1. Natural Killer Cells Andrew Makrigiannis Laboratory of Molecular Immunology IRCM

2. Natural Killer (NK) Cells Found predominantly in blood, liver, spleen, and lung. Rare in recirculating lymphocyte pool Non-T, Non-B lymphocyte Distinct Marker Combinations –CD3 -, Ig -, NK1.1 +, DX5 + Distinct Morphology –large granular lymphocytes Cytotoxic without prior sensitization Multiple roles in Innate Immunity –tumor surveillance –viral immunity –Th1 vs Th2 via IFN- 

3. IMPORTANCE Elevated NK cell-mediated activation or cytotoxicity seen in infections of: Arenavirus (Lymphocytic choriomeningitis virus) Herpesvirus (Murine cytomegalovirus and Herpes simplex virus) Orthomyxoviruses (Influenza virus) Picornavirus (Coxsackie virus) Peak NK cell responses in first several hours or days Peak T and B cell responses take more than a week to develop

4. IMPORTANCE Case study 1: Four related patients with specific NK cell deficiency resulting in Epstein Barr virus-driven lymphoproliferative disorder and viral-based respiratory illnesses. Case study 2: 13 year old with complete lack of NK cells –Initial overwhelming chicken pox infection and varicella pneumonia –Antibody and T cell responses were intact –Later developed primary life-threatening HCMV infection and a severe HSV infection. Other case studies: Low or no NK cell cytotoxic activity linked with increased sensitivity to: HSV, EBV, HCMV, papilloma virus. Also, NK cell defects occurn late in HIV infection.

5. NK CELL DEVELOPMENT (MAKING OF A KILLER)

6. NK CELL DEVELOPMENT In Vivo

7. NK CELL DEVELOPMENT In Vitro

8. NK CELL DEVELOPMENT

11. ‘LICENSE TO KILL’ ARMING OF NK CELLS

12. ‘LICENSING’ HYPOTHESIS

14. NK CELL FUNCTIONS

15. CYTOTOXIC MECHANISMS (THE ‘WEAPONS’)

16. PERFORIN -found exclusively in the cytoplasmic granules of killer cells -CTL and NK from perforin KO mice have major cytotoxic defects. -Perforin KO mice are more susceptible to tumours and infection. -pore forming protein of plasma membrane or endosome allowing granzymes to enter target cell cytoplasm -granulysin (humans) thought to function similarly to perforin

17. GRANZYME B -protease located in cytoplasmic granules of killer cells -cleaves after aspartate residues that have the sequence: Ile/Val-Gly/Met/Glu-X-Asp-X-Gly -caspase 3 and 8 are direct substrates for granzyme B -caspases are cysteine proteinases involved in apoptosis -other granzymes in granules (granzyme A, K, M)

18. GRANULE EXOCYTOSIS

20. GRANYZME B: APOPTOSIS

21. GRANZYME A KILLING

22. TRANSMEMBRANE DEATH RECEPTORS

23. NK CELL CYTOKINES Interferon gamma (IFN-  ): - activation, growth and differentiation of T, B, NK cells and macrophages -promotes Th1 differentiation -enhances MHC expression on APC -some direct anti-viral activity

24. NK CELL CYTOKINES Tumour necrosis factor alpha (TNF-  ): -paracrine and endocrine mediator of inflammation -regulates growth and differentation of a wide variety of cells -selectively cytotoxic for many transformed cells

25. NK CELL CYTOKINES Granulocyte/macrophage colony stimulating factor (GM-CSF): -survival and growth factor of hematopoeitic stem cells (HSCs) -differentiation and activating factor for granulocytic and monocytic cells -growth factor for endothelial cells, erythroid cells, megakaryocytes and T cells

26. RECEPTORS FOR TARGET CELL RECOGNITION (VICTIM SELECTION)

27. Antibody-dependent cell-mediated cytotoxicity (ADCC)

28. ACTIVATING RECEPTORS & ADAPTORS

29. ACTIVATING RECEPTORS

30. NKG2D MECHANISM

31. NKG2D LIGANDS

32. TUMOURS vs. NKG2D

33. CMV vs. NKG2D (part I)

34. Human C s-s C C NH2 C s-s C NH2 KIR Both MHC INHIBITORY RECEPTORS Mouse NH2 Ly49 NH2 CD94/NKG2A SHP-1 SHP-2 SHIP

35. MHC INHIBITORY RECEPTORS

36. normal cell H-2D d Ly49G granules NKG2D - NK cell tumor cell Rae1-  + NK cell INHIBITORY RECEPTOR FUNCTION

