Splenic Myeloid-derived Suppressor Cells Research Articles

Dexamethasone Prolongs Cardiac Allograft Survival in a Murine Model Through Myeloid-derived Suppressor Cells

BackgroundRecently, myeloid-derived suppressor cells (MDSCs) have attracted considerable attention because of their cancer-promoting and immunosuppressive effects. The glucocorticoid dexamethasone (Dex) is an important immunosuppressive agent used to treat autoimmune diseases and organ transplant rejection. However, the mechanism by which it modulates the immune system is not completely understood. Material and MethodsIn this study, we investigated the mechanisms by which Dex modulated the immune response in mice given an allogeneic cardiac transplant. ResultsDex injection significantly prolonged heart graft survival compared with phosphate-buffered saline–injected controls. Dex treatment increased the number of splenic MDSCs. Moreover, Gr-1high/CD11b+ MDSCs and CD3+/CD4+/Foxp3+ regulatory T cells (Tregs) were significantly increased in the Dex group compared with controls. Administration of anti–Gr-1 antibody (Ab) to the Dex group significantly shortened mouse heart graft survival. In addition, anti–Gr-1 Ab treatment significantly reduced Tregs in the Dex + anti–Gr-1 co-treatment group compared with the Dex group. These observations suggest that Dex treatment increased both MDSCs and Tregs, and that MDSCs regulated the incidence of Tregs in this immunosuppressive pathway. ConclusionAn important role of Dex in the prevention of the rejection of cardiac grafts in mice is to expand MDSCs and Tregs.

MDSCs are induced after experimental blunt chest trauma and subsequently alter antigen-specific T cell responses

Severe blunt chest trauma (TxT) induces a strong inflammatory response with posttraumatic immune suppression pointing to an impaired adaptive immune response. Since CD11b+Gr-1+-expressing myeloid-derived suppressor cells (MDSCs) are induced after inflammation and suppress T cell responses, MDSC induction and their impact on T cell functions was analysed in an experimental TxT model. MDSCs were induced preferentially in the lung until 24 hours after TxT. Although MDSC numbers were only faintly increased in the spleen, splenic MDSCs isolated after TxT strongly inhibited alloantigen-induced T cell proliferation in vitro. Suppressive activity correlated with increased expression of arginase-1 and iNOS. MDSCs also prevented antigen-induced T cell expansion in vivo, since staphylococcus enterotoxin B (SEB)-induced proliferation of vβ8+ T cells was impaired in TxT mice in the presence of CD11b+Gr-1+ cells. Surprisingly, MDSCs were not involved in shifting T cells into Th2 cells, characterized by the secretion of cytokines impairing cell-mediated immunity and promoting immunosuppression. Instead, the presence of CD11b+Gr-1+ cells was required for efficient IL-2, IFN-γ and TNFα production after antigenic stimulation, indicating, that elevation of MDSCs early after traumatic injuries might contribute to restrict the initial inflammatory response by alleviating T cell expansion, however, without impeding Th1 functions.

Daunorubicin conjugated with alpha-fetoprotein selectively eliminates myeloid-derived suppressor cells (MDSCs) and inhibits experimental tumor growth.

Failure of antitumor immunity in cancer was shown to be mediated by myeloid-derived suppressor cells (MDSCs), which are considered to be one of the key factors contributing to the development of malignant diseases. Therefore, the development of pharmacological approaches to effectively eliminate MDSCs in organisms carrying growing tumors is a promising pathway for potential treatment. For this purpose we propose alpha-fetoprotein (AFP) conjugated with a cytotoxic agent as a vector molecule, specifically recognizing MDSCs. The present study was aimed at examination of this suggestion using both in vitro and in vivo approaches. MDSCs, obtained from the spleen of Ehrlich carcinoma bearing mice, selectively bound AFP labeled with fluorescein isothiocyanate. AFP conjugated to daunorubicin (AFP-DR) and DR alone showed similar in vitro cytotoxicity against the granulocytic MDSC subpopulation. The monocytic MDSC subpopulation was resistant to treatment with DR, whereas it was completely depleted in the presence of AFP-DR. Treatment of mice bearing Ehrlich carcinoma with AFP-DR resulted in reduced numbers of splenic MDSCs, normalization of NK cell levels, and inhibition of tumor growth. The obtained results demonstrate that cytotoxic conjugates based on AFP are promising anticancer drugs, which, in addition to the direct effect on tumor cells expressing receptors to AFP, may contribute to elimination of MDSCs.

