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Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Killer-cell Immunoglobulin-like Receptor (KIR) genes encode receptors, which are mainly expressed on, and control functional activities of, Natural Killer (NK) cells. There exist six distinct activating KIR genes in humans, who differ from one another with respect to the repertoire of these genes. Because activated NK cells can potentially cause tissue destruction, we hypothesized that variation in the inherited activating KIR genes in humans is associated with their innate susceptibility/resistance to developing Crohn disease (CD). We performed case control studies on three independent Canadian CD patient cohorts (all of the Western European descent): two comprising children (Montreal having 193 cases and 245 controls, and Ottawa having 93 cases and 120 controls) and the third one comprising predominantly adults (Winnipeg having 164 cases and 200 controls). We genotyped cases and controls for activating KIR genes by PCR with gene-specific primers and investigated associations between the genes and cases using unconditional logistic regression. We observed strong associations between all the six KIR genes and CD in Ottawa children, with the strongest risk observed for the KIR2DS1 (p = 1.7 x10-10). Associations between all but the KIR2DS2 were replicated in the Montreal cohort with the strongest association evident for the KIR2DS5 (8.0 x 10-10). Similarly associations between five genes were observed in the adult Winnipeg cohort. In this cohort, strongest associations were evident with the KIR2DS5 (8.75 x 10-8). An overall analysis for all cohorts showed strong associations with four of the genes, with the strongest association evident for the KIR2DS5 (p = 1.35 x 10-17). In the combined analysis for four KIR genes, individuals carrying one or more of the KIR genes were at significantly higher risks for acquiring CD (p = 3.5 x 10-34). Activating KIR genes are associated with risk for developing CD in both children and adults.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger. Cancer is caused by a series of mutations that result in uncontrolled cell growth and division. Yet, the body's immune system can often detect and destroy abnormal cells before they cause tumors and disease. Natural killer cells are part of the immune system and have receptors on their surface that allow them to tell the difference between healthy host cells and host cells that are stressed or abnormal. Some of these receptors activate the natural killer cells when they bind to their target molecules. Other receptors have the opposite effect and inhibit the natural killer cells. Activation occurs when the signaling from the activating receptors is stronger than the signals from the inhibitory receptors. One of the well-studied activating receptors recognizes a number of proteins and molecules that are produced by abnormal or tumor cells, including a protein called ULBP1. This protein is absent from the surface of healthy cells but is found in abundance on tumor cells. However, it is still not clear what drives tumor cells to produce ULBP1 (or other molecules) that are recognized by natural killer cell receptors. Now, Gowen et al. report on a genetic screen that has revealed numerous genes that regulate the levels of ULBP1 in human cells. Many of these genes had independent effects that when added together accounted for most of the ULBP1 present on the cell surface. Gowen et al. then explored some of the ‘regulators’ encoded by these genes in more detail. One called ATF4, which had previously been linked to stress responses, was shown to increase the expression of the gene for ULBP1 in cancer cells. Another regulator called RBM4 instead acted in a different way and at a later stage in ULBP1 production. All together, these findings offer insight into the stress pathways that alert the immune system to abnormal cells. The next challenge will be investigating how these pathways might be exploited for cancer immunotherapy.

BackgroundIn a metastatic setting, systemically-administered therapies that overcome the immunosuppressive tumor microenvironment to promote T-cell recruitment and T-cell cytolytic function will be required to elicit durable anti-tumor immunity. To accomplish this, the STACT (S. Typhimurium-Attenuated Cancer Therapy) platform was developed. STACT is a live bacterial product that has been highly modified using precision genome editing for the following properties: (1) enhanced tolerability after IV dosing, (2) tumor-specific enrichment, (3) phagocytosis by tumor-resident antigen-presenting cells (APCs) with a lack of epithelial cell infectivity, (4) multiplexed genetic cargo delivery, and (5) attenuation of bacterial pathways that impair CD8+ T-cell function. An extensive screening campaign was performed to identify ideal encoded immunomodulatory payload combinations delivered by STACT for efficacy against T-cell excluded tumors.MethodsChromosomal edits to the STACT platform strain were made using PCR. A panel of immunomodulatory proteins, including cytokines, type I interferon (IFN)-inducing factors, co-stimulatory receptors, checkpoint antibodies and TGFβR-Fc decoys were tested for combinatorial potency using STACT. An engineered STING (eSTING) was designed through an extensive protein engineering campaign to identify optimal variants. Combinations were evaluated in primary human APCs using in vitro functional assays, where STACT IL-15Rα-IL-15 (IL-15) + eSTING (ACTM-838) emerged as a lead candidate. ACTM-838 was then evaluated in multiple murine tumor models for therapeutic efficacy and mechanism, as well as tolerability in rodents and primates after systemic administration.ResultsCombinatorial target profiling led to the discovery of ACTM-838, a STACT encoding IL-15 + eSTING. In vitro, ACTM-838 payloads synergistically produced high levels of type I IFN and T-cell recruitment and activation factors from primary human APCs. In vivo, ACTM-838 demonstrated a high degree of complete tumor responses that were entirely CD8+ T-cell dependent. In an autochthonous breast cancer model that lacks any significant lymphocyte infiltrate, ACTM-838 was able to uniformly enrich in each spontaneous lesion to high levels after IV dosing and resulted in significant CD8+ T-cell infiltration. In primates, ACTM-838 was well-tolerated, rapidly cleared, and elicited minimal cytokine response after IV dosing.ConclusionsACTM-838 is a highly attenuated, precision genome-engineered bacterial immunotherapy that delivers IL-15 + eSTING to phagocytic APCs of the solid tumor microenvironment after systemic administration. In preclinical studies, ACTM-838 promotes CD8+ T-cell mediated tumor clearance in T-cell excluded tumors and elicits durable anti-tumor immunity, and is well tolerated in primates. Based on these data, ACTM-838 was nominated for clinical development and has entered cGMP manufacturing and IND-enabling studies.Ethics ApprovalAll animals were used according to protocols approved by an Institutional Animal Care and Use Committee and maintained in specific pathogen-free conditions in a barrier facility.

Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D-activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, a shed form of MULT1, a high-affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are always inhibitory and suggest a new approach for cancer immunotherapy.

In recent years, interest in anti-cancer therapeutic monoclonal antibodies (mAb) has been renewed. Several of these reagents have been approved for therapy in a variety of cancer patients and many others are in different stages of development. It is believed that multiple mechanisms are involved in the anti-cancer effects of these reagents. However, several in vitro and in vivo studies have demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) is their predominant mode of action against cancer cells. The requirement for a direct interaction between mAb and receptors for the Fc region of the antibodies (FcR) has been demonstrated for anti-tumor effects of these antibodies. Consequently, FcR-bearing immune effector cells play an important role in mediating their effects. It is not surprising that cancer cells have developed different strategies to evade these antibodies. Several strategies are proposed to potentiate the mAb-mediated ADCC in cancer patients. They may enhance anti-cancer therapeutic effects of these regents.

Various cytokines have been evaluated as potential anticancer drugs; however, most cytokine trials have shown relatively low efficacy. Here, we found that treatments with IL-12 and IL-18 or with a mutant form of IL-2 (the \"superkine\" called H9) provided substantial therapeutic benefit for mice specifically bearing MHC class I-deficient tumors, but these treatments were ineffective for mice with matched MHC class I+ tumors. Cytokine efficacy was linked to the reversal of the anergic state of NK cells that specifically occurred in MHC class I-deficient tumors, but not MHC class I+ tumors. NK cell anergy was accompanied by impaired early signal transduction and was locally imparted by the presence of MHC class I-deficient tumor cells, even when such cells were a minor population in a tumor mixture. These results demonstrate that MHC class I-deficient tumor cells can escape from the immune response by functionally inactivating NK cells, and suggest cytokine-based immunotherapy as a potential strategy for MHC class I-deficient tumors. These results suggest that such cytokine therapies would be optimized by stratification of patients. Moreover, our results suggest that such treatments may be highly beneficial in the context of therapies to enhance NK cell functions in cancer patients.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Background. Concentrations of interleukin (IL)-18 increase in the circulation of human immunodeficiency virus (HIV)-infected persons. However, nothing is known concerning the regulation of IL-18-binding protein (IL-18BP), which neutralizes IL-18 in vivo. This issue is addressed in the present study. Methods. Serum samples obtained from healthy subjects and HIV-infected patients were analyzed by enzymelinked immunosorbent assay to determine their IL-18 and IL-18BP contents. Human monocyte-derived macrophages (MDMs) were infected in vitro with HIV type 1 (HIV-1), and the production of these 2 cytokines by these cells was measured. Finally, we determined the effect of IL-18 on HIV-1 replication in human cells. Results. In contrast to IL-18 levels, IL-18BP levels decreased in the serum of HIV-infected patients. This decrease resulted in enhanced levels of free IL-18 in the serum of such patients. The infection increased production of IL-18 but decreased that of IL-18BP in MDMs. IL-10 and transforming growth factor-β, concentrations of which are increased in HIV-infected persons, also decreased production of IL-18BP by human MDMs. Finally, recombinant human IL-18 enhanced HIV-1 replication in human CD4+ T cells. Conclusions. Production of IL-18 and its antagonist becomes imbalanced in HIV-1-infected persons. The infection and the cytokine milieu play a role in this decreased production. The increased biological activities of IL-18 may enhance viral replication in human CD4+ T cells.

IL-21 is a relatively newly discovered multifunctional and pleiotropic cytokine. It is produced primarily by CD4 + T cells, the principal targets of the virus, and therefore this cytokine has special relevance to HIV infection. Here we show for the first time that serum levels of this cytokine are significantly reduced in HIV-infected AIDS patients and correlate significantly with their CD4 + T-cell counts. These data suggest that the cytokine levels could act as a valuable biomarker for the progression of AIDS.