Reduction In Myeloid-derived Suppressor Cells Research Articles

Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in Mouse Multiple Myeloma Model

AbstractAbstract 5010 Background:Multiple myeloma (MM) is a B-cell malignancy that has remained essentially incurable by conventional therapy, highlighting the urgent need for novel treatment strategies. Lenalidomide (LEN) is thalidomide analog belonging to the class of immunomodulatory drugs which the activity are related with immunomodulatory properties. LEN augments both the adaptive and innate immune system via the co-stimulation of T cells, NK and NKT cells. In addition, LEN can inhibit the frequency and function of immunosuppressor cells. Therefore, LEN could be used to enhance immune response against MM. Cellular therapy with dendritic cells (DCs) is emerging as a useful immunotherapeutic modality to treat MM. The purpose of this study was to investigate the immunomodulatory effects of lenalidomide in combination with DCs vaccine to treat MM in vivomouse model. Methods:We used the MOPC315 myeloma murin model to evaluate tumor-specific cytotoxic T lymphocytes responses by a DC vaccine in combination with LEN. After tumor growth, LEN (50 mg/kg/day) was injected intraperitoneally at three consecutive days to cover the DC vaccination. The tumor growth inhibition effect and the antitumor activity of splenocytes from vaccinated mice were evaluated to reveal the synergistic effect of DCs and LEN. Results:The combination of LEN and DC vaccine efficiently inhibited tumor growth in mouse MM model when compared to single therapeutic agent. These vaccinated mice exhibit the reduction of myeloid-derived suppressor cells (MDSC) and regulatory T cell (Treg) in spleen. Inhibition of MDSC and Treg resulted in the increasing proportion of CD4+ and CD8+T cell in the spleen. High ratio of Th1- to Th2-type cytokines was induced by LEN plus DC vaccine. LEN also enhance the innate immune response by modulating NK cell number and function. In addition, LEN also can enhance the population of effector memory T cells in the spleen of vaccinated mice. Furthermore, the treatment of LEN can down-regulate the levels of VEGF and TNF-a on tumor tissues of vaccinated mice. Conclusion:These results suggest that a treatment combining the immunomodulatory drug lenalidomide with DC vaccine can improve antitumor immunity in mouse MM model by inhibiting immunosuppressor cells and recovering effector cells, as well as superior polarization of the Th1/Th2 balance in favor of Th1 type immune response. Disclosures:No relevant conflicts of interest to declare.

The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses.

Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.

The expression of membrane-anchored GM-CSF reduces the tumorigenicity of murine breast cancer cells and correlates with a reduction in MDSCs in vivo (46.36)

Abstract Currently under preclinical and clinical investigation are several cancer vaccines using whole tumor cells modified to secrete GM-CSF. However, a potential drawback of such vaccines is the risk of systemic toxicity. To address this concern, we generated a GPI-anchored form of mouse GM-CSF (GPI-mGM-CSF) to be stably expressed on the surface of tumor cells. To determine the immunogenicity of breast cancer cells expressing the GPI-mGM-CSF, we used the TUBO cell line, a highly tumorigenic, non-metastatic tumor cell line that constitutively expresses the HER-2/neu oncogene which is overexpressed in 30% of human breast cancer patients. BALB/c mice were challenged with 1e5 TUBO-WT or TUBO-mGM-CSF cells. At the conclusion of the experiment, mice were sacrificed, spleens were harvested and splenocytes were isolated for flow cytometry analysis following RBC lysis. Splenocytes were stained for the prevalence of immune suppressive cells including myeloid derived suppressor cells (MDSCs). We observed minimal, transient growth of tumors in mice challenged with TUBO-mGM-CSF compared to TUBO-WT. Additionally, we obsesrved a significant reduction in spleen weight and prevalence of CD11b+Gr-1+ MDSCs in these mice. These studies suggest that the addition of a membrane anchored form of GM-CSF to tumor cells can induce an antitumor immune response and inhibit the development of immune suppressive cells such as MDSCs.

Use of gemcitabine- (Gem) and fluropyrimidine (FP)-based chemotherapy to reduce myeloid-derived suppressor cells (MDSCs) in pancreatic (PC) and esophagogastric cancer (EGC).