Abstract 243: Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces MDSC accumulation

Abstract Obesity and physical inactivity increase breast cancer risk, while the prevention of weight gain by diet and exercise can be protective. Numerous biological mechanism(s) have been proposed to explain the beneficial effects of weight maintenance; however few studies have examined the immune response to energy balance. We have previously shown that diet and exercise-induced weight maintenance (WM) achieved via a 10% restriction in calories and access to voluntary running wheels in combination with a whole tumor cell vaccine (VAX) significantly reduced mammary tumor growth and metastases in the 4T1 mammary tumor model. This WM-induced reduction in tumor growth occurred concurrently with an elevation in tumor specific IFN-γ production and a reduction in the number of myeloid-derived suppressor cells (MDSCs). However, because tumor size is positively correlated with immune suppression, the goal of the current study was to investigate if mice in the WM+VAX intervention had enhanced anti-tumor immune responses and/or fewer MDSCs controlling for tumor size. Female BALB/c mice were randomized into weight gain (WG) and WM groups (n=8/group) and had access to voluntary running wheels or standard cages, respectively. WG mice were fed ad libitum while WM mice were energy-restricted by 10% to maintain a stable body weight. After 8 weeks on the intervention, all mice were orthotopically injected with 5x104 4T1.2 cells into the fourth mammary fat pad and continued on their intervention. Once injected, both WG and WM mice were further randomized into vaccination (VAX) and vehicle control (VEH) groups (n=4/gr) and administered 1x106 irradiated 4T1.2 cells (VAX) or HBSS (VEH) at day 7 post-tumor implantation. Primary tumor growth was quantified, and mice were sacrificed when tumor volume reached 0.1-0.2 cm3. WM mice weighed significantly less than WG mice over the course of the study (p<0.001). Mice in both WM+VEH and WM+VAX groups took a significantly longer number of days post tumor implantation to reach a tumor size of 0.1-0.2 cm3 (26.3 ± 3.3 d, p=0.003; 23.8 ± 0.5 d, p=0.039, respectively) compared to the WG+VEH group (18.3 ± 2.5 d). Despite the fact that mice were sacrificed when the tumor volumes were standardized at 0.1-0.2 cm3, the combination of WM+VAX resulted in a significant reduction of splenic MDSC accumulation (p=0.049) and enhanced CD4+ T cell proliferation ex vivo. These results suggest that diet and exercise-induced WM was highly effective in delaying primary mammary tumor growth. The combination of WM and an allogenic whole tumor cell vaccine reduced pro-tumorigenic MDSC accumulation and enhanced effector T cell function. Furthermore, these data suggest that diet and exercise may be changing the tumor microenvironment at an early stage of tumor growth favoring tumor clearance. This work is supported by R21 CA209144; T32AI074551. Citation Format: Yitong Xu, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces MDSC accumulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2017-243

Abstract 1260: Diet and exercise-induced weight maintenance may be preventing mammary tumor growth and metastatic burden by enhancing antitumor immunity and/or reducing protumorigenic factors

