Tumor-induced Myeloid-derived Suppressor Cells Research Articles

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Myeloid-Derived Suppressor Cells: Facilitators of Cancer and Obesity-Induced Cancer

Immature myeloid cells at varied stages of differentiation, known as myeloid-derived suppressor cells (MDSC), are present in virtually all cancer patients. MDSC are profoundly immune-suppressive cells that impair adaptive and innate antitumor immunity and promote tumor progression through nonimmune mechanisms. Their widespread presence combined with their multitude of protumor activities makes MDSC a major obstacle to cancer immunotherapies. MDSC are derived from progenitor cells in the bone marrow and traffic through the blood to infiltrate solid tumors. Their accumulation and suppressive potency are driven by multiple tumor- and host-secreted proinflammatory factors and adrenergic signals that act via diverse but sometimes overlapping transcriptional pathways. MDSC also accumulate in response to the chronic inflammation and lipid deposition characteristic of obesity and contribute to the more rapid progression of cancers in obese individuals. This article summarizes the key aspects of tumor-induced MDSC with a focus on recent progress in the MDSC field.

Autocrine IL6-Mediated Activation of the STAT3-DNMT Axis Silences the TNFα-RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells.

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 TNFα-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 lineage-specific 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. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα 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 TNFα expression. This results in decreased TNFα-induced and RIP1-dependent 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.

Therapies for tuberculosis and AIDS: myeloid-derived suppressor cells in focus

The critical role of suppressive myeloid cells in immune regulation has come to the forefront in cancer research, with myeloid-derived suppressor cells (MDSCs) as a main oncology immunotherapeutic target. Recent improvement and standardization of criteria classifying tumor-induced MDSCs have led to unified descriptions and also promoted MDSC research in tuberculosis (TB) and AIDS. Despite convincing evidence on the induction of MDSCs by pathogen-derived molecules and inflammatory mediators in TB and AIDS, very little attention has been given to their therapeutic modulation or roles in vaccination in these diseases. Clinical manifestations in TB are consequences of complex host-pathogen interactions and are substantially affected by HIV infection. Here we summarize the current understanding and knowledge gaps regarding the role of MDSCs in HIV and Mycobacterium tuberculosis (co)infections. We discuss key scientific priorities to enable application of this knowledge to the development of novel strategies to improve vaccine efficacy and/or implementation of enhanced treatment approaches. Building on recent findings and potential for cross-fertilization between oncology and infection biology, we highlight current challenges and untapped opportunities for translating new advances in MDSC research into clinical applications for TB and AIDS.

Autocrine IL6 activates the STAT3-DNMT axis to silence the TNFa-RIP1 necroptosis pathway to sustain myeloid-derived suppressor cell survival and accumulation

Abstract Accumulation of myeloid-derived suppressor cells (MDSCs) is a hallmark of cancer. However, the underlying mechanism of MDSC accumulation in the tumor microenvironment (TME) remain incompletely understood. We report that MDSC accumulation is regulated by the TNFα-RIP1-mediated necroptosis. We determined that inhibition of DNMTs with Decitabine (DAC) abolished MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes (CTLs) in tumor-bearing mice. DAC-induced decrease of MDSC accumulation is correlated with increased IRF8 expression in MDSCs. However, DAC also abolished MDSC-like cell accumulation in IRF8 KO mice, indicating that DNA methylation does not regulate MDSC lineage differentiation but mediates MDSC accumulation at post differentiation stage. We determined that DAC decreased MDSC accumulation through increasing cell death and identified RIP1-dependent necroptosis as target of DNA methylation in MDSCs. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter is hypermethylated in tumor-induced MDSCs in vivo. Consequently, DAC dramatically increased TNFα level in MDSCs and neutralizing TNFα significantly decreased MDSC cell death. Furthermore, recombinant TNFα induced MDSC cell death in a does- and RIP1-dependent manner. IL6 which is expressed in MDSCs in tumor-bearing mice and human colorectal cancer patients. Our data shows that the autocrine IL6 activates the STAT3-DNMT axis to epigenetically silence the TNFα-RIP1 necroptosis pathway to sustain MDSC survival and accumulation in cancer. Targeting the TNFα-RIP1 necroptosis is potentially an effective approach to supress MDSCs to activate tumor-reactive CTLs in the TME.

