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Supplementary Figure 1 - PDF file 73K, Tumor cells express high levels of PD-L1

Supplementary Figure 2 - PDF file 20K, Expression of PD-1 is upregulated on CAR T cells following antigen stimulation with e0771-Her2+ tumor cells

upplementary Figure 3 - PDF file 17K, Gr1+ cells express PD-1 and PD-L1

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression.Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach.Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen.Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR.

Supplementary Figure Legends - PDF file 66K, Supplementary Figure legends

PDF file - 296K, A)Regulation of Hif-1� and Hif-2alpha by hypoxia in breast cancer cells. B) Schematic of injection schedule

PDF file 3180K, Normal NK cell homeostasis in NLRP3-/- mice.

PDF file 1235K, Caspase-1 and IL-1 do not affect the development of lung metastasis.

PDF file - 221K, A) Pre-metastatic niche induced by hypoxia increases melanoma metastasis. B) Factors identified in HCM of breast cancer cells

PDF file-862K.NLRP3 inflammasome promotes the formation of MCA-induced fibrosarcomas in BALB/c mice.

PDF file - 49K, A) Analysis of other immune cell population in the pre-metastatic niche. B) CD69 expression on NK cells in pre-metastatic niche. C) Confirmation of NK cell ablation with anti-asialoGM1

PDF file - 33K, A/B) Reduction of CD11b+/Ly6Cmed but not CD11b+/Ly6Chigh cells in pre-metastatic niche induced by HCM after MCP-1 neutralization

PDF file 1781 K, CD11b+ Gr-1int cells do not suppress or enhance NK cell cytotoxic function.

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PDF file 1.8 MB, Cytokine profile of CD11b+ Gr-1int cells.

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PDF file 806K, Host NLRP3 does not affect B16F10 subcutaneous growth.

PDF file 2632K, CD11b+ Gr-1int cells express NLRP3.

Chemokines are chemotactic cytokines that regulate the migration of immune cells. Chemokines function as cues for the coordinated recruitment of immune cells into and out of tissue and also guide the spatial organization and cellular interactions of immune cells within tissues. Chemokines are critical in directing immune cell migration necessary to mount and then deliver an effective anti-tumor immune response; however, chemokines also participate in the generation and recruitment of immune cells that contribute to a pro-tumorigenic microenvironment. Here, we review the role of the chemokine system in anti-tumor and pro-tumor immune responses and discuss how malignant cells and the tumor microenvironment regulate the overall chemokine landscape to shape the type and outcome of immune responses to cancer and cancer treatment.

CD8(+) T cells responding to chronic infection adapt an altered differentiation program that provides some restrain on pathogen replication yet limits immunopathology. This adaptation is imprinted in stem-like cells and propagated to their progeny. Understanding the molecular control of CD8(+) T cell differentiation in chronic infection has important therapeutic implications. Here, we found that the chemokine receptor CXCR3 was highly expressed on viral-specific stem-like CD8(+) T cells and that one of its ligands, CXCL10, regulated the persistence and heterogeneity of responding CD8(+) T cells in spleens of mice chronically infected with lymphocytic choriomeningitis virus. CXCL10 was produced by inflammatory monocytes and fibroblasts of the splenic red pulp where it granted stem-like cells access to signals promoting differentiation and limited their exposure to pro-survival niches in the white pulp. Consequently, functional CD8(+) T cell responses were greater in Cxcl10(−/−) mice and were associated with a lower viral set point.

The NLRP3 inflammasome plays a crucial role in the innate immune response to pathogens and exogenous or endogenous danger signals. Its activity must be precisely and tightly regulated to generate tailored immune responses. However, the immune cell subsets and cytokines controlling NLRP3 inflammasome activity are still poorly understood. Here, we have shown a link between NKT-cell-mediated TNF-α and NLRP3 inflammasome activity. The NLRP3 inflammasome in APCs was critical to potentiate NKT-cell-mediated immune responses, since C57BL/6 NLRP3 inflammasome-deficient mice exhibited reduced responsiveness to α-galactosylceramide. Importantly, NKT cells were found to act as regulators of NLRP3 inflammasome signaling, as NKT-cell-derived TNF-α was required for optimal IL-1β and IL-18 production by myeloid cells in response to α-galactosylceramide, by acting on the NLRP3 inflammasome priming step. Thus, NKT cells play a role in the positive regulation of NLRP3 inflammasome priming by mediating the production of TNF-α, thus demonstrating another means by which NKT cells control early inflammation.