Abstract Two lifestyle factors that increase cancer risk and progression are weight gain and sedentary behavior. Possible mechanisms underlying this relation include changes in metabolic, inflammatory, and immune mediators. Few studies have examined the effect of body weight and exercise on the efficacy of immunotherapeutic strategies. An emerging immunotherapeutic strategy is PD-1 checkpoint blockade, which selectively targets the membrane protein programmed cell death-1 (PD-1) on T cells to promote a sustained, antitumor effector response. Thus, the goal of the current study was to determine if preventing weight gain through diet (10% reduction in calories) and exercise (voluntary running wheel activity) will improve the response to the dual administration of a whole tumor cell vaccine and PD-1 checkpoint blockade. Female BALB/c mice were randomized to sedentary, weight gain (WG) or exercising, weight maintenance (WM) groups (n=32/group). After 8 weeks, all mice were orthotopically injected with 5x104 luciferase-transfected 4T1.2 cells into the fourth mammary fat pad and continued on their intervention for 35 days. After injection, WG and WM mice were randomized into vaccination (VAX) or vehicle (VEH) groups, and 1x106 irradiated 4T1.2 cells or HBSS vehicle control, respectively, was administered at day 7 post-tumor injection. Mice were further randomized (n=8/group) to receive anti-PD-1 (10 mg/kg/mouse) or isotype control at day 9 and 12 post-tumor injection. All WM groups, regardless of immunotherapy intervention, weighed significantly less than WG groups over the course of the study (p<0.001). Mean tumor volume (p<0.001) and tumor weight at sacrifice (p=0.076) were significantly lower with PD-1 treatment in the WG but not WM groups. Furthermore, metastatic burden in the lung (p=0.032) and the number of splenic myeloid-derived suppressor cells (p=0.058) was reduced with VAX+PD-1 treatment in the WG but not WM groups. Thus, the combination of VAX+PD1 was effective at reducing primary tumor growth and metastatic burden and improving immune outcomes only in mice that gained weight over the course of the study. However, diet and exercise alone was effective in reducing tumor growth and metastatic burden. The lack of responsiveness to vaccination + anti-PD-1 treatment in WM mice suggests that WM achieved through diet and exercise may be enhancing antitumor immunity and/or reducing protumorigenic factors (i.e. similar mechanisms mediated by vaccination + anti-PD-1 therapy). These data demonstrate that preventing weight gain through diet and exercise may be an important recommendation to maintain prolonged antitumor effector responses and improve clinical outcomes. Citation Format: William J. Turbitt, Yitong Xu, Andrea M. Mastro, Connie J. Rogers. Diet and exercise-induced weight maintenance may be preventing mammary tumor growth and metastatic burden by enhancing antitumor immunity and/or reducing protumorigenic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1260. doi:10.1158/1538-7445.AM2017-1260

Dexamethasone Prolongs Cardiac Allograft Survival in a Murine Model Through Myeloid-Derived Suppressor Cells

Abstract Background Recently, myeloid-derived suppressor cells (MDSCs) have attracted considerable attention because of their cancer-promoting and immunosuppressive effects. The glucocorticoid dexamethasone (Dex) is an important immunosuppressive agent used to treat autoimmune diseases and organ transplant rejection. However, the mechanism by which it modulates the immune system is not completely understood. Material and Methods In this study, we investigated the mechanisms by which Dex modulated the immune response in mice given an allogeneic cardiac transplant. Results Dex injection significantly prolonged heart graft survival compared with phosphate-buffered saline–injected controls. Dex treatment increased the number of splenic MDSCs. Moreover, Gr-1high/CD11b+ MDSCs and CD3+/CD4+/Foxp3+ regulatory T cells (Tregs) were significantly increased in the Dex group compared with controls. Administration of anti–Gr-1 antibody (Ab) to the Dex group significantly shortened mouse heart graft survival. In addition, anti–Gr-1 Ab treatment significantly reduced Tregs in the Dex + anti–Gr-1 co-treatment group compared with the Dex group. These observations suggest that Dex treatment increased both MDSCs and Tregs, and that MDSCs regulated the incidence of Tregs in this immunosuppressive pathway. Conclusion An important role of Dex in the prevention of the rejection of cardiac grafts in mice is to expand MDSCs and Tregs.

Differential effects of low-dose fludarabine or 5-fluorouracil on the tumor growth and myeloid derived immunosuppression status of tumor-bearing mice

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and play a key role in the suppression of the innate and adaptive immunity. Chemotherapeutic strategies have been developed to deplete or deactivate MDSCs in different tumor models. The pyrimidine analog, 5-fluorouracil (5-FU) is found to reduce the tumor size by depleting MDSCs. Here, we asked whether the purine analog, fludarabine (Flu), could exert similar effects. Employing a lymphoma model, we demonstrated that in mice with advanced tumors (where MDSC-induced suppression was present), treatment with a single low-dose Flu (25, 50, 100mg/kg) elevated the numbers of splenic MDSCs and serum arginase activity, and simultaneously, increased the tumor growth (only the highest dose). On the other hand, in mice with palpable tumors (where the MDSC-induced suppression was in progress), treatment with Flu had no significant effects on the tumor growth or the number of splenic MDSCs. In contrast to Flu, treatment with low-dose 5-FU, irrespective of tumor stage, caused tumor regression which coincided with significant reductions in the numbers of splenic MDSCs and blood neutrophils, but increases in the ratios of splenic CD4+ T and CD8+ T cells to suppressive MDSCs. Finally, in healthy mice (where MDSC-induced immuosuppression did not exist), 5-FU, but not Flu induced significant decreases in the number of myeloid cells in the bone marrow, naturally occurring splenic MDSCs and thymocytes. In conclusion, Flu exacerbates MDSC-induced immunosuppression in a tumor stage-dependent manner, whereas 5-FU alleviates the suppressive effects of MDSC at all stages of tumor development.

