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Background Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown. Methods Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed. Results IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. Conclusion IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance. Electronic supplementary material The online version of this article (10.1186/s40425-019-0635-8) contains supplementary material, which is available to authorized users.

Tumor cells respond to IFN-γ of activated T cells to upregulate programmed death-ligand 1 (PD-L1) in the tumor microenvironment as an adaptive immune resistance mechanism. Tumor cells also express oncogene-driven PD-L1. PD-L1 is also expressed on myeloid-derived suppressor cells (MDSCs). It is known that both type I and II IFNs upregulate PD-L1 expression in MDSCs. However, the molecular mechanism underlying PD-L1 expression in MDSCs is still largely unknown. We report in this article that MDSCs exhibit constitutive STAT1 phosphorylation in vitro without exogenous IFNs, indicating a constitutive active JAK-STAT signaling pathway in mouse MDSCs in vitro. Furthermore, IFN-α and IFN-β but not IFN-γ are endogenously expressed in the MDSC cell line in vitro and in tumor-induced MDSCs in vivo. Neutralizing type I IFN or inhibiting the JAK-STAT signaling pathway significantly decreased constitutive PD-L1 expression in MDSCs in vitro. However, neither IFN-α expression level nor IFN-β expression level is correlated with PD-L1 expression level in MDSCs; instead, the level of IFN receptor type I (IFNAR1) is correlated with PD-L1 expression levels in MDSCs. Consequently, knocking out IFNAR1 in mice diminished PD-L1 expression in tumor-induced MDSCs. Therefore, we determined that 1) PD-L1 expression in MDSCs is activated by type I IFN through an autocrine manner and 2) the expression level of PD-L1 is controlled at least in part by the IFNAR1 level on MDSCs. Our data indicate that MDSCs may maintain their PD-L1 expression via autocrine type I IFN to exert their suppressive activity in the absence of IFN-γ from the suppressed T cells in the tumor microenvironment.

SUMMARY IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation.

Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSC). MDSCs use NO to nitrate both the T cell receptor and STAT1, thus inhibiting T cell activation and the anti-tumor immune response. The molecular mechanisms underlying iNOS expression and regulation in tumor-induced MDSCs are unknown. We report here that deficiency in IRF8 results in diminished iNOS expression in both mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo. Strikingly, although IRF8 was silenced in tumor-induced MDSC, iNOS expression was significantly elevated in tumor-induced MDSC, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Furthermore, tumor-induced MDSC exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSC showed increased SETD1B expression as compared to their cellular equivalents in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, the target of SETD1B, was enriched at the nos2 promoter in tumor-induced MDSC, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSC. Our results show how tumor cells use the SETD1B-H3K4me3 epigenetic axis to bypass a normal role for IRF8 expression in activating iNOS expression in MDSC, when they are generated under pathological conditions.