Quercetin promotes the survival of granulocytic myeloid-derived suppressor cells via the ESR2/STAT3 signaling pathway

Quercetin is a natural product that has been shown to induce tumor apoptosis and necrosis through multiple mechanisms. Tumor-induced myeloid-derived suppressor cell (MDSC) expansion negatively regulates the immune response by inhibiting T cell function through signal transducer and activator of transcription 3 (STAT3) activation, thereby facilitating tumor escape from host immune surveillance. Thus MDSC is an attractive target for cancer immunotherapy to enhance cytotoxic T cell responses. However, the effects of quercetin on MDSC are poorly understood. Here, we demonstrate that quercetin treatment enhanced mouse- and human- derived granulocytic-myeloid-derived suppressor cells (G-MDSC) survival and promoted the secretion of T cell-suppressive factors in vitro. Bioinformatics analysis further showed that quercetin was highly correlated with the estrogen receptor signaling pathway, which was confirmed by quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis. These findings highlight the potential advantages and feasibility of quercetin in reinforcing the suppressive property of G-MDSC. Thus impact of G-MDSC should be taken into consideration when quercetin is applied to tumor therapy.

Activated hepatic stellate cells regulate MDSC migration through the SDF-1/CXCR4 axis in an orthotopic mouse model of hepatocellular carcinoma.

Hepatic stellate cells (HSCs) are important stromal cells and pivotal mediators involved in the pathogenesis and immunosuppression of hepatocellular carcinoma (HCC). The liver has been demonstrated to be a site for accumulation of tumor-induced myeloid-derived suppressor cells (MDSCs). We previously reported that HSCs induced an increase in the number of MDSCs in HCC. However, how MDSCs are recruited in HCC remains largely unclear. In the present study, we found that HSC-conditioned medium (HSC-CM) induced bone marrow-derived cell and splenocyte migration, especially MDSC migration. Using chemokine-neutralizing antibodies and chemokine receptor inhibitors, we found that HSCs promoted MDSC migration through the SDF-1/CXCR4 axis. Subsequently, we used an orthotopic mouse liver tumor model to determine how HSCs mediated MDSC migration to HCC in vivo. The in vivo results indicated that pretreatment of MDSCs with a CXCR4 inhibitor or injection with SDF-1-knocked down HSCs inhibited MDSC migration to the spleen and liver of the tumor-bearing mice. Together, our findings indicate a central role for HSCs in MDSC migration mediated by the SDF-1/CXCR4 axis, thus revealing a potentially effective approach for modulating the tumor microenvironment by targeting HSCs in HCC.

Immunobiology of cholangiocarcinoma.

SummaryCholangiocarcinoma (CCA) represents a heterogeneous group of epithelial tumours that are classified according to anatomical location as intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). Although surgical resection and liver transplantation following neoadjuvant therapy are potentially curative options for a subset of patients with early-stage disease, the currently available medical therapies for CCA have limited efficacy. Immunotherapeutic strategies such as immune checkpoint blockade (ICB) harness the host immune system to unleash an effective and durable antitumour response in a subset of patients with a variety of malignancies. However, response to ICB monotherapy has been relatively disappointing in CCA. CCAs are desmoplastic tumours with an abundant tumour immune microenvironment (TIME) that contains immunosuppressive innate immune cells such as tumour-associated macrophages and myeloid-derived suppressor cells. A subset of CCAs may be classified as immune ‘hot’ tumours with a high density of CD8+ T cells and enhanced expression of immune checkpoint molecules. Immune ‘hot’ tumour types are associated with higher response rates to ICB. However, the suboptimal response rates to ICB monotherapy in human clinical trials of CCA imply that the preponderance of CCAs are immune ‘cold’ tumours with a non-T cell infiltrated TIME. An enhanced comprehension of the immunobiology of CCA, particularly the innate immune response to CCA, is essential in the effort to develop effective combination immunotherapeutic strategies that can target a larger subset of CCAs.

