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

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

2. Lecture outline Role of cytotoxicity Cells that mediate cytotoxicity Activation requirements for cytotoxicity Effector molecules Signaling pathways Consequences of cytotoxicity Development of cytotoxic T cell memory

3. 1. Role of cytotoxicity Pathogens Intracellular pathogens Viruses, bacteria (Listeria), protozoa (Toxoplasma, Trypanosoma) Tumors Transplantation Rejection of MHC-mismatched grafts Homeostasis Central tolerance: thymus Peripheral tolerance: T cell activation-induced cell death

4. 2. Cells that mediate cytotoxicity: NK cells

7. 3. Activation requirements of cytotoxicity: NK cells

8. 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

9. Inhibitory and Activating signals

10. Ly49 (mouse) H-2K, H-2D KIR (human) HLA-A, HLA-B, HLA-C ILT-2 HLA-G CD94/NKG2 Qa-1b HLA-E NKG2D Rae-1 MIC-A,MIC-B NK cell recognition molecules

11. Ly49 Family (mouse) Most Ly49 members are inhibitory receptors, some are activating receptors Expressed on NK cells and some activated T cells 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

12. KIR family (humans) KIR (Killer cell Ig-like receptors): Immunoglobulin (Ig)-like domains Contrast to Ly49 members, which have lectin-like extracellular 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 T cells Both recognize determinants of MHC Class I expressed by target cell Inhibitory receptors have cytoplasmic ITIMs Immunoreceptor Tyrosine-based Inhibitory Motif Activating receptors bind to ITAM-bearing DAP12 adaptor proteins Immunoreceptor Tyrosine-based Activation Motif Crucial for cell surface stability

14. Additional functions of theLy49/KIR family: Ly49/KIR expression can prevent apoptosis Fas pathway of apoptosis Induction of c-FLIP (Fas-ligand inhibitory protein) Fas-L Decreased Fas-L expression Ly49/KIR expression can attenuate effector functions ITIM-mediated decrease in tyrosine phosphorylation Decreased cytokine production, cytotoxicity

15. NK cell recognition molecules:NKG2D Expressed by NK cells and T cells Requires association with DAP10 adaptor protein Bind to ligands on target cells associated with structural homology to MHC Class I molecules Target cell ligands are induced by stress or infection Rae-1?-?, H60, MULT-1 (mouse) MIC-A,B; ULBP1-4 (human) Consists of 1 gene: activating Enhance cytokine production and cytotoxicity Human and mouse CMV prevent NKG2D-dependent immunity Tumors can frequently secrete NKG2D ligands; or, secrete TGF-b which downregulates NKG2D expressionHuman and mouse CMV prevent NKG2D-dependent immunity Tumors can frequently secrete NKG2D ligands; or, secrete TGF-b which downregulates NKG2D expression

16. NK cell recognition molecules:CD94/NKG2 Inhibitory and activating receptors expressed on NK, T cells CD94/NKG2A heterodimer: Inhibitory NKG2A has a cytoplasmic ITIM CD94/NKG2C heterodimer: Activating NKG2C must associate with DAP12 Binds to non-classical MHC molecules: HLA-E on target cell HLA-E (Qa-1b in mouse) Class Ib Binds leader peptides derived from other MHC class I alleles (HLA-A,B,C (humans), H-2 (mouse), HLA-G

17. Signaling molecules ITIMs ITAMs DAP10, DAP12

18. Signaling molecules: Activation Activating signals ITAM motif: Immunoreceptor tyrosine- based activation motif Adaptor proteins: DAP-12 DAP-10 Increases phosphorylation Increased transcription of cytokine and chemokine genes, degranulation

19. DAP molecules Transmembrane proteins Non-covalent interactions with activating receptors ITAM-bearing �opposites attract� Asp- in transmembrane domain of DAP; Lys+ in transmembrane domain of activating receptor Other ITAM-bearing molecules: CD3? (T cells) 

20. Signaling molecules: Inhibition Inhibitory signals ITIM motif: immunoreceptor tyrosine- based inhibitory motif Activates phosphatases Decreased phosphorylation Suppresses transcription of cytokine and chemokine genes

24. Steps leading to cytotoxicity Antigen recognition and conjugate formation CTL activation (lymph nodes) Migration to infected tissues (blood) Recognition of infected cell (non-lymphoid tissues) Delivery of the lethal hit Detachment of CTL Target cell death

25. Molecules that promote CTL cytotoxicity Costimulatory molecules CD28 expressed on na�ve T cells CD137 (4-1BB) expressed on activated T cells CD27 expressed on na�ve T cells effector CTL in non-lymphoid tissues Cytokines Interleukin-12 IFN-?

