Suppressive Function Of Myeloid-derived Suppressor Cells Research Articles

MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes.

Myeloid-derived suppressor cells (MDSCs) are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2], [3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF) is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4], [5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist—4-IPP [4], [6], [7], [8], [9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333) published by Yaddanapudi et al. (2015) in Cancer Immunology Research [10].

Olive Leaf Extract Reduces Myeloid-Derived Suppressor Cells, and Modulates the Function of Residual Cells in Experimental Model of Melanoma

Background: Immunosuppression in melanoma is mediated by increased accumulation of Myeloid Derived Suppressor Cells (MDSCs). Olive Leaf Extract (OLE) has been developed as a natural anti-inflammatory, anti-oxidant, anti-proliferative and antiapoptotic agent on cancer immunotherapy. Objective: To investigate whether OLE could inhibit MDSCs, enhance anti-tumor activities and consequently increase the survival rate of the murine melanoma model. Methods: The C57BL/6 mice were inoculated subcutaneously with B16/F10 melanoma tumor cell lines. Induced mice were orally treated with 500 mgkg-1 of olive extract per kg of body weight for 8 consecutive days. The frequency and function of MDSCs and induction of inflammatory mediators as well as tumor growth and survival rate were assessed in treated and untreated mice. Results: The results of current study revealed that the optimal dose of OLE (500 mgkg-1) reduced the tumor growth (40%), and prolonged mice survival (25%) by significant decreasing (P<0.05) the number (over 50%), and suppressive function of MDSCs (over 60%) (P<0.05). OLE was also significantly (P<0.05) down regulated the induction of inflammatory agents in melanoma-bearing mice (over 50%) at the applied dose (500 mgkg-1). Conclusion: Therefore, these results altogether provided some evidence that regulation of immunosuppression were the possible therapeutic effects of OLE in tumor cells.

Abstract 1671: Immunosuppressive myeloid-derived suppressor cells expressing PDL1 are increased in human melanoma tumor tissue

Abstract Despite the ability to elicit active immune responses, most immunotherapies fail to eliminate tumors in human cancer patients. One reason immunotherapy has not met expectations may be due to immunosuppression in the local environment of the tumor. Immunosuppressive myeloid-derived suppressor cells (MDSCs) are increased in the peripheral blood of melanoma patients and the frequency of MDSCs correlates with overall survival and disease progression. Furthermore, cytokines related to the recruitment and differentiation of MDSCs are also increased in melanoma patient serum and changes in these cytokine levels may correlate with disease progression. We find an increased frequency of regulatory T cells, PD-1-expressing T cells, and PD-L1 expressing MDSCs in primary melanoma tumors, suggesting that PD-1/PD-L1 interactions may play a role in immunosuppression in the microenvironment of the tumor. We are currently investigating the mechanisms of immunosuppression used by human MDSCs and whether the suppressive function of MDSCs can be blocked with antibodies targeting PD-L1. In conclusion, we have established MDSCs as an important player in immunosuppression in melanoma patients suggesting that combinatorial treatments that reduce the frequency or activity of MDSCs may improve the effectiveness of future immunotherapies. Citation Format: Kimberly Rae Jordan, Virginia Borges, Martin D. McCarter. Immunosuppressive myeloid-derived suppressor cells expressing PDL1 are increased in human melanoma tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1671. doi:10.1158/1538-7445.AM2014-1671

Baccatin III, a precursor for the semisynthesis of paclitaxel, inhibits the accumulation and suppressive activity of myeloid-derived suppressor cells in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) mediate tumor-associated immune suppression in both cancer patients and tumor-bearing animals. Reduction or elimination of MDSCs reduces the rate of tumor progression and improves cancer therapies that employ mechanisms of immunity. Here we show that baccatin III, which is the precursor for the semisynthesis of paclitaxel, exerts anti-tumor immunomodulatory activity in very low doses (0.05-0.5mg/kg), although it is regarded as an inactive derivative of paclitaxel. Oral administration of baccatin III significantly reduced the growth of tumors induced by engrafting BALB/c mice with either 4 T1 mammary carcinoma or CT26 colon cancer cells. Baccatin III (0.5mg/kg) did not exert anti-tumor activity in athymic nude mice. Baccatin III decreased the accumulation of MDSCs in the spleens of the tumor-bearing mice. Furthermore, MDSCs isolated from baccatin III-treated mice, compared with those isolated from vehicle-treated mice, had a significantly reduced suppressive effect on T cells treated with the anti-CD3 and anti-CD28 monoclonal antibodies. Moreover, these cells produced significantly reduced amounts of reactive oxygen species and nitric oxide. These results suggest that baccatin III reduced tumor progression by inhibiting the accumulation and suppressive function of MDSCs.