37. CMV vs. NK CELLS (part II) m157 (MHC-like)

38. MOUSE NK CELL RECEPTORS

39. Natural Killer Gene Complex (NKC) Nkrp1Nkg2Ly49 Mouse chr. 6 Human chr. 12 (Yokoyama and Plougastel, 2003, Nat. Rev. Immunol., 3:304)

40. NKC GENE ORGANIZATION

41. Stalk CRD 1 2 3 4 5 6 7 TM IC NKC GENE AND PROTEIN STRUCTURE

42. NKC RECEPTOR PROTEIN STRUCTURE

43. A, B, C, E, F, G, I, J, M, O, S, T, and V COOH MURINE INHIBITORY LY49 NH 2 2 SH2 PTPase SHP-1  Cytotoxicity  Cytokines ITIM

44. COOH R 54 R NH 2 2 D D Y x x L (x)6-8 Y x x L Y x x L Y x x L -PO 4 4 Zap70 DAP12 Syk SH2 Cat SH2 Cat D, H, L, P, R, U, and W MURINE ACTIVATING LY49  Cytotoxicity  Cytokines  Chemokines

45. Why are Ly49s important? Functional analogues of KIRs Regulate NK cell reactivity to self –Inhibitory Ly49 - Autoimmunity –Activating Ly49 - Pathogens Resistance to MCMV, Ectromelia, HSV, and Leishmania have been mapped to the Natural Killer Gene Complex.

46. Class I MHC Binding by C57BL/6 Ly49 Family Members AD d, k B? C D b, d, k, K b,d (peptide receptive) D D d, r, sp E? F ? GD d, L d Hm157 (MCMV) IDbDb J? Ly49Putative Ligand

47. Centromere Telomere Organization of the Ly49 Gene Family in B6 Mice Functional gene Pseudogene Transcriptional orientation a c m j l g i n h k d f x e q

48. Generation of the Complex Ly49 Repertoire Functional Pseudogene Y Y Y Y Y Y Y Y Y

49. The ‘129’ Inbred Mouse Strain Embryonic stem cells used for gene deletion models. 129 mice more susceptible to intracellular pathogens than B6 mice. 129 mice show impaired allogeneic and xenogeneic graft rejection. 129 NK cells show unusual reactivity with anti-Ly49 mAb. Many substrains: 129P3, 129X1, 129S1-6 etc.

50. The 129S6 Ly49 Cluster Sequence

51. Ly49 Subfamilies

52. 0.05 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 ui 129 k B6 n B6 h B6 u 129 i B6 i 2 129 j B6 c B6 i 1 129 q 2 129 q 3 129 q 1 129 q B6 b 129 b B6 r 129 d B6 lr 129 pd 129 x B6 p 129 m B6 l BALB o 129 v 129 a B6 g 129 g B6 t 129 Exon 2 i B6 ui 129 k B6 n B6 h B6 u 129 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 g 129 g B6 t 129 r 129 d B6 lr 129 pd 129 x B6 p 129 m B6 l BALB o 129 v 129 a B6 q 2 129 q 3 129 q 1 129 q B6 b 129 b B6 i 2 129 j B6 c B6 i 1 129 0.05 Total coding region 0.05 q 2 129 q 3 129 q 1 129 q B6 i B6 i 2 129 j B6 c B6 i 1 129 ui 129 k B6 n B6 h B6 u 129 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 b 129 b B6 r 129 d B6 lr 129 pd 129 x B6 p 129 g 129 g B6 t 129 m B6 l BALB o 129 a B6 Exon 3 0.05 b 129 b B6 m B6 l BALB g 129 g B6 t 129 o 129 v 129 a B6 q 2 129 q 3 129 q 1 129 q B6 ui 129 k B6 n B6 h B6 u 129 i B6 i 2 129 j B6 c B6 i 1 129 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 r 129 d B6 lr 129 pd 129 x B6 p 129 Exon 4 0.05 m B6 l BALB g 129 g B6 t 129 o 129 v 129 a B6 r 129 d B6 lr 129 pd 129 x B6 p 129 ui 129 n B6 h B6 u 129 i B6 i 2 129 j B6 c B6 i 1 129 k B6 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 Exon 6 0.05 ui 129 n B6 h B6 u 129 i B6 i 2 129 j B6 c B6 i 1 129 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 o 129 v 129 a B6 r 129 d B6 lr 129 pd 129 x B6 p 129 m B6 l BALB g 129 g B6 t 129 Exon 5 0.05 ui 129 n B6 h B6 u 129 i B6 i 2 129 j B6 c B6 i 1 129 k B6 s 129 f B6 e 129 e B6 ec 2 129 ec 1 129 m B6 l BALB g 129 g B6 t 129 o 129 v 129 a B6 r 129 d B6 lr 129 pd 129 x B6 p 129 Exon 7 v 129 Phylogenetic Analysis Reveals Hybrid Ly49 Genes

53. Hybrid Ly49 Stalk CRD TMIC Stalk CRD TMIC Stalk CRD TMIC Stalk CRD TM IC Stalk CRD TMIC Ly49HLy49L Ly49GLy49DLy49C

54. Ly49 Haplotype Evolution

55. The BALB/c Mouse - Common animal model for immune studies - Susceptible to MCMV - Decreased ability to kill CHO tumor cells - Known BALB/c Ly49: a, c, g, and l

56. C57BL/6 129 efdkhigl j ax m cn q1q1 eq2q2 e/c 2 l/rste/c 1 ruu/ii1i1 gp/di2i2 ov p q qeiglc 0 100 200 300 400 500600 xa BALB/c 700 kb q3q3 Ly49 Gene Content in Three Divergent Mouse Haplotypes functionalpseudogeneunknown

57. 0 100 200 300 400 500 600 700 kb CEN TEL  o i2i2 p pd g i1i1 ui u r ec 1 q3q3 t s lr ec 2 q2q2 v e 129S6  a c m j l g i n h k d f x e q q1q1 C57BL/6  a c l g i y e q BALB/c Three Ly49 Haplotypes

58. 0 100 200 300 400 500 600 700 kb CEN TEL  a c m j l g i n h k d f x e q C57BL/6  a c l g i y e q BALB/c CHO MCMV

59. 1 2 3 4 5 Human KIR Haplotypes (Trowsdale et al., 2001, Immunological Reviews, 181:20)

60. qe [] ig [] ca Framework Ly49 Genes

62. Non-MHC Inhibition of NK cells

64. AGCDFE AGCBFE CD69 CD94/ NKG2 CD69 CD94/ NKG2 B6 BALB QDKHLM CD69 CD94/ NKG2 B6 EXNF G I C J A QL CD69 CD94/ NKG2 BALB EY G I CA Nkrp1/Clr Ly49 99.710094.1 98.495.993.897.996.592.699.699.1 98.710095.7 Cen Tel

65. THERAPEUTIC POTENTIAL

66. Copyright ©2002 American Society of Hematology. Copyright restrictions may apply. Farag, S. S. et al. Blood 2002;100:1935-1947 Figure 1.

67. Copyright ©2002 American Society of Hematology. Copyright restrictions may apply. Farag, S. S. et al. Blood 2002;100:1935-1947 Figure 4.

68. FURTHER READING Figures and tables were taken from the following: What does it take to make a natural killer? Nat Rev Immunol. 2003 May;3(5):413-25. Natural killer cells, viruses and cancer. Nat Rev Immunol. 2001 Oct;1(1):41-9. How do natural killer cells find self to achieve tolerance? Immunity. 2006 Mar;24(3):249-57. Immune functions encoded by the natural killer gene complex. Nat Rev Immunol. 2003 Apr;3(4):304-16. Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol. 2002 Jun;2(6):401-9. Natural killer cell receptors: new biology and insights into the graft- versus-leukemia effect. Blood. 2002 Sep 15;100(6):1935-47.