2588 Background: Pre-clinical data demonstrates that Gem and FP treatment leads to substantial falls in MDSCs in murine cancer models. We prospectively evaluated whether such a phenomena occurs in patients (pts) with cancers treated with these agents. Methods: Peripheral blood was collected from pancreatic and esophago-gastric cancer pts prior to and following Gem or FP based therapy. PBMC was harvested with subsequent flow-cytometric analysis of HLADR- Linlow/- CD33+ CD11b+ MDSCs. Percentage change in MDSC value was compared to independent evaluation of tumor volume percentage change, measured using the RECIST v1.1 criteria for target lesion assessment. Results: 16 PC pts receiving Gem based treatment and 23 EGC pts treated with FP based therapy were analyzed. There was a statistically significant reduction in pre and post treatment MDSC % (p<0.0001). Of the 21 pts having a >10% decrease in tumor volume, 20 had a fall in MDSCs, 60% of whom the MDSCs fell by >30%. There was a statistically significant reduction in MDSC % in the remaining 18 pts who had a ≤10% decrease in tumor volume or progressive disease (p=0.0016). 4 of 7 pts with a 0-10% tumor volume decrease had a >25% fall in MDSCs. None of the 11 pts with an increase in tumor volume had a >8% increase in MDSCs. Of these, 3 pts had substantial falls in MDSCs >30%, including an EGC pt where MDSCs fell from a value above the upper limit of normal (ULN) (defined by controls, n=47), to within the normal range. In all pts with initial MDSCs above the ULN, Gem or FP therapy reduced MDSCs to within normal range, independent of response. Conclusions: Gem and FP therapy significantly reduces or stabilizes MDSCs in PC and EGC, independent of response. These findings have important significance for programs integrating chemotherapy with immunotherapies such as cancer vaccines.

Reduction of myeloid-derived suppressor cells in peripheral blood by local hyperthermia in patients with advanced solid tumors.

e21058 Background: Circulating levels of myeloid-derived suppressor cells (MDSCs) correlate with clinical cancer stage, metastatic tumor burden and tumor immune evasion. The potential contribution of MDSCs to thermotherapy has not been explored. The aim of this study was to investigate the impact of treatment of local hyperthermia on the levels of MDSCs in peripheral blood. Methods: Thirty patients with advanced solid tumors treated with local hyperthermia were enrolled. Peripheral blood samples were obtained before and after treatment immediately. Circulating MDSCs (identified as HLA-Dr-/low, CD11b+, CD33+ cells) were measured by flow cytometry. Meanwhile lymphocyte subpopulations in peripheral blood samples (T-helper-/T4-cells, T-suppressor-/T8-cells, natural-killer-/NK-cells, CD19+cells) were also analyzed using the same method. The association between MDSCs and the other lymphocyte subpopulation was examined by ultivariate linear regression analysis. Results: The percentage of MDSCs and other lymphocyte subpopulations before and after treatment are shown in the Table1. MDSCs were significantly reduced after local hyperthermia, so were the T-suppressor cells and the NK cells. But the T-helper cells and the ratio of T-helper/T-suppressor cells were increased. The numbers of CD3+ cells and CD19+ cells were not significantly affected by the treatment. This reduction of MDSCs is not associated with any other lymphocyte subtype. Conclusions: It was demonstrated that local hyperthermia can reduce abnormal accumulation of circulating MDSCs. The reduction of MDSCs was not associated with the other lymphocyte subpopulation. The percentage of MDSCs and lymphocyte subpopulations before and after treatment. MDSC CD3+CD4+ CD3+CD8+ CD4+/CD8+ CD3+ NK CD19+ B# 7.71±5.92 38.84±10.72 28.26±10.05 1.56±0.71 65.65±13.49 23.77±11.37 5.69±5.13 A* 6.45±4.85 43.80±10.89 26.92±9.93 1.98±1.26 68.80±14.23 19.37±10.58 6.37±6.25 P value 0.042 0.003 0.006 0.004 0.052 0.004 0.150 B# Before Local Hyperthermia. A* After Local Hyperthermia.

Abstract 3650: Anti-MIF gene therapy of murine mammary tumor results in decreased tumor metastasis and reduction of MDSC (Myeloid derived suppressor cells)

Abstract Introduction: Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine which has been shown to be secreted by a variety of tumors and results in autocirne tumor growth. The purpose of this study is to determine the effect of down-regulation of MIF expression on primary tumor growth, metastasis and cellular mediators of immune suppression. Methods: Ten days post 4T1-luciferase tumor cells injection into Balb/c mice, nanoparticles with anti-MIF siRNA and controls (PBS and nanoparticles with scrambled siRNA) were directly injected into established tumor daily for 7 days. At different time points, tumor sizes were measured, tumor metastases in live mice were imaged, tumors and blood samples were harvested. The gene expression in tumor slides and 4T1 cells from tumor digest were investigated by FACS and immunofluorescence microscopy. The total number of circulating MDSCs and MDSCs expressing suppressive cytokines were determined by FACS. Results: We have developed a non-viral gene carrier system that contains a biodegradable β-glucan (10 Kda) backbone and protonable imidazole at the reducing end. The nanoparticles at N/P of 8 lead to the high uptake efficiency by 4T1 cells. The imidazole has buffering capacity to induce endosomal escape. Once released into cytosol, the biodegradable glucan shell of nanoparticles is slowly oxidized and degraded so that the siRNAs are sustained released and the target gene MIF is sustained reduced. Applying this intratumoral anti-MIF gene therapy in vivo, we have made the following observations: 1) MIF protein is highly expressed in 4T1 mammary tumors and can be efficiently blocked in vivo; 2) Reduction of MIF production in established tumor in vivo results in reduced primary tumor size and metastasis; 3) Reduction of MIF production in 4T1 tumor cells in vitro results in reduced MIF secretion in 4T1-conditioned medium and enhanced tumor cell apoptosis and death; 4) Reduction of MIF production in established tumor results in a significant reduction in the total number of circulating MDSCs by 37.6%. The MDSCs expressing immune suppressive cytokines including TGF-β, IL-10 and IL-1β was reduced by 43%, 47% and 70%. Significance: Although MIF appears to have recognized autocrine growth activity in a variety of tumors, development of therapeutics directed at MIF have been problematic because it serves a number of functions in normal biological processes. Targeted reduction of MIF by intratumoral injection of siRNA using a non-viral nanoparticle technology alleviates the difficulty of gene delivery. In addition, since the down-regulation of tumor-secreted MIF may reduce mammary tumor-induced immune suppression by reducing MDSCs, the local therapy can result in a favorable systemic antitumor effect by enhancing host immunity. This study represents a first step in developing this therapeutic approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3650. doi:10.1158/1538-7445.AM2011-3650

Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice

Background and aimsNew options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models....

Abstract 5311: Myeloid-derived suppressor cells and immune cell responsiveness in tumor-bearing mice

Abstract Conditions that inhibit IFN-induced signal transduction and gene transcription can dampen the host immune system and its ability to recognize and eliminate tumors leading to impaired immunoediting. We hypothesized that in the setting of cancer, myeloid-derived suppressor cells (MDSC) could impair the responsiveness of immune effector cells to interferons. We used the murine adenocarcinoma cell line C26 which induces MDSC generation when implanted in C2DF1 mice. C26-bearing mice had significantly elevated levels of GR1+CD11b+ MDSC (p&amp;lt;0.0001) and plasma IL-6 as compared to controls (p&amp;lt;0.0001). The ability of IFNa or IFNy to stimulate phosphorylation of the STAT1 transcription factor (P-STAT1, a direct measure of IFN action) was measured by intracellular flow cytometry. Induction of P-STAT1 following a 15 minute stimulation of splenocytes with IFNa (102-104 U/mL) was significantly reduced in C26-bearing mice (Mean P-STAT1 = 18.8; Range = 4.0 - 33.4) as compared to control mice (Mean P-STAT1 = 37.9; Range = 27.0 - 50.0; p&amp;lt;0.0001). A similar decrease in P-STAT1 in splenocytes of C26-bearing mice was observed following IFNy stimulation (1 ng/mL; 15 min; p=0.002). This decrease in IFN responsiveness was also observed in cells derived from inguinal lymph nodes following IFN-stimulation. The level of IFNa induced P-STAT1 was inversely correlated with the plasma IL-6 levels (Pearson correlation = −0.635; p=0.019). However, direct exogenous treatment of mice with IL-6 did not decrease splenic IFN response. In vitro co-culture experiments revealed that MDSC inhibited IFN-responsiveness of splenocytes from normal mice. Reduction of MDSC with either gemcitabine or anti-GR1 antibody treatment restored IFN responsiveness in splenocytes from C26-bearing mice as compared to tumor-bearing mice treated with vehicle or isotype control antibody. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide (NO). The elevation in NO in C26-bearing mice was associated with increased levels of nitration on STAT1. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. Taken together, these data suggest a novel link between MDSC and a decreased molecular response of the immune effectors responsible for promoting functional anti-tumor responses. 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 5311.

Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients

Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Reduction of Myeloid-Derived Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Established Metastatic Disease

More than 60% of STAT6(-/-) mice immunologically reject spontaneous metastatic mammary carcinoma and survive indefinitely if their primary tumors are removed, whereas 95% of STAT6-competent BALB/c mice succumb to metastatic disease. BALB/c and STAT6-deficient mice with primary tumors have elevated levels of Gr1(+)CD11b(+) myeloid suppressor cells (MSCs), which inhibit T cell activation. After removal of primary tumor, MSC levels revert to baseline in STAT6-deficient mice, but remain elevated in BALB/c mice. The decrease is IFN-gamma dependent, as is the reduction in metastatic disease. Neither BALB/c nor STAT6-deficient MSCs produce inducible NO synthase; however, both produce arginase and reactive oxygen species. STAT6-deficient mice produce M1 macrophages, which contain high levels of NO and are tumoricidal, whereas BALB/c mice produce M2 macrophages, which make arginase and are not tumoricidal. Immunity in STAT6-deficient mice requires the activation of NO-producing M1 macrophages that are tumoricidal, the reduction in MSC levels to baseline after surgical removal of primary tumor, and the activation of tumor-specific T cells. These mechanisms occur in STAT6(-/-) mice because STAT6 deficiency prevents signaling through the type 2 IL-4Ralpha, thereby blocking the production of arginase and promoting the synthesis of NO.