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Recent evidence suggests that NK cells require priming to display full effector activity. Here, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signalling-deficient NK cells were found to be unable to secrete IFN-γ in response to ex vivo stimulation with IL-12. This was not due to a co-stimulatory role of IL-18 because blocking IL-18 signalling during the ex vivo stimulation with IL-12 did not alter IFN-γ production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-γ upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-γ transcription by NK cells was comparable in IL-18 signalling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-γ mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Key Points• Blockade of inhibitory KIRs with MHC class I antigens on lymphoma cells by anti-KIR antibodies augments NK-cell spontaneous cytotoxicity.• In combination with anti-CD20 mAbs, anti-KIR induces enhanced NK cell-mediated, rituximab-dependent cytotoxicity against lymphoma.

IntroductionNatural killer (NK) cells participate in innate immune responses by virtue of their ability to recognize, without prior specific sensitization, microbe-infected, transformed, and allogeneic cells, while sparing most autologous healthy cells. 1-4 NK cell activation can elicit 2 different effector functions: cytotoxicity of target cell and/or secretion of a large array of cytokines and chemokines. 5 NK cell activation is regulated by the dynamic integration of negative and positive signals. 6 Although the ligands, the mode of action, and the function of inhibitory NK receptors have been widely dissected, 7 the biologic function of NK cell-activating receptors as well as their downstream signaling circuits are only partially understood.Noncovalent association with signaling ITAM-bearing subunits is required for the function and often the stable cell-surface expression of several NK cell-activating receptors. In NK cells, ITAM-dependent activating receptors can be divided in 2 groups, according to their association with DAP12 (also known as KARAP and TYROBP), or with CD3 or FcR␥ ITAM-bearing polypeptides. 4,8 In humans, activating killer cell Ig-like receptors (KIR-S), CD94/NKG2C-E, and the NKp44 NCR (natural cytotoxicity receptor) associate with DAP12, whereas the NKp46 and NKp30 NCRs as well as the CD16 pair with CD3 and FcR␥. In mice, DAP12-dependent NK cell receptors include the activating Ly49 molecules, CD94/NKG2C-E, NKG2D-S, CD200R4, and PIRL-␤, whereas mouse NCR NKp46 (MAR-1), as well as CD16 and NKRP1-C, associates with FcR␥. Among these molecules, human NCRs are involved in natural cytotoxicity against several tumor cell lines 9 and have also been reported to interact with viral products. [10][11][12] NKG2D is an activating receptor expressed on both human and mouse NK and T cells, which recognizes nonclassic MHC class I molecules. The cell-surface expression of these ligands is often the consequence of cellular stress, such as DNA damage. 13,14 NKG2D ligands include MIC (MHC class I chain related) and ULBP (UL-16 binding protein) molecules in humans as well as Rae (retinoic acid early inducible 1), H60, and MULT-1 (murine UL16-binding protein-like transcript) proteins in mice. Alternative splicing of mouse Nkg2d, also known as Klkr1, generates 2 different transcripts: a long isoform, NKG2D-L, which associates only with the DAP10 subunit, as previously reported for human NKG2D, and a short one, NKG2D-S, which can couple with both DAP10 and DAP12. 15,16 Analysis of DAP12-deficient mice has pointed out that 2 DAP12-dependent mouse NK cell receptors, NKG2D-S and Ly49H, are involved in antitumor and antiviral NK cell activity, respectively. 15,17 Engagement of ITAM-dependent receptors initiates in NK cells a signal transduction cascade recapitulating some of the early steps that have been dissected in T cells. 6,18,19 First, members of src protein tyrosine kinase (PTK) family induces phosphorylation of both ITAM tyrosine residues, allowing the consequent recruitment and activation of Syk and/or ZAP...

anti-tumor therapy ͉ innate immunity ͉ pre-clinical model ͉ tolerance

Natural killer (NK) cells mediate anti-lymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of NK cell's efficacy by spontaneous cytoxicity. Using a killer cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with MHC class I antigens on lymphoma by anti-KIR antibodies prevents a tolerogenic interaction and augments NK cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in syngeneic and KIR transgenic murine lymphoma models. Specifically targeting murine NK cells in vitro, anti-Ly49C/I F(ab')2 increased anti-CD20 mAb-mediated NK cell degranulation as measured by CD107a mobilization and interferon-γ release, as well as increased cytotoxicity as assessed by chromium release. In the syngeneic EL4-huCD20 lymphoma model, anti-Ly49C/I F(ab')2 enhanced the anti-lymphoma activity of anti-CD20 mAb in vivo (Fig 1A-1B) and was NK cell-dependent with efficacy abrogated by NK cell depletion with anti-Asialo-GM1. To validate these observations and the potential efficacy of a fully human anti-KIR mAb (IPH2101, lirilumab), we demonstrated, in vitro, dose-dependent KIR2DL3 saturation and tumor lysis following blockade of KIR2DL3/HLA-C with lirilumab. In the transgenic KIR murine model, lirilumab therapy improved survival in an NK cell-dependent manner in both a prophylactic and therapeutic HLA+ (221 HLA-Cw3) lymphoma model. In combination, lirilumab therapy synergistically enhanced rituximab's anti-lymphoma efficacy in vivo in an NK cell-dependent manner (Fig 2A-C). These results support a therapeutic strategy of combination, rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor targeting therapy with an NK cell agonist thus stimulating the post-rituximab anti-lymphoma immune response. Disclosures: Thielens: Innate Pharma: Employment, Equity Ownership. Sola:Innate Pharma: Employment, Equity Ownership. Chanuc:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment. Bonnafous:Innate Pharma: Employment, Equity Ownership. Vivier:Innate Pharma: Membership on an entity’s Board of Directors or advisory committees. Romagne:Innate Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Andre:Innate-Pharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership.

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