26. 4. Effector molecules of cytotoxicity Granule exocytosis pathway Perforin, Granzymes Activates caspase-dependent and caspase-independent pathways Used by CTL and NK cells NK cells have pre-formed granules CTL require de novo synthesis, 1-3 days before maximum synthesis Trigger apoptosis Fas/Fas-L pathway Receptor-mediated death, caspase-dependent Used by CTL and NK cells

28. The lytic granule Specialized lysosomal compartment Contents: Perforin Granulysin (humans only) Granzymes (pH) Fas-L Calreticulin Serglycin Cathepsins (lysosomal enzymes) C: function B: function

30. Perforin Disrupts the membrane of the target cell Required for all granzyme delivery Hypotheses on mechanism: Pores allow entry of granzymes into target cell Outside-->inside Facilitates the entry of granzymes from endosomal compartment into cytosol Homology with Streptolysin and Listeriolysin Inside-->inside

31. Hypothetical models of perforin mode-of-action

32. 5. Signaling Pathways: Caspases Cytosolic proteases Cleave at aspartic acid residues Initiator caspases: Caspases-2, 8, 9 Effect on effector caspases Effector caspases: Caspases-3, 6, 7 Effector caspases activate DNases, which lead to DNA �cuts� and apoptosis

33. Granzyme B Cleaved by Cathepsin C, generates Granzyme B* Constituent of the lytic granule (active form) Serine protease Activates caspase-dependent and independent pathways that lead to apoptosis

34. Granzyme B pathways

35. Granzyme A: Caspase independent Cleaved by Cathepsin C, generates Granzyme A* Constituent of the lytic granule (active form) Synergistic with Granzyme B Different kinetics of killing Different targets of attack

36. Granzyme A pathways

37. Granzyme A Grz A targets the SET complex: Induces changes in mitochondrial membrane potential Increased susceptibility to oxidative damage No mitochondrial damage No Cytochrome C release Degrades proteins associated with the SET complex: Degrades IGAAD-->GAAD Introduces single-strand DNA �nicks� HI degradation: opens up chromatin to exonuclease attack

38. Fas/Fas-L Part of the death receptor pathway TNF-receptor family Fas-L (CD95L) is part of the lytic granule Expressed by effector cell Effector cell can be a CTL or CD4+ Th1 cell Fas is expressed on target cell Target cell can be an activated lymphocyte (fratricide) Fas may be expressed on same cell (suicide) Role in lymphocyte homeostasis Activation-induced cell death (AICD) (periphery) Termination of immune responses

39. Fas/Fas-L Immune privilege Eye Brain Testis Uterus Fas ligand on target tissues Fas on lymphocytes

40. Fas-Fas-L

41. 6. Consequences of cytotoxicity Apoptosis is inside-out death. Damage to DNA and mitochondrial components, usually does not lead to inflammation Normal aspect of cell growth and development Triggered by radiation, growth factor withdrawl

42. Features of apoptosis Breakdown of nuclear envelope Cleavage of DNA DNA condensation Disruption of electron transport system in mitochondria Exposure of phosphatidylserine (PS) residues on extracellular portion of cell membrane PS residues provide recognition and clearance of apoptotic bodies

43. Perforin and granzyme deficiencies Perforin: Viral, tumor clearance (mice) Granzyme B: Defective killing Perforin mediated cell membrane damage still occurs Requires prolonged contact with target Perforin-dependent (Grz A) and independent (Fas) Granzyme A Delayed killing Granzyme A x Granzyme B Equivalent to perforin KO

44. Fas/Fas-L deficiencies lpr mutation: deficient in Fas (CD95) gld mutation: deficient in Fas-L severe autoimmune disease hyper-proliferative syndrome Autoimmune lymphoproliferative syndrome (ALPS)

45. Similarities between CTL and NK cells Both CTL and NK cells have same effector molecules for killing target cells Perforin, Granzymes, Fas-L Both require formation of an immunological synapse Antigen/recognition receptors Adhesion molecules Both use actin rearrangements to cause directional migration of effector molecules to synapse and thus target cell Both cause apoptosis of target cell

46. Differences between CTL and NK cells NK cells: innate immune response (early, no memory) pre-formed effector molecules Patrolling and �sensing� host tissues, serve as immediate effector cells CTL: adaptive immunity (late, memory) must synthesize effector molecules de novo na�ve cells, restricted to spleen and lymph nodes, must undergo differentiation first

47. 7. Development of CTL memory

53. Cytokines promote memory

54. Memory T cell subsets Central memory (TCM) Development favored by cytokine stimulation (ie. IL-15) Express more IL-2 relative to IFN-? High capacity for self-renewal Found primarily in secondary lymphoid tissues Express CCR7 and CD62L (L-selectin) CD44hi Effector memory (TEM) Express more IFN-? relative to IL-2 Provide rapid defense against re-infection Found primarily in non-lymphoid tissues Usually don�t express CCR7 or CD62L CD44hi

55. Memory T cell subsets

56. Memory CTL subsets in vivo

58. Reading Janeway text (6th edition): NK cells: 90-92, 187, 213, 226-227 232-233 CTL: 340-347, 351-356 Memory : 235, 303, 450-451, 447