Myeloid-derived suppressor cells are increased in frequency but not maximally suppressive in peripheral blood of Type 1 Diabetes Mellitus patients.

Type 1 Diabetes Mellitus (T1D) results from the destruction of insulin-producing beta cells in the pancreas by autoreactive T cells. Myeloid derived suppressor cells (MDSCs) are a recently identified immune cell subset that down-regulate T cells. Whether defects in MDSC numbers or function may contribute to T1D pathogenesis is not known. We report here that MDSCs are unexpectedly enriched in peripheral blood of both mice and patients with autoimmune diabetes. Peripheral blood MDSCs from T1D patients suppressed T cell proliferation in a contact-dependent manner; however, suppressive function could be enhanced with in vitro cytokine induction. These findings suggest that native T1D MDSCs are not maximally suppressive and that strategies to promote MDSC suppressive function may be effective in preventing or treating T1D.

Cutting Edge: Flt3 Ligand Mediates STAT3-Independent Expansion but STAT3-Dependent Activation of Myeloid-Derived Suppressor Cells

The Flt3-Flt3 ligand (Flt3L) pathway is critically involved in the differentiation and homeostasis of myeloid cells, including dendritic cells (DC); however, its role in the expansion and function of myeloid-derived suppressor cells (MDSC) has not been determined. In this article, we describe the ability of Flt3L to expand and activate murine MDSC capable of suppressing allograft rejection upon adoptive transfer. Although Flt3L expands and augments the stimulatory capacity of myeloid DC, MDSC expanded by Flt3L have increased suppressive activity. Although STAT3 is considered the central transcription factor for MDSC expansion, inhibition and genetic ablation of STAT3 did not block, but rather augmented, Flt3L-mediated MDSC expansion. MDSC suppressive function, preserved when STAT3 inhibition was removed, was reduced by genetic STAT3 deletion. Both STAT3 inhibition and deletion reduced Flt3L-mediated DC expansion, signifying that STAT3 had reciprocal effects on suppressive MDSC and immunostimulatory DC expansion. Together, these findings enhance our understanding of the immunomodulatory properties of Flt3L.

Baccatin III inhibits the accumulation and suppressive activity of myeloid‐derived suppressor cells in tumor‐bearing mic (LB584)

Myeloid‐derived suppressor cells (MDSCs) mediate tumor‐associated immune suppression in both cancer patients and tumor‐bearing animals. Reduction or elimination of MDSCs reduces the rate of tumor progression and improves cancer therapies that employ mechanisms of immunity. Here we show that baccatin III, which is the precursor for the semisynthesis of paclitaxel, exerts antitumor immunomodulatory activity in very low doses (0.05 ‐ 0.5 mg/kg), although it is regarded as an inactive derivative of paclitaxel. Oral administration of baccatin III significantly reduced the growth of tumors induced by engrafting mice with either 4T1 mammary carcinoma or CT26 colon cancer cells. Antitumor activity was immune‐mediated because baccatin III was not directly cytotoxic. Baccatin III decreased the accumulation of MDSCs in the spleens of the tumor‐bearing mice. Furthermore, MDSCs isolated from baccatin III‐treated mice, compared with those isolated from vehicle‐treated mice, had a significantly reduced suppressive effect on T cells treated with the anti‐CD3 and anti‐CD28 monoclonal antibodies. Moreover, these cells produced significantly reduced amounts of reactive oxygen species and nitric oxide. These results demonstrate that baccatin III reduced tumor progression by inhibiting the accumulation and suppressive function of MDSCs.