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2) Expressed in the Host Promotes Lymphoma Cells Growth By Controlling Myeloid Derived Suppressor Cells Expansion

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive cells that generate from myeloid progenitors under pathological conditions including cancers.In response to tumor-derived factors, myeloid progenitors differentiate intoimmature myeloid cells and accumulate as monocyte and granulocyte-MDSC (M- and G-MDSC), and correlate with poor survival in patients. Promoting the differentiation of MDSCs towards mature dendritic cells and macrophage is a potential way to eliminate MDSCs and restore anti-tumor immunity. In this study, we demonstrate that the enzyme Calcium/Calmodulin dependent Kinase Kinase 2 (CaMKK2) as a potential therapeutic target to control the growth of lymphoma by regulating the expansion of MDSCs.CaMKK2 is a crucial kinase in the calcium-signaling cascade. Activated by the Ca2+/CaM complex, CaMKK2 can phosphorylate the down stream targets including CaMKI, CaMKIV, and 5' AMP-activated kinase (AMPK). Beyond central nerve system, CaMKK2 is expressed in hematopoietic stem and progenitors cells, and some hematopoietic mature cells including macrophages and monocytes. In adipogenesis, granulopoiesis, and muscle development, CaMKK2 inhibits the differentiation of progenitors towards mature cells. We reasoned that CaMKK2 might have a similar function in myeloid cells, controlling terminal differentiation of tumor-induced MDSCs.We first confirmedthe expression of CaMKK2 in tumor-induced MDSCs. EG.7-OVA lymphoma cells were injected s.c. in the flank of Camkk2-egfp reporter mice, and MDSCs subsets from the tumors and spleens of the mice were assessed by flow cytometry. We found that Camkk2 promoter was highly active in Ly6Chigh/Ly6Gdim subset (M-MDSC), but low in Ly6Cdim/Ly6Ghigh subset (G-MDSC) and Ly6Cneg/Ly6Gneg/F4/80dim/neg/MHC-IIdim/neg cells. (Figure 1) Camkk2 promoter was not active in lymphocytes as previous reported.To determine the function of CaMKK2 expressed in the host, E.G7 cells with luciferase expression were injected into the flank of wild type (WT) and Camkk2-/- mice, and monitored by tumor size and bioluminescent imaging. (Figure 2 A&B) In WT mice injected with E.G7-OVA cells, tumors were detectable by bioluminescence by day 7, and gradually increased in photon signal during the following two weeks. Although tumors successfully engrafted in Camkk2-/- mice, bioluminescent signal gradually decreased after 21 days and eventually disappeared after 32 days. (Figure 2A) In LyM-Cre+Camkk2loxP mice which Camkk2 is selectively deleted in myeloid cell compartment, when challenged with E.G7-OVA cells, similar E.G7 tumor grow suppression was observed as in the Camkk2-/- global knockout model. (Figure 2C)We next demonstrated that Camkk2 deletion decreased MDSCs accumulation in EG7 bearing mice. In normal conditions, Camkk2-/- mice had comparable CD11b+and Ly6ChighLy6Gdim percentage in the spleenswith WT mice, but slightly higher Ly6Cdim Ly6Ghigh percentage. A significant increase of MDSCs percentage was detected in EG.7-bearing mice compared to tumor-free mice; while in Camkk2-/- mice, the MDSC accumulation in E.G7-OVA spleens and tumors showed a significant decrease in percentage compared to WT ones. (Figure 3 A & B) To evaluate whether the failure of MDSC expansion in tumor-bearing Camkk2-/- mice regulated tumor suppression, MDSCs were isolated from the spleens of WT and Camkk2-/- E.G7-OVA-bearing mice, adoptively transferred into Camkk2-/- mice, and followed by tumor inoculation. As expected, lymphoma cells failed to growth in Camkk2-/- control mice without MDSC injection. However, tumors developed in all Camkk2-/- mice that received either WT or Camkk2-/- MDSCs, with no significant difference. (Figure 3C) These results indicated that restoring the number of MDSC was sufficient to revert the protective effect of Camkk2 deletion on tumor growth. Interestingly, this data also suggested that WT and Camkk2-/- MDSCs have a comparable immunosuppressive activity at single-cell level.In conclusion, our results identifies for the first time CaMKK2 as an important component of molecular machinery that mediates the detrimental effect of lymphoma cells on myeloid development. Of note, our data also pinpoint CaMKK2 as a new pharmacological target to limit the expansion of tumor-induced MDSC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IFNAR1 Controls Autocrine Type I IFN Regulation of PD-L1 Expression in Myeloid-Derived Suppressor Cells.

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.