Immunosuppressive myeloid-derived suppressor cells are increased in splenocytes from cancer patients

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that are increased in the peripheral blood of cancer patients and limit productive immune responses against tumors. Immunosuppressive MDSCs are well characterized in murine splenic tissue and are found at higher frequencies in spleens of tumor-bearing mice. However, no studies have yet analyzed these cells in parallel human spleens. We hypothesized that MDSCs would be increased in the spleens of human cancer patients, similar to tumor-bearing mice. We compared the frequency and function of MDSC subsets in dissociated human spleen from 16 patients with benign pancreatic cysts and 26 patients with a variety of cancers. We found that total MDSCs (Linneg CD11bpos CD33pos HLA-DRneg), granulocytic MDSCs (additional markers CD14neg CD15pos), and monocytic MDSCs (CD14pos CD15neg) were identified in human spleen. The monocytic subset was the most prominent in both spleen and peripheral blood and the granulocytic subset was expanded in the spleen relative to matched peripheral blood samples. Importantly, the frequency of CD15pos MDSCs in the spleen was increased in patients with cancer compared to patients with benign pancreatic cysts and was associated with a significantly increased risk of death and decreased overall survival. Finally, MDSCs isolated from the spleen suppressed T cell responses, demonstrating for the first time the functional capacity of human splenic MDSCs. These data suggest that the human spleen is a potential source of large quantities of cells with immunosuppressive function for future characterization and in-depth studies of human MDSCs.

Pak2 Regulates MDSC Development and Function

Myeloid derived suppressor cells (MDSCs) are a heterogeneous cell population at various stages of differentiation that can increase under various pathologic conditions such as cancer, infection or inflammation, displaying suppressive function. It is well recognized that MDSCs contribute to tumor evasion by suppressing cell-mediated immunity. Based on the differential expression of Ly6C and/or Ly6G in mice, MDSCs are characterized as granulocytic (CD11b+Ly6G+Ly6Clow) or monocytic MDSCs (CD11b+Ly6Glow/−Ly6Chi). These subsets induce T-cell hyporesponsiveness and can have various functions and distribution depending on their environment. Although much research has focused on the tumorigenic effects of MDSCs, studies on the regulation of their development during hematopoiesis remain limited.p21-activated kinases (Paks) are serine/threonine kinases that regulate diverse cellular activities including cytoskeletal remodeling, cell motility, proliferation, apoptosis and mitosis. Despite active research on pharmacological inhibition of group I Paks in treating solid tumors, few studies have examined the role of Paks in modulating normal hematopoiesis. Knowledge of the role of Pak2 in regulating long-term hematopoiesis and lineage commitment remains limited. Utilizing a conditional Pak2-KO murine model, we have previously demonstrated that Pak2 disruption in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and CD11b+Gr1+ cell expansion in mice. Compared to mice reconstituted with wild type (WT) bone marrow (BM), mice transplanted with Pak2-KO BM displayed a significantly higher percentage of granulocyte-monocyte progenitors (GMPs) in the BM and higher numbers of CD11b+Gr1+ cells in the spleen.In this study, we demonstrated that CD11b+Gr1high cells isolated from the spleens of mice with Pak2-KO BM displayed significantly greater suppressive function on T cell proliferation in vitro, consistent with MDSC phenotype. There was a near 2-fold increase in the numbers of both granulocytic and monocytic splenic MDSCs in mice reconstituted with Pak2-KO BM. At HSPC level, Pak2-KO BM yielded greater than 3-fold more colonies in response to GM-CSF but not G-CSF or M-CSF when compared to WT cells, indicating selective hypersensitivity to GM-CSF. In parallel experiments, Pak2-KO and WT BM C-kit+ cells that were enriched for hematopoietic progenitor cells (HPCs) were cultured in liquid culture in the presence of GM-CSF. Pak2-KO BM C-kit+ cells yielded greater than 2-fold higher numbers of CD11b+Gr1+ MDSCs that displayed potent suppression on CD8+ T cell proliferation. These data demonstrate that Pak2 disruption increases HPC sensitivity to GM-CSF signaling and drives lineage commitment toward granulocyte-monocyte lineage thus promoting MDSC development. In addition, we have also found that Pak2 deficient MDSCs are more proliferative and more resistant to apoptosis when compared to WT CD11b+Gr1+cells, thus contributing to expansion of this population in vivo. Loss of Pak2 decreases MDSC sensitivity to apoptosis through differential regulation of multiple pro- and anti-apoptotic gene expression. Furthermore, Pak2 disruption down regulates the expression of IRF8, a well-described myeloid transcription factor. Together, our data indicate that loss of Pak2 promotes HPC myeloid lineage commitment and CD11b+Gr1+ MDSC proliferation while suppressing apoptotic cell death in these cells. Further studies are ongoing to determine the interaction between Pak2 and IRF8. DisclosuresNo relevant conflicts of interest to declare.