Targeting S1P1 Receptor Protects against Murine Immunological Hepatic Injury through Myeloid-Derived Suppressor Cells

Although FTY720 may alter migration and homing of lymphocytes via sphingosine-1-phosphate (S1P) receptors, our recent studies indicated that FTY720 directly controls the differentiation of Th1 cells to regulatory T cells (Tregs) by targeting S1P1. However, the pharmacological function of FTY720 in immunological hepatic injury remains unknown. In this study, the role and regulatory signaling pathway of S1P receptor were investigated using a pharmacological approach in immune-mediated hepatic injury (IMH). In the context of IMH, FTY720 significantly ameliorated mortality and hepatic pathology. In FTY720-treated mice, recruited CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) mediate protection against IMH and are functional suppressive immune modulators that result in fewer IFN-γ-producing Th1 cells and more Foxp3(+) Tregs. In agreement, FTY720-treated MDSCs promote the reciprocal differentiation between Th1 cells and Tregs in vitro and in vivo. Mechanistically, FTY720 treatment induced inducible NO synthase expression and NO production in MDSCs. Pharmacologic inhibition of inducible NO synthase completely eliminates MDSC suppressive function and eradicates their inducible effects on T cell differentiation. Finally, the mTOR inhibitor, rapamycin, photocopies the effects of FTY720 on MDSCs, implicating mTOR as a downstream effector of S1P1 signaling. This study identifies MDSCs as an essential component that provides protection against IMH following FTY720 or rapamycin treatment, validating the S1P1-mTOR signaling axis as a potential therapeutic target in hepatic injury.

A copper chelate selectively triggers apoptosis in myeloid-derived suppressor cells in a drug-resistant tumor model and enhances antitumor immune response

Myeloid-derived suppressor cells (MDSCs), one of the major orchestrators of immunosuppressive network are present in the tumor microenvironment suppress antitumor immunity by subverting Th1 response in tumor site and considered as a great obstacle for advancement of different cancer immunotherapeutic protocols. Till date, various pharmacological approaches have been explored to modulate the suppressive functions of MDSCs in vivo. The present study describes our endeavor to explore a possibility of eradicating MDSCs by the application of a copper chelate, namely copper N-(2-hydroxy acetophenone) glycinate (CuNG), previously found to be a potential immunomodulator that can elicit antitumorogenic Th1 response in doxorubicin-resistant EAC (EAC/Dox) bearing mice. Herein, we demonstrated that CuNG treatment could reduce Gr-1+CD11b+ MDSC accumulation in ascitic fluid and spleen of EAC/Dox tumor model. Furthermore, we found that CuNG mediated reduction in MDSCs is associated with induction of Th1 response and reduction in Treg cells. Moreover, we observed that CuNG could deplete MDSCs by inducing Fas-FasL mediated apoptotic cell death where death receptor Fas expression is enhanced in MDSCs and FasL is provided by activated T cells. However, MDSC expansion from bone marrow cells and their differentiation was not affected by CuNG. Altogether, these findings suggest that the immunomodulatory property of CuNG is attributed to, at least in part, by its selective cytotoxic action on MDSCs. So, this preclinical study unveils a new mechanism of regulating MDSC levels in drug-resistant cancer model and holds promise of translating the findings into clinical settings.

Reprogramming tumor microenvironment in cancer immunotherapy (P4274)

