer: • CD94/NKG2E heterodimer • Activating • NKG2C must associate with DAP12 • Recognize HLA-E (Qa-1b in mouse) on target cell • Binds leader peptides derived from other MHC class I alleles (HLA-A,B,C (humans), H-2 (mouse), HLA-G CD94/NKG2 interactions exhibit peptide specificity!

15. NKG2D: Activating receptor Structurally linked but not encoded by the MHC locus

16. Pan NK cell activating receptors

17. Summary

18. CTL differentiation into cytotoxic T cells

21. Requirements for generating CD8+ cytotoxic T cells • Extrinsic factors: • Antigen-bearing DC (Signal 1) • Co-stimulation (Signal 2) • CD27L/OX40L and CD27L/4-1BBL • CD40/CD40L • CD4 help • Cytokines (Signal 3) • Inverse relationship b/w inflammation and help • Intrinsic factors • Transcription factors • T-bet, Eomes

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27. Mechanisms of cytotoxicity

30. Effector molecules of cytotoxicity • Used by NK cells and CTL! • Granule exocytosis pathway • Perforin, Granzymes • Trigger apoptosis • Caspase-dependent/independent • Fas/Fas-L pathway • Receptor-mediated death, caspase-dependent

31. Granzymes, perforin Cause apoptosis Calreticulin Inhibitor of perforin Serglycin Complex with granzymes Cathepsins Cat C Processes granzymes Cat B Protection Lytic granules:Secretory lysosomes

32. Mediators of cytotoxicity

33. Delivery of granules

34. Hours 20-30 minutes The immunological synapse

35. Movement of granules

36. Summary Membrane reorganization Cytoskeletal polarization Docking Exocytosis Binding Entry C2 domain of perforin acts as a lipid-recognition domain; pH-dependent

37. Effects of Granzyme B

38. Granzyme B mode of action Bcl-2

39. Granzyme A mode of action

41. The granzymes synergistically promote cytotoxicity in a perforin-dependent manner

42. Fas-Fas-L

43. Similarities between CTL and NK cells • Importance of MHC class I molecules • Peptide requirements • CD8, Ly49/KIR, CD94/NKG2 • Formation of immunological synapse • Effector molecules, lytic granules

44. Differences between CTL and NK cells • NK cells: • Innate • Pre-formed effector molecules • Surveillance • CTL: • Adaptive • Must synthesize effector molecules de novo • Restricted circulation • Synapse

45. Transition of cytotoxic T cells into memory cells

46. Annual Reviews

47. Marking memory

48. Linear progression

49. Asymmetric model