Abstract A128: Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response

Abstract The success of cancer immunotherapy is limited by multiple mechanisms of tumor-induced immunosuppression often a result of suppressive myeloid cells. M2-like tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC) constitute this immunosuppressive tumor microenvironment (TME) and have repeatedly been associated with poorer prognoses. Therapies designed to overturn this suppressive immune microenvironment could substantially enhance the efficacy of multiple immunotherapeutic approaches. Imprime PGG (Imprime) is a yeast β-1,3/1,6 glucan that has shown compelling efficacy in multiple Phase II trials with tumor-targeting and anti-angiogenic antibodies. As a pathogen associated molecular pattern (PAMP), Imprime activates myeloid cells (monocytes/macrophages, neutrophils, dendritic cells). Our previous ex vivo human and in vivo mouse studies have shown that Imprime promotes repolarization of M2 macrophages to an anti-tumor, M1-like orientation and enhances DC maturation, driving T cell expansion and the production of interferon gamma (IFNγ). We now show that Imprime can modulate the function of immature myeloid cells- the myeloid derived suppressor cells. MDSCs were generated from human cord blood by purifying CD34+ cells and culturing with GMCSF or GCSF ± tumor conditioned media. After 9 days, HLA-DR cells were depleted. The remaining cells were isolated and treated with Imprime or vehicle control. We found that incubating tumor-conditioned, vehicle-treated MDSCs with CD3/CD28-activated CD8 or CD4 T cells inhibited T cell proliferation. Imprime treatment significantly reduced this inhibition. Phenotypically, Imprime treatment elicited substantially increased expression of the co-stimulatory molecules CD80 and CD86 in both monocytic (Mo) and granulocytic (PMN) MDSCs, suggesting that these MDSCs now exhibited APC-like characteristics. In in vivo murine tumor models, Imprime was also able to modulate MDSC phenotype and function, which coincided with enhanced anti-tumor efficacy. Specifically, in the H441 human NSCLC xenograft model, Imprime when combined with 10mg/kg DC101 (murine anti-VEGFR2 Ab), significantly repressed tumor growth compared to the DC-101 alone or vehicle groups. Moreover, spleens from Imprime + DC101 treated-mice had reduced numbers of immunosuppressive, splenic Mo- or PMN-MDSC compared to either DC101 alone or vehicle control. The splenic Mo- or PMN-MDSCs from Imprime + DC101 treated mice that were present expressed significantly higher CD86. The Mo-MDSCs also expressed higher levels of iNOS, a key marker for activated macrophages. In another xenograft NSCLC model, H1299, the splenic MDSC after treatment with Imprime and α-VEGF-A Ab, bevacizumab (bev), showed increased iNOS expression and reduced Arg-1 expression- a shift typifying the M1 polarization state. Collectively, these data show that Imprime treatment can reshape the suppressive immune microenvironment of the tumor and elicit a robust anti-tumor immunity by targeting multiple myeloid lineage cells. Citation Format: Kathryn Fraser, Anissa Chan, Xiohong Qiu, Nadine Ottoson, Adria Bykowski Jonas, Jeremy Graff, Nandita Bose. Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A128.