Abstract Type-1 immune effector cells (CTLs, Th1 and NK cells) express high levels of CXCR3 and CCR5 and depend on the relevant chemokine ligands (CXCL9, CXCL10 and CCL5) for entry into tumor microenvironments. We observed that local production of prostaglandin (PG)E2 suppresses the induction of immune effector cell and selectively inhibits the production of the effector cell-attracting chemokines within peritoneal tumors (ovarian and colorectal cancers and different forms of peritoneal carcinomatosis). COX2 is also the key factor driving the MDC/CCL22- and SDF1/CXCL12-mediated accumulation of Tregs and myeloid-derived suppressor cells (MDSCs) in tumor tissues, orchestrating local inhibition of CTL function. COX2 blockers antagonized the accumulation and suppressive function of MDSCs and proved to be a necessary (but not sufficient) element of strategies aimed at restoring local immunosurveillance. While TLR-ligands used as stand-alone vaccine adjuvants can amplify the PGE2-driven suppressive events, their combination with COX2 inhibitors and IFNα allows to selectively induce effector cell-attracting chemokines within tumors (bu tnot marginal tissues) and suppress the MDSC- and Treg-attracting chemokines (CXCL12 and CCL22). Our current trials evaluate the effectiveness of tumor-selective conditioning of tumor microenvironments to promote the entry of spontaneously-arising and vaccination-induced CTLs into tumors and counteracting local immunosuppression.

Myeloid-derived suppressor cell subsets inhibit T- and B-cell responses in murine Retrovirus-induced immunodeficiency (P6179)

Abstract Compared to tumor microenvironments, myeloid-derived suppressor cells (MDSCs) have been less well studied in infectious diseases, particularly for retroviruses inducing immunodeficiency. We have found the development of a large population of immunosuppressive MDSCs after LP-BM5 retrovirus infection which causes murine acquired immunodeficiency. These MDSCs express a cell-surface marker signature (CD11b+Gr-1+Ly6C+) and inhibit not only T-cell, but B-cell, responsiveness by a substantially iNOS-dependent mechanism. MDSC immunosuppressiveness parallels the extent of disease in LP-BM5-infected w.t. vs. k.o. mice, differentially susceptible to disease. Retroviral load indicate that the suppressive Ly6C+CD11b+MDSC subset is LP-BM5 positive, although significantly lower than non-fractionated splenocytes from infected mice. Further fractionation of the MDSCs by FACS indicated, whereas all four subsets defined by Ly6C versus CD11b expression suppressed B-cell responses, only the Ly6Cbright CD11b+ subset suppressed T-cells, indicating distinct versus overlapping targeting of the two adaptive immune arms. These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression, and, as the total MDSC suppressive function mirrors that of LP-BM5-induced disease, support a pathogenic effector role for these retrovirus-induced MDSCs.

Abstract 4746: Modulation of suppressive myeloid populations by tasquinimod.

Abstract Purpose: Suppressive myeloid cells such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are a major barrier of cancer immunotherapy. These populations suppress immune responses, promote angiogenesis, and facilitate cancer progression and metastasis. Tasquinimod is an experimental anti-angiogenic agent tested in prostate cancer. In the current study, we investigated the novel immunomodulatory effects of tasquinimod. We focused on the modulation of myeloid populations by tasquinimod since tasquinimod binds to the inflammatory protein S100A9, which potentially activates MDSCs. Thus, we studied whether tasquinimod affects the induction/function of MDSCs and TAMs, and whether this agent can facilitate vaccine therapy in prostate and kidney cancer models. Experimental Design: The effects of tasquinimod on myeloid cells were tested in Myc-CaP and TRAMP transplantable prostate cancer models, and the RENCA orthotopic model of renal cell carcinoma. Splenocytes, blood cells, and cells from tumor samples were isolated from tumor bearing mice. The samples were subjected to staining with MDSC markers (Gr1 and CD11b) and macrophage markers (F4/80 and CD206), and to flow cytometry analysis. An immunotherapy combining tasquinimod with a modified survivin peptide vaccine, SurVaxM, was administered in Myc-CaP and TRAMP models. Tumor growth, MDSCs, and TAMs accumulation and their function after different treatments were monitored. Tasquinimod's effect on metastases is currently being tested in a LuCaP 23.1 orthotopic prostate cancer model. Results: Tasquinimod treatment reduced peripheral Gr1−CD11b+ monocytes, but did not affect the number of peripheral Gr1+CD11b+ MDSCs. However, tumor infiltrating Gr1+CD11b+ MDSCs significantly decreased following tasquinimod treatment (60% reduction, p = 0.03). In addition, Gr1−CD11b+ monocytes gained the macrophage marker, F4/80, in tumors, and tasquinimod reduced the M2 macrophages (immunosuppressive macrophages) in both peripheral sites and tumors (56% reduction, p = 0.04). Tasquinimod had a modest effect on the suppressive function of MDSCs. In the vaccine therapy, tasquinimod, combined with SurVaxM, improved antitumor immune responses and reduced tumor growth (48% inhibition, p = 0.03), as compared to vehicle and single treatments (no inhibition by SurVaxM, 15% inhibition by tasquinimod). Conclusions: Our study suggests that tasquinimod does not affect the expansion of Gr1+CD11b+ MDSCs induced by tumor, but interferes with the migration of MDSCs to tumor sites. Moreover, tasquinimod targets M2 macrophages, possibly by depleting the monocytic precursor populations such as Gr1−CD11b+ monocytes. By inhibiting MDSCs and TAMs, tasquinimod has an immunomodulatory potential to target cancer progression and metastasis, and to improve immunotherapies. Citation Format: Li Shen, Michael Ciesielski, Kiersten M. Miles, Robert Fenstermaker, Roberto Pili. Modulation of suppressive myeloid populations by tasquinimod. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4746. doi:10.1158/1538-7445.AM2013-4746