Arginase-1 is neither constitutively expressed in nor require for myeloid-derived suppressor cell (MDSC)-mediated inhibition of T cell proliferation

Abstract Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit T cell proliferation by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition of T cell proliferation. Employing Percoll density gradient centrifugation, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and exhibit potent inhibition on T cell proliferation induced via TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. These MDSC, though suppressive toward T cell proliferation, do not hinder TCR-induced IFNγ secretion or granzyme B expression. Despite effectively inhibiting T cell proliferation, both G- and M-MDSC exhibit no arginase-1 expression; however, arginase-1 can be induced by exposure to TCR-activated T cells or their culture medium, but not by T cells activated by other means or growing tumor cells. Western blot analysis revealed that TCR-activated T cells confer arginase-1 expression in MDSC by secreting cytokines that manifest as two distinct signaling-relay axes, IL-6-to-IL-4 or GM-CSF/IL-4-to-IL-10. Specifically, IL-6 signaling increases IL-4R on MDSC, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R on MDSC, allowing IL-10-mediated induction. Surprisingly, our data demonstrate that the induction of arginase-1 is not conducive, per se, for MDSC-mediated inhibition of T cell proliferation, as neither supplementation of L-arginine nor arginase-1 inhibitor rescue T cell proliferation. Instead, direct cell contact is required for the inhibition and is not dampened by PD-L1 blockade or SIRPα deficiency.

Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell-mediated inhibition of T-cell proliferation.

Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit Tcells by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and demonstrated potent inhibition in T-cell proliferation activated by TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase-1 expression and/or exert their inhibitory effects independent of arginase-1 activity. However, arginase-1 expression in MDSC can be induced by exposure to TCR-activated Tcells or their culture medium, but not Tcells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR-activated Tcells as orchestrating two signaling-relay axes, IL-6-to-IL-4 and GM-CSF/IL-4-to-IL-10, leading to arginase-1 expression in MDSC. Specifically, IL-6 signaling increases IL-4R, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R, allowing IL-10-mediated induction. Surprisingly, our study indicates that induction of arginase-1 expression is not conducive to the critical MDSC-mediated inhibition toward Tcells, which is rather dependent on direct cell contacts undiminished by PD-L1 blockade or SIRPα deficiency.

SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells.

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 antitumor 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 MDSCs, iNOS expression was significantly elevated in tumor-induced MDSCs, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathologic conditions. Furthermore, tumor-induced MDSCs exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs showed increased SETD1B expression as compared with 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 MDSCs, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. 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 MDSCs when they are generated under pathologic conditions. Cancer Res; 77(11); 2834-43. ©2017 AACR.

SETD1B compensates loss of IRF8 expression to activate iNOS expression in myeloid-derived suppressor cells through an epigenetic mechanism

Abstract 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 (MDSCs). MDSCs use NO to nitrate the T cell receptor and STAT1 to inhibit T cell activation and the anti-tumor immune response. The molecular mechanism for iNOS expression regulation in tumor-induced MDSCs is unknown. We show that IRF8 deficiency results in diminished iNOS expression in mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo, indicating that IRF8 is an essential transcriptional activator for iNOS expression in myeloid cells under physiological conditions. Though IRF8 is silenced in tumor-induced MDSCs, iNOS expression is dramatically elevated in tumor-induced MDSCs suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Tumor-induced MDSCs exhibit diminished STAT1 and NF-κB Rel protein level, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs show increased SETD1B expression compared to their equivalent in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, a product of SETD1B enzymatic activity, is enriched at the nos2 promoter in tumor-induced MDSCs. Inhibition/silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our data determine that IRF8 is an essential transcriptional activator of iNOS in myeloid cells under physiological conditions. Tumor cells use the SETD1B-H3K4me3 epigenetic axis to compensate for loss of IRF8 expression to activate iNOS expression in MDSCs under pathological conditions.

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells

Abstract Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating immune cells in human colon carcinoma, and 25–67% CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) from peripheral blood of human colon cancer patients are PD-L1+. Using a spontaneous colon cancer mouse model, we observed that PD-L1+MDSCs increased significantly in tumor-bearing mice as compared to MDSCs from tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas, bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNg is highly expressed in cells of the tumor tissues and IFNg neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNg-activated pSTAT1 does not bind to the cd274 promoter in CD11b+Gr1+PD-L1+ myeloid cells. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to an unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a less degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.

IRF8 deficiency in colonic epithelial cells promotes inflammation-mediated colon tumorigenesis and myeloid-derived suppressor cell differentiation.