Suppressive effects of low-dose 5-fluorouracil, busulfan or treosulfan on the expansion of circulatory neutrophils and myeloid derived immunosuppressor cells in tumor-bearing mice

As reported previously, large numbers of neutrophils appear in the circulation during tumor development. However, the relationship between these cells and myeloid-derived suppressor cells (MDSCs), as well as their susceptibility to myelosuppressive drugs have not been yet investigated. Here, we employed a lymphoma model to characterize tumor-associated circulating neutrophils, including their sensitivity to 5-fluorouracil (5-FU), busulfan (Bu) or treosulfan (Treo). Tumor-bearing mice exhibited pronounced elevations in the numbers of splenic MDSCs and circulating neutrophils, MDSCs, and granulocytic-MDSCs (G-MDSCs). Although these cells were not affected by 5-FU at a dose of 17mg/kg, 50 and 100mg/kg were equally effective in causing dramatic tumor regression, normalizing neutrophil number, and significantly reducing the numbers of splenic MDSCs, B cells, and bone marrow myeloid cells. Treatment with Bu (10, 30 or 60mg/kg) only reduced the number of circulating neutrophils, with no effects on these other parameters. At a concentration of 500mg/kg, Treo was ineffective, whereas, doses of 1500 and 3000mg/kg comparably reduced the tumor size as well as the numbers of circulatory neutrophils and bone marrow myeloid cells. Finally, in comparison to 5-FU alone, a combined treatment with low-dose 5-FU and Treo resulted in a more pronounced reduction in the numbers of circulatory neutrophils and bone marrow myeloid cells, together with longer survival time. We conclude that tumor-associated circulatory neutrophils represent blood MDSCs/G-MDSCs that are highly sensitive to 5-FU and Treo, but not Bu. Moreover, the efficacy of 5-FU can be potentiated by Treo.

Abstract 1448: MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer

Abstract Carcinogen-associated oral cavity cancers are a heterogeneous group of aggressive cancers with a high recurrence rate after definitive treatment and a poor 5-year survival. The genetic alteration rate of these cancers tends to be high, and many oral cancers express immune checkpoint molecules in the tumor microenvironment with potential to respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. Here, we investigated the role of myeloid derived suppressor cells (MDSCs) in a highly aggressive but poorly immunogenic syngeneic model of carcinogen-induced oral cavity cancer (MOC2). Performing a time course analysis of immune infiltrates, MOC2 tumors demonstrated robust recruitment of CXCR2+CSFR1+CCR2− MDSCs that peaked at 15 days following tumor transplantation. This intratumoral accumulation of MDSCs preceded draining lymph node and splenic MDSC accumulation by 6 and 9 days respectively. The accumulation of MDSCs corresponded to a sharp decrease in the presence of tumor infiltrating CD8+ T-lymphocytes (TIL), CD4+ TIL, FoxP3+CD4+ Tregs and CD3−NK1.1+ NK cells. As CD8+ TIL numbers decreased, cell surface expression of the lymphocyte activation markers CD69, CD44 and ICOS decreased with the checkpoint molecule CTLA-4 increased. Phenotypically, the great majority of tumor-infiltrating MDSCs were granulocytic MDSCs (Ly6GhiLy6Cint), while few were monocytic MDSCs (Ly6GloLy6Chi). In a CFSE-based functional T-lymphocyte suppression assay, sorted peripheral and tumor-infiltrating MDSCs strongly suppressed T-lymphocyte proliferation at MDSC:T-lymphocyte ratios as low as 1:32. Hypothesizing that decreased tumor MDSCs would impact primary tumor growth, MOC2 tumors were allowed to engraft to 100mm3 and depletion of granulocytic MDSCs was performed with a rat anti-mouse Ly6G depleting antibody (clone RB6-8C5). This depletion resulted in a statistically significant delay in MOC2 primary tumor growth. Flow cytometric analysis of tumor tissues revealed that MDSC depletion was transient with rapid rebound of MDSC tumor infiltration within days of depleting antibody administration. This data highlights the critical role that MDSCs play in local immunosuppression and suggest that the syngeneic MOC model represents a powerful tool to study MDSC pathobiology. Functional inhibition or elimination of MDSCs from the tumor microenvironment represents an exciting adjuvant therapy that may enhance the response to checkpoint inhibition in patients with oral cavity cancer. Citation Format: Paul E. Clavijo, Ruth Davis, Zhong Chen, Carter Van Waes, Clint T. Allen. MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1448.