CD96, CD226 (DNAM-1) and TIGIT belong to an emerging family of receptors that interact with nectin and nectin-like proteins. CD226 activates natural killer (NK) cell-mediated cytotoxicity, whereas TIGIT reportedly counterbalances CD226. In contrast, the role of CD96, which shares the ligand CD155 with CD226 and TIGIT, has remained unclear. In this study we found that CD96 competed with CD226 for CD155 binding and limited NK cell function by direct inhibition. As a result, Cd96(-/-) mice displayed hyperinflammatory responses to the bacterial product lipopolysaccharide (LPS) and resistance to carcinogenesis and experimental lung metastases. Our data provide the first description, to our knowledge, of the ability of CD96 to negatively control cytokine responses by NK cells. Blocking CD96 may have applications in pathologies in which NK cells are important.

Summary Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A 2A /A 2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A 2A −/− mice were strongly protected against tumor metastasis, indicating that host A 2A receptors enhanced tumor metastasis. A 2A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A 2A blockade was due to enhanced NK cell function. Interestingly, A 2B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73 + tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A 2A or A 2B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A 2A /A 2B receptor antagonists have already entered clinical trials in other therapeutic settings.

The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell–mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ–dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow–derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b+Gr-1intermediate (Gr-1int) myeloid cells within the lung tumor microenvironment of NLRP3−/− mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b+Gr-1int myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell–mediated control of carcinogenesis and metastases and identify CD11b+Gr-1int myeloid cells that promote NK cell antimetastatic function. Cancer Res; 72(22); 5721–32. ©2012 AACR.

Chronic inflammation is a risk factor for tumor development. However, understanding the effect of the immune system on tumor development has only been significantly advanced over the past two decades. We now appreciate that the immune system, in addition to tumor-suppressive function by eliminating nascent transformed tumor cells, can also exert selection pressure on tumor cells and facilitate tumor growth by providing a favorable tumor microenvironment. Yet, the distinctions between tumor-promoting inflammation and tumor-suppressive immunity are still not clear due to the dual role of some cytokines and other molecules in the immune system. The danger signal hypothesis has shaped our view of the role of immunity in cancer development, but still little is known about the exact role of danger signal receptors in cancer progression. In this review, we introduce the processes of cancer immunoediting and inflammation-induced cancer and discuss what is currently known about the role of danger signal receptors in cancer development and progression.

Chemokines are chemotactic cytokines that control the migration of cells between tissues and the positioning and interactions of cells within tissue. The chemokine superfamily consists of approximately 50 endogenous chemokine ligands and 20 G protein–coupled seven-transmembrane spanning signaling receptors. Chemokines mediate the host response to cancer by directing the trafficking of leukocytes into the tumor microenvironment. This migratory response is complex and consists of diverse leukocyte subsets with both antitumor and protumor activities. Although chemokines were initially appreciated as important mediators of immune cell migration, we now know that they also play important roles in the biology of nonimmune cells important for tumor growth and progression. Chemokines can directly modulate the growth of tumors by inducing the proliferation of cancer cells and preventing their apoptosis. They also direct tumor cell movement required for metastasis. Chemokines can also indirectly modulate tumor growth through their effects on tumor stromal cells and by inducing the release of growth and angiogenic factors from cells in the tumor microenvironment. In this Masters of Immunology primer, we focus on recent advances in understanding the complex nature of the chemokine system in tumor biology with a focus on how the chemokine system could be used to augment cancer immunotherapeutic strategies to elicit a more robust and long-lasting host antitumor immune response. Cancer Immunol Res; 2(12); 1125–31. ©2014 AACR.

International audience; The immune system is routinely confronted with cell death resulting from the physiological turnover of renewable tissues, as well as from pathological insults of several types. We hypothesize the existence of a mechanism that allows the immune system to discriminate between physiological and pathological instances of cell death, but the factors that determine whether cellular demise is perceived as a neutral, tolerogenic or immunogenic event remain unclear 1. Infectious insults are accompanied by so-called microbe-associated molecular patterns (MAMPs), i.e., viral or bacterial products that activate immune cells through a panel of pattern-recognition receptors (PRRs) 2. Moreover, intracellular pathogens generally trigger adaptive mechanisms aimed toward the re-establishment of homeosta-sis, including the unfolded protein response (UPR) and autophagy 3,4. In mammals, MAMPs coupled to the activation of stress responses Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN- and IFN- receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy. npg