Abstract IRF8 is a member of the nine IRF family proteins that was originally identified as a key transcription factor for myeloid cell lineage differentiation. IRF8 deficiency leads to deregulation of myelopoiesis and accumulation of CD11b+Gr1+ immature myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs). It has since been documented that IRF8 also plays important roles in other immune cells, including B, Natural Killer, Dendritic and T cells. IRF8 expression and function has recently been extended to non-hematopoietic cells, including epithelial cancer cells and heart cells. IRF8 is often silenced by its promoter DNA methylation in cancer cells. However, low level of IRF8 is expressed in cancer cells and IRF8 is IFNg inducible in cancer cells. IRF8 acts as an apoptosis inducer. To determine the function of IRF8 in spontaneous tumor development, we generate IRF8 deficient mice with IRF8 KO only in colonic epithelial cells (termed IRF8 CKO mice). Unlike conventional IRF8-deficient mice, IRF8 CKO mice phenotypically resemble WT mice. However, IRF8 CKO exhibited greater susceptibility to AOM-DSS-induced colon cancer formation as compared to WT mice. Although no differences were observed in MDSC profiles between WT and IRF8 CKO mice, tumor-bearing IRF8 CKO mice have significantly higher level of CD11b+Gr1+MDSCs than tumor-bearing WT mice. Our data determine that IRF8 functions in colonic epithelial cells as a spontaneous colon cancer suppressor and also functions in colon carcinoma cells to mediate MDSC differentiation in tumor-bearing host in vivo.

Mechanisms overseeing myeloid-derived suppressor cell production in neoplastic disease.

Perturbations in myeloid cell differentiation are common in neoplasia, culminating in immature populations known as myeloid-derived suppressor cells (MDSCs). MDSCs favor tumor progression due to their ability to suppress host immunity or promote invasion and metastasis. They are thought to originate from the bone marrow as a result of exposure to stromal- or circulating tumor-derived factors (TDFs). Although great interest has been placed on understanding how MDSCs function, less is known regarding how MDSCs develop at a transcriptional level. Our work explores the premise that MDSCs arise because cancer cells, through the production of certain TDFs, inhibit the expression of interferon regulatory factor-8 (IRF8) that is ordinarily essential for controlling fundamental properties of myeloid cell differentiation. Our interest in IRF8 has been based on the following rationale. First, it is well-recognized that IRF8 is a 'master regulator' of normal myelopoiesis, critical not only for producing monocytes, dendritic cells (DCs), and neutrophils, but also for controlling the balance of all three major myeloid cell types. This became quite evident in IRF8-/- mice, whereby the loss of IRF8 leads to a disproportionate accumulation of neutrophils at the expense of monocytes and DCs. Second, we showed that such myeloid populations from IRF8-/- mice exhibit similar characteristics to MDSCs from tumor-bearing mice. Third, in a reciprocal fashion, we showed that enforced expression of IRF8 in the myeloid system significantly mitigates tumor-induced MDSC accumulation and improves immunotherapy efficacy. Altogether, these observations support the hypothesis that IRF8 is an integral negative regulator of MDSC biology.

Arsenic trioxide inhibits tumor-induced myeloid-derived suppressor cells and enhances T-cell activity.

Myeloid-derived suppressor cells (MDSCs), one of the major orchestrators of the immunosuppressive network, are associated with immune suppression and considered a prime target for cancer immunotherapy. At present, various strategies have been explored to deplete and/or inactivate MDSCs in vivo. In this study, we investigated the effect of arsenic trioxide (ATO) on MDSCs derived from tumor-bearing mice. This study examined the in vitro and in vivo effects of ATO administration on MDSCs from C57/j mice bearing either the B16 or H22 tumor. The MDSCs were then characterized for phenotype, gene expression and function. Administration with ATO in vitro significantly induced MDSC differentiation, inhibited their proliferation and triggered apoptosis. Treatment with ATO in these murine tumor models significantly inhibited tumor growth and splenomegaly, decreased the percentages of MDSCs in the spleen, promoted their differentiation, reduced tumor necrosis factor-α and interleukin-10 levels and weakened the immune inhibition activity of MDSCs on T cells. In addition, we observed the underlying mechanism involved in the regulation of MDSCs by ATO, which included a panel of cytokines and signaling pathways. The findings showed the immunoregulatory effects of ATO by inducing apoptosis, promoting differentiation and inhibiting the function of MDSCs, suggesting that ATO has potential clinical benefit as it selectively attenuates MDSC-induced immunosuppression.

Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes.

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity.