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Critical immune-suppressive pathways beyond programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) require greater attention. Nectins and nectin-like molecules might be promising targets for immunotherapy, since they play critical roles in cell proliferation and migration and exert immunomodulatory functions in pathophysiological conditions. Here, we show CD155 expression in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. Cd155-/- mice displayed reduced tumor growth and metastasis via DNAM-1 upregulation and enhanced effector function of CD8+ T and NK cells, respectively. CD155-deleted tumor cells also displayed slower tumor growth and reduced metastases, demonstrating the importance of a tumor-intrinsic role of CD155. CD155 absence on host and tumor cells exerted an even greater inhibition of tumor growth and metastasis. Blockade of PD-1 or both PD-1 and CTLA4 was more effective in settings in which CD155 was limiting, suggesting the clinical potential of cotargeting PD-L1 and CD155 function.

and T.C.M.F. performed all of the retrovirus transductions and confocal microscopy. J.U. developed the PEPCK tetramer and provided advice on its use. N.G. and W.R.H. produced the Plasmodium peptide-MHC I tetramer and helped design the PbT-I cell-killing assays.

◥ Purpose: Natural killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors (IR) regulate NK-cell-mediated tumor control. The IR T-cell immunoglobulin and ITIM domain (TIGIT) and its counterreceptor CD226 exert opposite effects on NK-cell-mediated tumor reactivity.Experimental Design: We evaluated the frequency, phenotype, and functions of NK cells freshly isolated from healthy donors and patients with melanoma with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of IL15 and TIGIT blockade in increasing NK-cell-mediated cytotoxicity in vitro and in two mouse models.Results: NK cells are present at low frequencies in metastatic melanoma, are dysfunctional, and downregulate both TIGIT and CD226 expression. As compared with TIGIT À NK cells, TIGIT þ NK cells exhibit higher cytotoxic capacity and maturation, but paradoxically lower cytotoxicity against CD155 þ MHC class Ideficient melanoma cells. Membrane bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK-cell-mediated tumor reactivity. IL15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK-cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the antimetastatic activity of IL15, while CD226 blockade decreases the effects of IL15 and TIGIT blockade. Conclusions: Our findings support the development of novel combinatorial immunotherapy with IL15 and TIGIT blockade to promote NK-cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8 þ T-cell-mediated immunity.

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RA-inducible gene I (RIG-I/DDX58) has been shown to activate IFN-β promoter stimulator 1 (IPS-1) on recognizing cytoplasmic viral RNAs. It is unclear how RIG-I functions within the IFN and/or RA signaling process in acute myeloid leukemia (AML) cells, however, where obvious RIG-I induction is observed. Here, we show that the RIG-I induction functionally contributes to IFN-α plus RA-triggered growth inhibition of AML cells. Interestingly, although RIG-I induction itself is under the regulation of STAT1, a major IFN intracellular signal mediator, under circumstances in which it does not stimulate IPS-1, it conversely augments STAT1 activation to induce IFN-stimulatory gene expression and inhibit leukemia cell proliferation. Thus, our results unveil a previously undescribed RIG-I activity in regulating the cellular proliferation of leukemia cells via STAT1, which is independent of its classic role of sensing viral invasion to trigger type I IFN transcription.

Tumor metastases are responsible for death in the majority of cancer patients. Here we have explored the role of the ectonucleotidase CD39 in select models of tumor metastases and further tested the therapeutic anticancer activity of the NTPDase inhibitor sodium polyoxotungstate (POM-1). CD39 was expressed on tumor-infiltrating regulatory T cells (Treg), myeloid cells and some NK cells, and it was upregulated on these cells within tumors early after inoculation in vivo. NK cell numbers and effector functions were increased in globally CD39-deficient mice and also in WT mice treated with POM-1. Dosing with POM-1 suppressed experimental and spontaneous metastases in four different tumor models and was well tolerated. This anti-metastatic activity was completely abrogated in mice, that were depleted of NK cells, had IFNγ neutralized or were deficient in CD39 expression in bone marrowderived cells. POM-1 was highly effective in suppressing metastases when used in combination with BRAFi/MEKi or anti-PD-1/anti-CTLA-4 or IL-2. These data highlight the importance of the CD39 pathway in suppressing NK cell-mediated anti-tumor immunity and validate further the development of CD39based therapies in the clinic. ARTICLE HISTORY

RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection-namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of Rig-I ؊/؊ myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes. knockout mice ͉ myelopoiesis ͉ RA-inducible gene I R A-induced granulocytic differentiation of acute promyelocytic leukemia (APL) represents a successful application of tumor differentiation-inducing therapy in the treatment of human malignancies. It is also an excellent model for studying the cellular and molecular mechanisms controlling RA-induced, and probably physiological cues-regulated myelopoiesis. Previously, we reported the isolation of a number of genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Among the up-regulated genes, a particularly interesting one was RIG-I, encoding a putative DExD/H box-containing RNA helicase (1). Recently, RIG-I has also been identified in the screening experiment for the molecules that mediate viral RNA-induced type I IFN generation (2). Upon the occupancy of its DExD/H box-containing carboxyl terminal region by foreign invading RNAs, or even RNase L-cleaved small self-RNAs (3), the amino-terminal double-CARD motif of RIG-I binds onto IPS-1/MAVS/VISA/Cardif, resulting in the recruitment and phosphorylation of certain downstream cytoplasmic factors, including IKK␥ and IKKrelated kinases TBK1/IKK. These immediate events, in turn, relay signals via phosphorylation cascade and nuclear translocation of transcription factors such as NF-B and IRF3 to coordinate the formation of IFN-␤ promoter enhancesome within the nucleus (4, 5). As expected, melanoma differentiation antigen 5 (MDA-5), a close structural and functional analogue of RIG-I, was also found to mediate a viral infection-induced type I IFN transcription through the IPS-1 pathway (6, 7). Notably, MDA-5 activity has been implicated in a biological setting without any signs of active viral infection. The overexpression of full-length of MDA-5, but not that of the CARD domain-or ATPase domain-deleted mutant, induced melanoma cells to apoptosis (8). Now that RIG-I expression can be highly induced by regulatory cues other than type I IFN, such as RA and IFN-␥, we postulated that additional biological funct...