Subsets of myeloid-derived suppressor cells in tumor-bearing mice

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of cells that play a critical role in tumor associated immune suppression. In an attempt to identify a specific subset of MDSC primarily responsible for immunosuppressive features of these cells, 10 different tumor models were investigated. All models showed variable but significant increase in the population of MDSC. Variability of MDSC expansion in vivo matched closely the effect of tumor cell condition medium in vitro. MDSC consists of two major subsets of Ly6G(+)Ly6C(low) granulocytic and Ly6G(-)Ly6C(high) monocytic cells. Granulocytic MDSC have increased level of reactive oxygen species and undetectable level of NO whereas monocytic MDSC had increased level of NO but undetectable levels of reactive oxygen species. However, their suppressive activity per cell basis was comparable. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. We performed a phenotypical and functional analysis of several surface molecules previously suggested to be involved in MDSC-mediated suppression of T cells: CD115, CD124, CD80, PD-L1, and PD-L2. Although substantial proportion of MDSC expressed those molecules no differences in the level of their expression or the proportion, positive cells were found between MDSC and cells from tumor-free mice that lack immune suppressive activity. The level of MDSC-mediated T cell suppression did not depend on the expression of these molecules. These data indicate that suppressive features of MDSC is caused not by expansion of a specific subset but more likely represent a functional state of these cells.     

Swertianolin ameliorates immune dysfunction in sepsis via blocking the immunosuppressive function of myeloid-derived suppressor cells

In this study, we studied the long-term proliferation trajectory of myeloid-derived suppressor cells (MDSCs) in murine sepsis model and investigated whether swertianolin could modulate the immunosuppressive function of MDSCs. A murine sepsis model was established by cecal ligation and perforation (CLP), according to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. The bone marrow and spleen of the mice were collected at 24 h, 72 h, 7 and 15 d after sepsis induction. The proportions of monocytic- MDSCs (M-MDSCs; CD11b⁺LY6G^–LY6C^hi) and granulocytic-MDSCs (G-MDSC, CD11b⁺ Ly6G⁺ Ly6C^low) were analyzed by flow cytometry. Then, we have investigated whether swertianolin could modulate the immunosuppressive function of MDSCs in in vitro experiments. G-MDSCs and M-MDSCs increased acutely after sepsis with high levels sustained over a long period of time. G-MDSCs were the main subtype identified in the murine model of sepsis with polymicrobial peritonitis. Furthermore, it was found that swertianolin reduced significantly interleukin-10 (IL-10), nitric oxide (NO), reactive oxygen species (ROS), and arginase production in MDSCs, while reducing MDSC proliferation and promoting MDSC differentiation into dendritic cells. Swertianolin also improved T-cell activity by blocking the immunosuppressive effect of MDSCs. Both subsets of MDSCs significantly increased in the bone marrow and spleen of the mice with sepsis, with GMDSCs being the main subtype identified. Swertianolin effectively regulated the functions of MDSCs and reduced immune suppression.Key words: Sepsis, myeloid-derived suppressor cells (MDSCs), immunosuppression, swertianolin     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice

Summary The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E₁. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.     

IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-gamma and Th1 cells. Because Arg1 and iNOS share L-arginine as a common substrate, our results indicate that L-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells

Immunostimulatory CpG oligonucleotides (ODN) activate cells that express TLR9 and have been shown to improve the host's response to tumor Ags. Unfortunately, the immunosuppressive microenvironment that surrounds many cancers inhibits Ag-specific cellular responses and thus interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near tumor sites where they inhibit the activity of Ag-specific T and NK cells. Current studies indicate that the delivery of CpG ODN directly into the tumor bed reduces the immunosuppressive activity of monocytic (CD11b(+), Ly6G(-), Ly6C(high)) MDSC. Monocytic MDSC express TLR9 and respond to CpG stimulation by 1) losing their ability to suppress T cell function, 2) producing Th1 cytokines, and 3) differentiating into macrophages with tumoricidal capability. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and they support intratumoral injection as the optimal route for their delivery.     

髓系抑制细胞在1型糖尿病患者中作用的研究

髓系抑制细胞(myeloid-derived suppressor cells, MDSC)是来源于髓系的一类未成熟细胞群,初期对肿瘤的研究中发现,这类细胞具有抑制免疫应答,并促进炎性细胞因子及趋化因子分泌的作用,近年来对这类细胞在治疗自身免疫性疾病等方面的作用进行了研究。发现MDSC具有治疗自身免疫性疾病的功效;但也有研究显示,MDSC不仅无治疗效果,甚至还会起到相反的作用,这可能与其促炎因子过度分泌或诱导辅助性T细胞17(T helper cell, Th17)分化有关。1型糖尿病(type 1 diabetes, T1D)是一种常见的自身免疫性疾病,如何有效治疗该疾病一直是内分泌研究领域的热点。现就MDSC的来源、功能及其在T1D中的作用进行阐述,旨在为T1D的治疗提供新思路。     

Immunosuppression of T lymphocyte function by fractionated serum from tumor-bearing mice.

Sera from mice with transplanted 3-methylcholantrene-induced tumors have been shown previously to inhibit the function of normal lymphoid cells. When chromatographed on Sephadex G-150, the fraction eluting with immunoglobulin has been shown to inhibit the proliferative response of normal spleen cells to concanavalin A and to inhibit the in vitro antibody response to a T-dependent antigen, but has a lesser effect on the antibody response to a T-independent antigen. This paper deals with studies on the mode of action of the serum factor. The immunoglobulin containing fraction of serum from tumor-bearing mice inhibited the in vitro generation of both allogeneic and syngeneic cytotoxic lymphocytes. Time course studies demonstrate that the serum fraction inhibits the generation of antibody-producing and cytotoxic lymphocytes if added during the first 2 days of a 5-day culture. Serum fractions added after day 2 had no effect on the in vitro response. The serum factor appears to inhibit the generation of specific T cell function during the proliferative stage of development but has no effect on the differentiation stage which leads to either antibody-producing cells or cytotoxic lymphocytes.     

ADRB3 induces mobilization and inhibits differentiation of both breast cancer cells and myeloid-derived suppressor cells

Metastatic tumors are mainly composed of neoplastic cells escaping from the primary tumor and inflammatory cells egressing from bone marrow. Cancer cell and inflammatory cell are remained in the state of immaturity during migration to distant organs. Here, we show that ADRB3 is crucial in cell mobilization and differentiation. Immunohistochemistry revealed ADRB3 expression is significantly more frequent in breast cancer tissues than in adjacent noncancerous tissues (92.1% vs. 31.5%). Expression of ADRB3 correlated with malignant degree, TNM stage and poor prognosis. Moreover, ADRB3 expression was markedly high in activated disseminated tumor cells, myeloid-derived suppressor cells (MDSCs), lymphocytes and neutrophil extracellular traps of patients. Importantly, ADRB3 promoted the expansion of MDSC through stimulation of bone marrow mobilization and inhibiting of the differentiation of immature myeloid cells. Furthermore, ADRB3 promoted MCF-7 cells proliferation and inhibited transdifferentiation into adipocyte-like cell by activating mTOR pathway. Ultimately, the MDSC-deficient phenotype of ADRB3 ^-/- PyMT mice was associated with impairment of mammary tumorigenesis and reduction in pulmonary metastasis. Collectively, ADRB3 promotes metastasis by inducing mobilization and inhibiting differentiation of both breast cancer cells and MDSCs.Subject terms: Breast cancer, Breast cancer     

基于髓源性抑制细胞的食药用菌多糖抗肿瘤免疫作用机制

髓源性抑制细胞(myeloid-derived suppressor cells,MDSCs)是一种异质性的免疫调节细胞。在癌症机体中,MDSCs是主要的免疫抑制细胞,通过多种途径诱导T淋巴细胞衰竭和凋亡,促进肿瘤细胞逃逸,从而导致肿瘤不受控制地生长,是癌症治疗的主要障碍。目前,MDSCs是癌症药物研究的热点和关键靶点。近年来,研究报道显示多糖可下调MDSCs在癌症患者及肿瘤实验动物体内数量和比例,并诱导免疫抑制功能丧失。食药用菌多糖是天然多糖的主要来源,可以通过多种途径激活肿瘤免疫应答,其抑制MDSCs功能的研究报道逐年增多,目前研究主要集中在香菇多糖、灵芝多糖等部分种类。因此,本文简要描述髓源性抑制细胞在癌症中的免疫抑制功能,然后详细地综述食药用菌多糖对髓源性抑制细胞作用的研究进展,以期为食药用菌多糖在肿瘤免疫药物开发及辅助增强(如免疫检查点抑制剂)等免疫治疗提供新思路。     