T helper type 17 cells contribute to anti‐tumour immunity and promote the recruitment of T helper type 1 cells to the tumour

T helper type 17 (Th17) lymphocytes are found in high frequency in tumour-burdened animals and cancer patients. These lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, have a well-defined role in the development of inflammatory and autoimmune pathologies; however, their function in tumour immunity is less clear. We explored possible opposing anti-tumour and tumour-promoting functions of Th17 cells by evaluating tumour growth and the ability to promote tumour infiltration of myeloid-derived suppressor cells (MDSC), regulatory T cells and CD4(+) interferon-γ(+) cells in a retinoic acid-like orphan receptor γt (RORγt) -deficient mouse model. A reduced percentage of Th17 cells in the tumour microenvironment in RORγt-deficient mice led to enhanced tumour growth, that could be reverted by adoptive transfer of Th17 cells. Differences in tumour growth were not associated with changes in the accumulation or suppressive function of MDSC and regulatory T cells but were related to a decrease in the proportion of CD4(+) T cells in the tumour. Our results suggest that Th17 cells do not affect the recruitment of immunosuppressive populations but favour the recruitment of effector Th1 cells to the tumour, thereby promoting anti-tumour responses.

Clinical Significance and Functional Studies of Myeloid-Derived Suppressor Cells in Chronic Hepatitis C Patients

Myeloid-derived suppressor cells (MDSCs) are known to accumulate under some pathologic conditions and suppress immune system in a variety of ways. This study aims to evaluate the significance of MDSCs in chronic Hepatitis C (CHC) patients. 14 CHC patients and healthy donors were enrolled and subject to antiviral therapy including Peg-INF-alpha and Ribavirin for 48 weeks. The peripheral blood mononuclear cells (PBMCs) were collected at different weeks post-therapy and MDSC frequency was analyzed by flow cytometry. The correlation between MDSCs level with CHC disease parameters was analyzed by Spearman's rank test. The suppressive function of MDSCs from CHC patients and the underlying mechanism was further evaluated. A significant elevation of MDSCs was observed in the peripheral blood of treatment-naive CHC patients compared with healthy donors. The level of MDSCs in CHC patients correlated with plasma HCV-RNA (r = 0.7164, p = 0.0039), blood aminotransaminase (r = 0.6116, p = 0.021), and activated CD38(+) T cells (CD4(+): r = 0.6649, p = 0.0095; CD8(+): r = 0.6189, p = 0.0189). Initiation of clinical therapy reduced MDSC levels as early as 4 weeks, while it rebounded at week 12 post-therapy in patients. CHC-derived MDSCs could suppress T cell function in an arginase-1-dependent manner, that was distinct from the HCV core protein-generated MDSCs as previously reported. Our study reveals a significant correlation between MDSC levels with HCV disease progression, and their response to antiviral therapy. The arginase-1-dependent mechanism of MDSCs from CHC patients indicates that arginase-1 may be promising target for HCV immunotherapy.