Despite the remarkable efficacy of immune checkpoint inhibitor (ICI) immunotherapy in various types of human cancers, colon cancer, except for the approximately 4% microsatellite-instable (MSI) colon cancer, does not respond to ICI immunotherapy. ICI acts through activating CTLs that use the Fas-FasL pathway as one of the two effector mechanisms to suppress tumor. Cancer stem cells are often associated with resistance to therapy including immunotherapy, but the functions of Fas in colon cancer apoptosis and colon cancer stem cells are currently conflicting and highly debated. We report here that decreased Fas expression is coupled with a subset of CD133 þ CD24 lo colon cancer cells in vitro and in vivo. Consistent of the lower Fas expression level, this subset of CD133 þ CD24 lo Fas lo colon cancer cells exhibits decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively enriches CD133 þ CD24 lo Fas lo colon cancer cells.CD133 þ CD24 lo Fas lo colon cancer cells exhibit increased lung colonization potential in experimental metastatic mouse models and decreased sensitivity to tumor-specific CTL adoptive transfer and ICI immunotherapies. Interestingly, FasL challenge selectively enriched this subset of colon cancer cells in microsatellite-stable (MSS) but not in the MSI human colon cancer cell lines. Consistent with the downregulation of Fas expression in CD133 þ CD24 lo cells, lower Fas expression level is significantly correlated with decreased survival in patients with human colon cancer.Implications: Our data determine that CD133 þ CD24 lo Fas lo colon cancer cells are capable to evade Fas-FasL cytotoxicity of tumor-reactive CTLs and targeting this subset of colon cancer cells is potentially an effective approach to suppress colon cancer immune evasion.(Continued.) D, The sorted CD133 þ CD24 lo Fas lo and CD133 þ CD24 hi Fas hi cells were injected intravenously into C57BL/6 mice (3 Â 10 5 cells/mouse, n ¼ 5). Fourteen days later, mice were sacrificed and India ink was perfused into the lung. The ink-inflated lungs were fixed. Shown are tumor-bearing lungs. The tumor nodule number was counted and presented at the right. E, Sorted CD133 þ CD24 lo Fas lo (n ¼ 6) and CD133 þ CD24 hi Fas hi (n ¼ 7) MC38.met cells were injected into FasL-deficient fasl gld mice (3 Â 10 5 cells/mouse) intravenously. Fourteen days later, mice were sacrificed and India ink was perfused into the lung. The ink-inflated lungs were fixed. Shown are tumor-bearing lungs. The tumor nodule number was counted and presented at the bottom. F, CD133 þ CD24 lo Fas lo and CD133 þ CD24 hi Fas hi CT26 cells were injected intravenously into BALB/c mice (2 Â 10 5 cells/mouse, n ¼ 5). Four days later, mice with treated with saline control or perforin-deficient pk03 CTLs (3 Â 10 5 cells/mouse). Mice were sacrificed on day 14 and analyzed as in B. G, CD133 þ CD24 lo Fas lo and CD133 þ CD24 hi Fas hi CT26 cells were injected intravenously into BALB/c mice (2 Â 10 5 cells/mouse, n ¼ 5). Four days later, mice were treated with IgG (200 mg/m...

◥Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFa-RIP1-mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineagespecific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genomewide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFa levels in MDSC in vitro, and neutralizing TNFa significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFa induced MDSC cell death in a dose-and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3-DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFa expression. This results in decreased TNFa-induced and RIP1dependent necroptosis to sustain survival and accumulation.Significance: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.

PD-1 is a co-repressive receptor that curbs T cell activation and thereby serves as a protection mechanism against autoimmunity under physiological conditions. Under pathological conditions, tumor cells express PD-L1 as an adaptive resistant mechanism to suppress PD-1 T cells to evade host immunosurveillance. PD-1 therefore is a key target in cancer immunotherapy. Despite the extensive studies of PD-1 expression regulation, the transcription machinery and regulatory mechanisms are still not fully understood. We report here that the NF-κB p50 homodimer is a transcription regulator of PD-1 in activated T cells. A putative κB sequence exists at the promoter. All five NF-κB Rel subunits are activated in activated T cells. However, only the p50 homodimer directly binds to the κB sequence at the promoter in CD4 and CD8 T cells. Deficiency in p50 results in reduced PD-1 expression in both CD4 and CD8 T cells . Using an mixed bone marrow chimera mouse model, we show that p50 regulates PD-1 expression in a cell-intrinsic way and p50 deficiency leads to decreased PD-1 expression in both antigen-specific CD4 and CD8 T cells . The expression levels of H3K4me3-specific histone methyltransferase increased significantly, resulting in a significant increase in H3K4me3 deposition at the promoter in activated CD4 and CD8 T cells. Inhibition of H3K4me3 significantly decreased p50 binding to the promoter and PD-1 expression in a T cell line. Our findings determine that the p50-H3K4me3 axis regulates transcription activation in activated T cells.