Phenotype,function and clinical implications of myeloid-derived suppressor cells in cancer patients

The involvement of a smouldering microenvironment is currently considered a cancer hallmark and a required step for tumour cells to disable specific immunity while promoting angiogenesis and stroma remodelling. Nevertheless, the molecular pathways driving such aberrant interactions in human cancer and their actual implication in disease progression are still poorly defined. Here, we will report about the remarkable efforts devoted by our group as well as many other scientists to dissect this process focusing on tumour-mediated activation of myeloid dysfunctional pathways occurring in cancer patients. Indeed, myeloid-derived suppressor cells (MDSC), playing a crucial role as cellular regulators of immune responses, have been extensively shown to restrain tumour immunity through a vast array of molecular mechanisms and to promote tumour progression in different murine models. Although in mice the phenotypic features of these cells were defined initially rather generally by Gr1⁺ and CD11b⁺ co-expression, more recent studies have unravelled the actual complexity of this population and the existence of different cell subsets. This complexity is even more remarked in the human setting, where heterogeneous populations of myeloid cells with variable phenotype and immunosuppressive features have been described in patients affected by different types of tumours. The lack of homogeneous properties of human MDSC has made these cells a controversial and still unacknowledged player in cancer-related immune suppression and disease progression. Nevertheless, with the efforts of the scientific community, MDSC will soon reveal their key role thereby becoming novel targets for innovative therapeutic strategies.     

Functional changes in myeloid-derived suppressor cells (MDSCs) during tumor growth: FKBP51 contributes to the regulation of the immunosuppressive function of MDSCs

Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were downregulated but protumor function-related genes were upregulated in both monocytic MDSCs (Mo-MDSCs) and polymorphonuclear granulocytic MDSCs (PMN-MDSCs) at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo-MDSCs and PMN-MDSCs isolated from the late time points. Experiments using small interfering RNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing inducible NO synthase, arginase-1, and reactive oxygen species levels and enhancing NF-κB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs.     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Hes-1-targeting siRNA inhibits the maturation of murine myeloid-derived dendritic cells

Activation of Notch by Jagged-1 may plays a pivotal role in maturation of dendritic cells (DCs), but the mechanism has not been completely defined. In the present study, Hes-1 (Hairy/enhancer-of-split)-targeting siRNA was used to confirm a role of Jagged-1-Notch signaling pathway activation in maturation of murine bone marrow-derived DCs and to search for a target that plays a critical role. The results showed that compared with the control, lipopolysaccharide or Zymosan A groups, Jagged-1 (a soluble Jagged 1/Fc chimera protein) effectively increased expression of Hes-1 and Deltex-1 mRNA, which could be reversed by DAPT (2, 4-diamino-5-phenylthiazole), a specific inhibitor of the Notch signaling pathway. Hes-1-targeting siRNA could successfully down-regulate the endogenous Hes-1 expression in the DCs. Concurrently, a significant down-regulation of CD40, CD80, CD86 and MHC-II expressions on the surface of the DCs was found with the reduction of IL-12 yielded by the DCs. Our results demonstrate that Hes-1-targeting siRNA can inhibit the maturation of the DCs induced by Jagged-1, indicating Hes-1 may be an important target of Notch signaling mediating the maturation of DCs.     

Suppression of generation of concomitant antitumor immunity by passively transferred suppressor T cells from tumor-bearing donors

Summary Infusion of normal recipient mice with suppressor T cells from donors bearing a progressive Meth A fibrosarcoma results in a diminished capacity of the recipients to generate concomitant and postexcision antitumor immunity. The passive transfer of suppressor cells which prevented the generation of immunity to the Meth A fibrosarcoma did not affect the capacity of the recipients to reject an allogeneic tumor. The data provides direct evidence in support of the hypothesis that suppressor T cells, generated at later stages of growth of Meth A fibrosarcoma, function to down-regulate an already acquired mechanism of concomitant immunity.This work was supported by Grants CA-16642 and CA-17794 from the National Cancer Institute, Grant RR-05705 from the Division of Research Resources, NIH, and a grant from R. J. Reynolds Industries, Inc     

Regulation of arginase I activity and expression by both PD-1 and CTLA-4 on the myeloid-derived suppressor cells