International audience; Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1β. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

The NLRP3 inflammasome plays a crucial role in the innate immune response to pathogens and exogenous or endogenous danger signals. Its activity must be precisely and tightly regulated to generate tailored immune responses. However, the immune cell subsets and cytokines controlling NLRP3 inflammasome activity are still poorly understood. Here, we have shown a link between NKT-cell-mediated TNF-α and NLRP3 inflammasome activity. The NLRP3 inflammasome in APCs was critical to potentiate NKT-cell-mediated immune responses, since C57BL/6 NLRP3 inflammasome-deficient mice exhibited reduced responsiveness to α-galactosylceramide. Importantly, NKT cells were found to act as regulators of NLRP3 inflammasome signaling, as NKT-cell-derived TNF-α was required for optimal IL-1β and IL-18 production by myeloid cells in response to α-galactosylceramide, by acting on the NLRP3 inflammasome priming step. Thus, NKT cells play a role in the positive regulation of NLRP3 inflammasome priming by mediating the production of TNF-α, thus demonstrating another means by which NKT cells control early inflammation.

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression. Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach. Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR . See related article by Morales-Kastresana, et al., [p. 5546][1] This article is featured in Highlights of This Issue, [p. 5543][2] [1]: /lookup/volpage/19/5546?iss=20 [2]: /lookup/volpage/19/5543?iss=20

Asbestos exposure can result in serious and frequently lethal diseases including malignant mesothelioma. The host sensor for asbestos induced inflammation is the NLRP3 inflammasome and it is widely assumed that this complex is essential for asbestos induced cancers. Here we report that acute interleukin 1beta production and recruitment of immune cells into peritoneal cavity were significantly decreased in the NLRP3 deficient mice after the administration of asbestos. However NLRP3 deficient mice displayed a similar incidence of malignant mesothelioma and survival times as wild type mice. Thus early inflammatory reactions triggered by asbestos are NLRP3 dependent but NLRP3 is not critical in the chronic development of asbestos induced mesothelioma. Notably in a two stage carcinogenesis induced papilloma model NLRP3 deficient mice showed a resistance phenotype in two different strain backgrounds suggesting a tumour promoting role of NLRP3 in certain chemically induced cancer types.Immunology and Cell Biology advance online publication 25 September 2012; doi:10.1038/icb.2012.46.

Hypoxia within a tumor acts as a strong selective pressure that promotes angiogenesis, invasion, and metastatic spread. In this study, we used immune competent bone marrow chimeric mice and syngeneic orthotopic mammary cancer models to show that hypoxia in the primary tumor promotes premetastatic niche formation in secondary organs. Injection of mice with cell-free conditioned medium derived from hypoxic mammary tumor cells resulted in increased bone marrow–derived cell infiltration into the lung in the absence of a primary tumor and led to increased metastatic burden in mammary and melanoma experimental metastasis models. By characterizing the composition of infiltrating bone marrow–derived cells, we identified CD11b+/Ly6Cmed/Ly6G+ myeloid and CD3−/NK1.1+ immune cell lineages as key constituents of the premetastatic niche. Furthermore, the cytotoxicity of natural killer (NK) cells was significantly decreased, resulting in a reduced antitumor response that allowed metastasis formation in secondary organs to a similar extent as ablation of NK cells. In contrast, metastatic burden was decreased when active NK cells were present in premetastatic lungs. Together, our findings suggest that primary tumor hypoxia provides cytokines and growth factors capable of creating a premetastatic niche through recruitment of CD11b+/Ly6Cmed/Ly6G+ myeloid cells and a reduction in the cytotoxic effector functions of NK cell populations. Cancer Res; 72(16); 3906–11. ©2012 AACR.

Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have proven fruitful using the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). In these different models, the effects of α-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell, B cell, or conventional CD4+ and CD8+ T cell activation/regulation as well as immunomodulatory cytokine production. In this study, we showed that γδ T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells (cytokine production and cytotoxic properties) and were required to achieve an optimal α-GalCer–induced immune response. Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the α-GalCer–induced IFN-γ production by γδ T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting their bystander effect on γδ T cells. In line with this, γδ T cells failed to directly recognize the CD1d/α-GalCer complex. We also provided evidence that γδ T cells increase their cytotoxic properties after α-GalCer injection, resulting in an increase in killing of tumor cell targets. Moreover, using cancer models, we demonstrated that γδ T cells were required for an optimal α-GalCer–mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with α-GalCer enhanced the adaptive immune response elicited by OVA, and this effect was strongly mediated by γδ T cells. We conclude that γδ T cells amplify the innate and acquired response to α-GalCer, with possibly important outcomes for the therapeutic effects of this compound.