Targeting myeloid-derived suppressor cells augments antitumor activity against lung cancer

Lung cancer evades host immune surveillance by dysregulating inflammation. Tumors and their surrounding stromata produce growth factors, cytokines, and chemokines that recruit, expand, and/or activate myeloid-derived suppressor cells (MDSCs). MDSCs regulate immune responses and are frequently found in malignancy. In this review the authors discuss tumor-MDSC interactions that suppress host antitumor activities and the authors’ recent findings regarding MDSC depletion that led to improved therapeutic vaccination responses against lung cancer. Despite the identification of a repertoire of tumor antigens, hurdles persist for immune-based anticancer therapies. It is likely that combined therapies that address the multiple immune deficits in cancer patients will be required for effective therapy. MDSCs play a major role in the suppression of T-cell activation and they sustain tumor growth, proliferation, and metastases. Regulation of MDSC recruitment, differentiation or expansion, and inhibition of the MDSC suppressive function with pharmacologic agents will be useful in the control of cancer growth and progression. Pharmacologic agents that regulate MDSCs may be more effective when combined with immunotherapies. Optimization of combined approaches that simultaneously downregulate MDSC suppressor pathways, restore APC immune-stimulating activity, and expand tumor-reactive T cells will be useful in improving therapy.

Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma.

Myeloid-derived suppressor cells (MDSC) have emerged as an immune-regulatory cell type that is expanded in tumor-bearing mice, but less is known about their immune-suppressive role in patients with cancer. To study the importance of MDSC in patients with melanoma, we characterized the frequency, phenotype, and suppressive function of blood myeloid-derived cells and tumor-infiltrating myeloid cells in 26 freshly resected melanomas. Blood and tumor-infiltrating myeloid cells (Lin(-) CD11b(+)) could be phenotypically and morphologically classified into monocytes/macrophages, neutrophils, eosinophils, and immature myeloid cells according to marker expression (CD14(+), CD14(-) CD15(hi), CD14(-) CD15(int), and CD14(-) CD15(-), respectively). In contrast to the expansion of MDSC reported in tumor-bearing mice, we found no differences in the frequency and phenotype of myeloid subsets in the blood of patients with melanoma compared with healthy donors. Myeloid cells represented 12% of the live cells in the melanoma cell suspensions, and were phenotypically diverse with high tumor-to-tumor variability. Interestingly, a positive association was found between the percentage of Tregs and granulocytic cells (Lin(-) CD11b(+) CD14(-)CD15(+)) infiltrating melanoma tumors. However, melanoma-infiltrating myeloid cells displayed impaired suppression of nonspecific T-cell proliferation compared with peripheral blood myeloid cells, in which monocytes and eosinophils were suppressive. Our findings provide a first characterization of the nature and suppressive function of the melanoma myeloid infiltrate and indicate that the suppressive function of MDSC in patients with melanoma seems far less than that based on murine tumor models.

Negative Regulation of Myeloid-derived Suppressor Cells in Cancer

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive function on immune response. In this review, we discuss recent studies about mechanisms of expansion and suppressive function of MDSCs during inflammation, infection and autoimmune diseases, as well as pro-angiogenic and pro-metastatic functions of these cells in tumor development. Further, we focus on novel studies of MDSCs and therapeutic approaches to eliminate these cells in cancer.

Resveratrol Prevents Endothelial Cells Injury in High-Dose Interleukin-2 Therapy against Melanoma

Immunotherapy with high-dose interleukin-2 (HDIL-2) is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS). In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) and FoxP3+CD4+ regulatory T cells (Treg). We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory T cell in the development of VLS.