Dynamics of the spleen and its significance in a murine H22 orthotopic hepatoma model.

The dynamics of the spleen during tumor progression remains incompletely understood. In this study, we established a murine H22 orthotopic hepatoma model and dynamically detected alterations in the percentages of immunocytes in the spleen. We observed a prominent myeloid-derived suppressor cell (MDSC) accumulation during the early response which persisted through all the stages of tumor growth. In addition, the percentage of regulatory T cells (Tregs) increased by week 2. Although the percentage of CD3(+)CD49b(+) natural killer T (NKT) cells increased by day 3, and that of CD3(+)CD4(+) T cells slightly increased by week 1, they decreased to either normal or lower levels compared with those of normal mice. The percentages of total CD3(+), CD3(+)CD4(+), and CD3(+)CD8(+) T cells decreased by week 2, and that of NK cells decreased by week 3. The activation of non-Treg CD4(+) T cells was scarce. Moreover, splenic MDSCs of tumor-bearing mice suppressed the activation of splenocytes. Therefore, a negative immune response gradually prevailed over a positive immune response during tumor growth. In addition, splenectomy was performed at the time of tumor inoculation, and we found that splenectomy could prolong the survival time, reduce the tumor weights, decrease the ascites volumes, and ameliorate the immune status of the tumor-bearing mice. Splenectomy also decreased the percentage of MDSCs and increased the percentages of CD8(+) T cells, NK, and NKT cells in tumor tissues. Additionally, splenectomy decreased the percentage of MDSCs and increased that of CD8(+) T cells in peripheral blood. Overall, our findings suggest that immune-negative cells are dominant in the spleen during tumor progression. Splenectomy could be helpful to improve the immune responses of tumor-bearing hosts.

Abstract A02: Imprime PGG modulates the myeloid component of the tumor microenvironment to coordinate an antitumor immune response

Abstract Imprime PGG (Imprime) is a soluble, yeast-derived 1,3-1,6 β-glucan that acts as a pathogen associated molecular pattern (PAMP), effectively binding to and activating cells of innate immune system to drive a coordinated anti-cancer immune attack. In a randomized phase 2 clinical study, stage IV NSCLC patients treated with Imprime plus the anti-VEGF antibody bevacizumab (bev), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime. We sought to explore a mechanistic understanding for this promising clinical activity. In vitro mechanistic studies using human whole blood from healthy volunteers have demonstrated that Imprime has the potential to alter the immunosuppressive nature of myeloid cells, including macrophages, myeloid-derived suppressor cells (MDSC) and neutrophils. Utilizing whole blood from human volunteers, we found that monocyte-derived M2 macrophages displayed M1-like characteristics, including reduced surface expression of CD163 and increased expression of CD86 and PD-L1. In mixed lymphocyte reactions, these Imprime treated myeloid cells triggered enhanced expansion of both CD4 and CD8 T cells and increased production of the potent anti-tumor cytokine interferon gamma indicating a shift toward Th1 cell polarization. We also found that Imprime treatment significantly reduced the ability of myeloid derived suppressor cells (MDSC) cultured from human cord blood to inhibit T cell proliferation. Furthermore, Imprime treatment enhanced neutrophil activity as evidenced by modulation of activation markers CD11b, CD62L, CD88, as well as enhanced reactive oxygen species production specifically in response to anti-CD20 antibody coated B cell lymphomas. We sought to evaluate whether Imprime treatment would affect the same myeloid subsets in vivo. Athymic nude mice were injected with the H1299 NSCLC cells. Once tumors reached a group mean of ~100mm3, mice were treated with vehicle, bev (5 mg/kg twice weekly IP), Imprime (1.2 mg/ mouse twice weekly IV) or bev+ Imprime. Spleen and tumor tissue were harvested at the end of the study and single cell suspensions were analyzed by flow cytometry, RT-PCR and ELISA. Murine macrophages harvested from spleens of mice treated with Imprime + bev showed a shift, when compared to splenic macrophages from bev-treated mice, to an M1-like phenotype- increased expression of the activation marker CD86 and inducible nitric oxide synthase (iNOS2) in concert with a significantly decreased Arginase-1 (Arg-1). Splenic MDSCs showed a similar increase in iNOS2 expression with significantly reduced Arg-1 expression. Remarkably, CD11b+ cells harvested from the tumor tissue of mice treated with both Imprime and bev showed a similar shift toward an activated phenotype, demonstrating significant increases in iNOS2 and PD-L1 expression, and significantly reduced expression of Arg-1, compared to cells examined from bev group. Tumor associated macrophages and neutrophils from Imprime + bev treated mice showed a significant upregulation of the activation marker CD86 compared to bev treatment alone, indicating a shift in polarization to a M1 and N1 activated phenotype within the tumor microenvironment. Furthermore, ELISA data from these tumors showed that Imprime treatment significantly reduced expression of the potent immunosuppressor, TGFβ, when compared to tumors from mice treated only with bev. Importantly, the greatest reduction of TGFβ was evident in the tumors most profoundly inhibited by Imprime plus bev treatment. Collectively, these data show for the first time that, in vivo, Imprime PGG treatment can activate myeloid cells within the tumor microenvironment to orchestrate a profound shift in this microenvironment that promotes tumor recognition and eradication. Citation Format: Kathryn Fraser, Nadine Ottoson, Xiaohong Qiu, Anissa SH Chan, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose. Imprime PGG modulates the myeloid component of the tumor microenvironment to coordinate an antitumor immune response. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A02.