Toll-like receptor-4-lipopolysaccharide (LPS)-mediated inflammation is used to delineate signals involved in cross-talk between antigen-presenting cells (APCs) and lymphocytes such as natural killer (NK) cells. Following APC stimulation and cytokine release, NK cells produce interferon (IFN)-γ. High levels of LPS induce endotoxicosis, a systemic inflammatory disease in which IFN-γ causes significant morbidity and mortality. Several studies have highlighted the role of interleukin (IL)-18, IL-1β, IL-17A and IFN-γ in the development of endotoxicosis, but whether these cytokines interact with each other is yet to be determined. Our data demonstrate that IL-18 and IL-17A have important roles in NK cell IFN-γ production during endotoxicosis. Importantly, we provide the first evidence that IL-18 also has a role in IL-17A production by T-cell receptor (TCR)-δ cells. Furthermore, we demonstrate that IL-18-deficient mice have a defect in γδ T-cell homeostasis and IL-1β production, both of which can contribute to the development of disease through induction of IL-17A. These results reveal novel requirements for IL-18 in innate immune cell homeostasis and activation, demonstrating that the role of IL-18 in innate immunity occurs at a level other than activation.

Lymphocyte perforin and serine protease granzymes are well-recognized extrinsic mediators of apoptosis. We now demonstrate that cytotoxic lymphocyte granule components profoundly augment the myeloid cell inflammatory cytokine cascade in response to TLR4 ligation. Whereas caspase-1–deficient mice were completely resistant to LPS, reduced serum cytokine production and resistance to lethal endotoxicosis were also obtained with perforin-deficient mice, indicating a role for granzymes. Consistently, a lack of granzyme M (GrzM) resulted in reduced serum IL-1α, IL-1β, TNF, and IFN-γ levels and significantly reduced susceptibility to lethal endotoxicosis. These altered responses were also observed in granzyme A-deficient but not granzyme B-deficient mice. A role for APC–NK cell cross-talk in the inflammatory cascade was highlighted, as GrzM was exclusively expressed by NK cells and resistance to LPS was also observed on a RAG-1/GrzM-double deficient background. Collectively, the data suggest that NK cell GrzM augments the inflammatory cascade downstream of LPS-TLR4 signaling, which ultimately results in lethal endotoxicosis. Most importantly, these data demonstrate that granzymes should no longer be considered solely as mediators of apoptosis, but additionally as potential key regulators of inflammation.

The Toll-like receptor 3 (TLR3) agonist poly(I:C) is a promising adjuvant for cancer vaccines due to its induction of potent antitumor responses occurring primarily through the activation of dendritic cells (DCs) and natural killer (NK) cells. However, little is known about the role of TLR3 sensing of endogenous ligands in innate tumor immunosurveillance. Here, we investigated whether TLR3 could modulate immune responses and facilitate tumor control without administration of an agonist. We observed only limited impact of TLR3 deficiency on spontaneous carcinogenesis and primary growth of B16F10, E0771 or MC38 tumors when injected subcutaneously to mice. Nevertheless, TLR3 was observed to limit experimental B16F10 lung metastasis, an immunologic constraint dependent on both IFNγ secretion and NK cells. Interestingly, we observed that NK cells derived from Tlr3 null (Tlr3-/- ) mice were hyporesponsive to cytokine stimulation. Indeed, compared with NK cells with intact TLR3, Tlr3-/- NK cells produced significantly reduced pro-inflammatory cytokines, including IFNγ, when incubated in the presence of different combinations of IL-12, IL-18 and IL-15. Bone-marrow chimera experiments established that competent NK cell responses required TLR3 sensing on radio-sensitive immune cells. Intriguingly, although CD8α DCs robustly express high levels of TLR3, we found that those cells were not necessary for efficient IFNγ production by NK cells. Moreover, the defective NK cell phenotype of Tlr3-/- mice appeared to be independent of the gut microbiota. Altogether, our data demonstrate a pivotal role of endogenous TLR3 stimulation for the acquisition of full NK cell functions and immune protection against experimental metastasis.