Regulatory T Cells Stimulate B7-H1 Expression in Myeloid-Derived Suppressor Cells in ret Melanomas

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells, and they promote an immunosuppressive environment in tumor-bearing hosts. To characterize MDSCs in melanoma, we examined the expression of inhibitory B7 molecules by CD11b(+)Gr1(+) cells isolated from mice with transplantable ret tumors. B7 molecules were expressed on CD11b(+)Gr1(+) cells, which also expressed CD124 and inducible nitric oxide synthase, thus verifying their relation to MDSCs. In developing melanomas, CD11b(+)Gr1(+) cells express only low levels of B7-H1. In contrast, B7-H1 is upregulated in large tumors, and functional analysis demonstrates that CD11b(+)Gr-1(+) cells suppress the proliferation of CD4(+) T cells through B7-H1. Depletion of regulatory T cells (Tregs) significantly downregulated the expression of B7-H1, B7-H3, and B7-H4 on MDSCs and reduced tumor growth, indicating a concerted immunosuppressive activity of Tregs and MDSCs. No differences in the suppressive function of MDSCs between CD25-depleted and non-depleted mice were recorded. Instead, tumor-derived MDSCs from Treg-depleted hosts produced less IL-10 and more IFN-γ as compared with Treg-harboring mice. These studies indicate that Tregs in tumors not only suppress effector T cells directly, but also modify the phenotype of tumor-infiltrating CD11b(+) cells to express inhibitory B7-H molecules and to produce IL-10.

Abstract 3848: Abrogation of TGF-β signaling promotes mammary carcinoma progression by enhanced Th17 response

Abstract We have shown that TGF-β signaling in carcinoma cells can suppress the expression of chemokines that are responsible for the expansion and recruitment of myeloid derived suppressor cells (MDSCs) in tumor bearing mice. Using mice with conditional deletion of the type II TGF-β receptor gene (Tgfbr2) in mammary epithelial cells we have shown that derivative MMTV-PyMT induced carcinomas were better able to recruit tumor-promoting MDSCs than carcinomas with intact TGF-β signaling. This recruitment correlated with higher levels of Cxcl1/5 expression in tumors lacking intact TGF-β signaling. Cxcl1/5 signal through the CXCR2 receptor and it has been shown that CXCR2 signaling in vitro was able to promote migration and invasion of MDSC. IL-17 can up-regulate secretion of Cxc chemokines by epithelial cells. Differentiation of Th17 cells in the tumor microenvironment depends on the balance of cytokines such as TGF-β, IL-6 and IL-23, which are primarily produced by tumor infiltrating myeloid cells. Based on this information, we hypothesize that loss of TGF-β signaling in carcinoma cells results in increased tumor volume through an enhanced Th17 response. In vivo studies were performed using a spontaneous tumor model MMTV-PyMT and MMTV-PyMT/Tgfbr2 KO. On 7-9 days after tumor formation we isolated tumor tissue and prepared single cell suspensions which were used for flow cytometry analysis and for FACS sorting. CD4+ cells were isolated by CD4 Microbeads from Miltenyi Biotec. For in vitro studies we used immortalized epithelial cell lines which were prepared from PyMT tumors of MMTV-WT and MMTV-KO mice. Levels of cytokines were measured by ELISA. We found that MDSCs don't express CXCR2 in the lung and tumor tissue compared with MDSCs from the spleen and bone marrow. Carcinoma cells without Tgbr2 produce more CXCL1/5 and ELISA data showed that release of these two chemokines is upregulated by IL-17. Also, we have found increased expression of IL-17r on knockout epithelial cells and these cells are 4 times more sensitive to low concentrations of IL-17. In parallel, we found that TGF-β decreases secretion of CXCL1/5 from normal epithelial cells but not from cells without Tgbr2. Levels of TGF-β and IL-6, which are important for differentiation of Th17 cells, are increased in tumor tissue without Tgbr2. These data are paralleled with an increased number of Th-17 cells and level of IL-17. In addition, we discovered a secondary effect of IL-17 in increasing the suppressive function of MDSC's on T cells through the upregulation of Arg, IDO and COX2. We conclude that the tumor microenvironment of mammary carcinomas with defective TGF-β signaling in epithelial cells have increased levels of IL-17 which enhance tumor progression thru increased MDSCs migration and enhanced suppressive function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3848.