An elevated number of Gr-1⁺CD11b⁺ myeloid-derived suppression cells (MDSCs) has been described in mice and human bearing tumor and associated with immune suppression. Arginase I production by MDSCs in the tumor environment may be a central mechanism for immunosuppression and tumor evasion. In this study and before, we found that Gr-1⁺CD11b⁺ MDSCs from ascites and spleen of mice bearing ovarian 18D carcinoma express a high level of PD-1, CTLA-4, B7-H1 and CD80 while other co-stimulatory molecules, namely CD40, B7-DC and CD86 are not detected. Further studies showed that PD-1 and CTLA-4 on the Gr-1⁺CD11b⁺ MDSCs regulated the activity and expression of arginase I. The blockage and silencing of PD-1, CTLA-4 or both PD-1 and CTLA4 molecules could significantly reduce arginase I activity and expression induced with tumor-associated factor. Similar results were also observed while their ligands B7-H1 and/or CD80 were blocked or silenced. Furthermore, CD80 deficiency also decreased the arginase I expression and activity. Antibody blockade or silencing of PD-1, CTLA-4 or both reduced the suppressive potential of PD-1+CTLA-4+MDSCs. Blockade of PD-1, CTLA-4 or both also slowed tumor growth and improved the survival rate of tumor-bearing mice. Thus, there may exist a coinhibitory and costimulatory molecules-based immuno-regulating wet among MDSCs. This research was supported by Nankai University grant, NSFC grant “30771967”, “985” grant,The Ministry of Science and Technology grant “2006AA020502”“06C26211200695”, Tianjin Grant “07JCZDJC03300” and “06ZHCXSH04800”.     

Chronic alcohol consumption enhances myeloid-derived suppressor cells in B16BL6 melanoma-bearing mice

We previously found that chronic alcohol consumption decreases the survival of mice bearing subcutaneous B16BL6 melanoma. The underlying mechanism is still not completely understood. Antitumor T cell immune responses are important to inhibiting tumor progression and extending survival. Therefore, we examined the effects of chronic alcohol consumption on the functionality and regulation of these cells in C57BL/6 mice that chronically consumed 20% (w/v) alcohol and subsequently were inoculated subcutaneously with B16BL6 melanoma cells. Chronic alcohol consumption inhibited melanoma-induced memory T cell expansion and accelerated the decay of interferon (IFN)-γ producing T cells in the tumor-bearing mice. Foxp3⁺CD4⁺CD25⁺ regulatory T cells were not affected; however, the percentage of myeloid-derived suppressor cells (MDSC) was significantly increased in the peripheral blood and spleen. T cell proliferation as determined by carboxyfluorescein succinimidyl ester labeling experiments in vitro was inhibited by alcohol consumption relative to control water-drinking melanoma-bearing mice. Collectively, these data show that chronic alcohol consumption inhibits proliferation of memory T cells, accelerates the decay of IFN-γ producing CD8⁺ T cells, and increases MDSC, all of which could be associated with melanoma progression and reduced survival.     

Depletion of regulatory T cells facilitates growth of established tumors: a mechanism involving the regulation of myeloid-derived suppressor cells by lipoxin A4

Regulatory T cells (Tregs) are thought to facilitate tumor development by suppressing protective antitumor immune responses. However, recent clinical and laboratory studies show that Tregs are a favorable element against cancer. In this study, we provide evidence that Tregs have both promoting and inhibiting effects on tumors, depending on the stage of tumor development. By using 0.5 mg cyclophosphamide, we constructed a murine liver cancer model in which Tregs were continuously and selectively depleted. Under such conditions, we found that tumor growth was inhibited at early stages but accelerated later on. Analysis of the tumor microenvironment disclosed that long-term Treg depletion by 0.5 mg cyclophosphamide treatment induced Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Ablation of MDSCs by anti-Gr-1 Ab blocked Treg depletion-induced promotion of tumor growth. Furthermore, lipoxygenases 5 and 12, two enzymes participating in the biosynthesis of the lipid anti-inflammatory mediator lipoxin A(4), were upregulated or downregulated by Treg depletion or adoptive transfer. Correspondingly, the levels of lipoxin A(4) were increased or decreased. Lipoxin A(4) thus regulated the induction of MDSCs in response to Treg depletion. These findings suggest that Tregs may play different roles at different stages of tumor growth: promoting early and inhibiting late tumor growth. Our study also suggests that the interplay among Tregs, MDSCs, and lipoxin A(4) tunes the regulation of tumor-associated inflammation.