Cross-talk among M , NK and cancer cells: M  cells help NK cells to attack tumor by stimulatory RAE-1 but escape from NK killing by inhibitory Qa-1 Zhigang Tian, Zhixia Zhou, Cai August 19, 2010, Shenzhen Institute of Immunology/School of Life Sciences University of Science & Technology of China Chinese Academy of Sciences Hefei, Anhui, China

Figure 2-49 NK cell: a professional killer of tumor

Nature review immunology, 2008;8:713 1.initiation 2.effector 3.termination Three main stages of cell lytic synapse SMAC: supramolecular activation cluster SMIC: supramolecular inhibitory cluster Two kinds of immunological synapse NK Cell Immunological Synapse

Nature review immunology, 2008;8:713

Annu. Rev. Immunol :225–74 Natural killer cell recognition of “missing self”

KIR : killer immunoglobulin-like receptors KLR: killer lectin-like receptors

MM NK Death Tumor cells Poly I:C 1. Macrophages increase cytolysis of NK cells against cancer cells + % Lysis of NK cells to M  :125:15:1 E/T ratio Untreated peritoneal M  poly I:C treated peritoneal M  :125:150:1 ** % Lysis of NK cells to tumor NK alone NK/M  (medium) NK/M  (poly I:C) * * E/T ratio

MM NK Death Tumor cells Poly I:C NKG2D ㈩ 2. Poly I:C-treated macrophages increase NKG2D expression of NK cells Fold Induction of NKG2D gene NK alone NK + RAW264.7 NK + RAW264.7 (poly I :C) * 36.48% 32.74% 50.71% NK only NK + RAW264.7 NK + RAW264.7 (poly I:C) NKG2D Events ㈩

MM Death Tumor cells Poly I:C 3. Increased NKG2D expression correlates to activation of NK cells NK only NK + M  NK + M  (poly I:C) NK + M  (poly I:C) + Isotype-control NK + M  (poly I:C) + anti-NKG2D Events 25.71% 25.13% 62.52% 55.03% 38.41% CD69 NK NKG2D ㈩ ㈩

MM NK Death Tumor cells Poly I:C NKG2D ㈩ 4. Increased NKG2D expression correlates to function of NK cells IFN - NK+MØ (poly I:C)+ anti-NKG2D MØ alone NK+MØ NK alone NK+MØ (poly I:C) NK+MØ (poly I:C)+Isotype-contyol MØ (poly I:C) IFN-  (pg/ml) * E:T ratio :125:150:1 M  (Medium) M  (poly I:C) M  (poly I:C)+Isotype-control M  (poly I:C)+Anti-NKG2D % Lysis to YAC-1 cells * * * ㈩

MM NK Death Tumor cells Poly I:C NKG2D ㈩ IFN - IL-15 IFN-  IL-12 IL Macrophage-derived cytokines play critical roles in NK cell activation IL-15IL-12 IL-18 IFN-  IFN-  ** * * * pg/ml Untreated M  poly I:C treated M  NK Events 32.03% 40.24% 54.79% 47.80% 31.05% 22.08% NK alone NK+M  NK M  (poly I:C) Isotype anti-IFN-  anti-IL-15 M  (poly I:C) NKG2D ㈩

6. Macrophage-derived cytokines play critical roles in NK cell function M  alone NK alone NK+ M  NK+ M  (poly I:C) NK+ M  (poly I:C)+Isotype-contyol NK+ M  (poly I:C)+ anti-Il-15 NK+ M  (poly I:C)+ anti-IFN-  IFN-  (pg/ml) * MM NK Death Tumor cells Poly I:C NKG2D ㈩ IFN - IL-15 IFN-  IL-12 IL-18 ㈩

MM NK Death Tumor cells Poly I:C NKG2D ㈩ RAE-1 IFN - 7. Up-regulation of NKG2D ligands on poly I:C-treated macrophages 0 µg 10  g/ml(polyI:C) 100  g/ml(polyI:C) RAE-1H60MULT-1 20  g/ml(polyI:C) * * * ** * * Fold Induction Untreated peritoneal M  poly I:C treated peritoneal M  RAE-1H60MULT-1 ** Fold Induction 0  g/ml 10  g/ml 20  g/ml 100  g/ml 27.99% % 53.35% 63.76% Events C57BL/6 BALB/c RAE % 16.78% 33.85% 74.76% 1.87% 10.69% 35.85% 63.77% Events RAW IL-15 IFN-  IL-12 IL-18 ㈩

MM NK Death Tumor cells Poly I:C NKG2D ㈩ RAE-1 IFN - 8. TLR3 mediates the up-regulation of NKG2D ligands by macrophages 0 µg 100 µg poly I:C 100 µg poly I:C+si-Control 100 µg poly I:C+si-TLR3 Fold Expression RAE-1 H60 MULT-1 * * * TLR3TLR4 Fold Induction ** Untreated M Ø poly I:C treated M  IL-15 IFN-  IL-12 IL-18 ㈩

MM NK Death Tumor cells Poly I:C NKG2D ㈩ RAE-1 IFN - IL-15 IFN-  IL-12 IL-18 Qa-1b NKG2A ㈠ 9. Qa-1 contributes to protect macrophages from NK cell-mediated lysis YAC-1 BALB/c M  BALB/c M  +poly I:C RAW264.7 RAW264.7+poly I:C Relative Expression Qa-1a Qa-1b ** * Fold Induction ** * RAE-1 H60 MULT-1 YAC-1 BALB/c M  BALB/c M  +poly I:C RAW264.7 RAW264.7+poly I:C NK only NK + RAW264.7 NK + RAW264.7 (poly I:C) Events 44.13% 37.43% 36.56% NKG2A ㈩

10. Qa-1 knock-down cause macrophages sensitive to NK cell killing :125:1 * poly I:C poly I:C+Isotype-control poly I:C+anti-Qa-1b untreated * % Lysis to RAW264.7 E/T ratio MM NK Death Tumor cells Poly I:C NKG2D ㈩ RAE-1 IFN - IL-15 IFN-  IL-12 IL-18 Qa-1b NKG2A ㈠ ㈩

MM NKG2D RAE-1 NK IFN-  IL-15 IFN-  IL-12 IL-18 Death Qa-1b NKG2A IFN - Tumor cells Poly I:C ㈩ ㈩ ㈠ M  cells help NK cells to attack tumor by RAE-1 but escape from NK killing by Qa-1 Conclusion Macrophages may activate NK cells to attack tumor by activating RAE-l-NKG2D recognition but protect themselves from cytolysis of NK cells via preferential inhibitory Qa-1-NKG2A recognition, by which the NK cells will constitutively be activated by macrophages to keep strong innate immunity against tumor. Cross-talk among M , NK and cancer cells

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20 NK receptor complex-ligand interactions

21 Klas Kärre in the laboratory at the Karolinska Institute in Nature immunology, 2008;9:477 The idea that NK cells can distinguish aberrant cells by recognizing ‘absence of the expected’, rather than ‘presence of the unexpected’ emerged more than 25 years ago. Klas Kärre recapitulates how the idea took shape, and the first five years of experimental work to test its general predictions. Natural killer cell recognition of missing self