MUC-1 Regulates MiR34a Expression in Acute Myeloid Leukemia Cells Resulting in an Accumulation of Granulocytic Myeloid-Derived Suppressor Cells

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu that blunts effector cell function and the generation of tumor specific immunity. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive tumor microenvironment that fosters immune tolerance and disease growth. The role of MDSCs in AML and the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. The C-terminus of MUC1 (MUC1-C) forms dimers and translocates to the nucleus where it mediates downstream signaling. The effect of MUC1-C mediated signaling on immune modulation in AML has not been well characterized. In prior studies, we have demonstrated that in vitro expansion of MDSCs is correlated with MUC1 expression by the AML cells. In the present study, we sought to characterize the effect of MUC1 on in vivo recruitment of MDSCs and examine the mechanism by which this is accomplished.The murine AML cell line TIB-49 was transplanted in C57BL/6J mice. Following establishment of disease, the mice were euthanized, along with healthy controls, and bone marrow and spleen CD11b+ Gr1+ MDSCs were quantified by flow cytometry. Engrafted mice had an average MDSC burden of 47% in the marrow and 8.7% in the spleen, compared with 35% and 2% in control mice, respectively. The increase in MDSCs was granulocyte predominant, consistent with our findings in patients with AML. Gr1+CD11b+ cells derived from the marrow and spleens of engrafted mice showed higher levels of Arginase-1 compared to MDSCs from control mice, suggesting an increase in immune suppressive phenotype.To investigate the role of MUC1 on the expansion of MDSCs, expression of MUC-1C, the signaling C-terminus of MUC1, was silenced in TIB-49 cells by stable expression of a MUC1-C shRNA as determined by Western Blot analysis. MUC1 silenced or control vector transduced TIB-49 cells were transplanted into C57BL/6J mice. Following establishment of disease, the mice were euthanized, and bone marrow and spleens were quantified for MDSCs. Control AML engrafted mice had an average splenic MDSC burden 2-fold higher than MUC1 silenced AML engrafted mice (n=4). We have developed a cell-penetrating peptide (GO-203) that disrupts homodimerization of the MUC1-C subunit necessary for its downstream signaling. C57BL/6J mice were challenged with TIB-49 AML cells and after 24 hours were treated daily with GO-203. Mice treated with the MUC1 inhibitor had a 2-fold decrease in splenic MDSCs, compared to control mice (n=3) at time of analysis following disease establishment.Noncoding RNAs have emerged as a critical biologic effector of oncogenic pathways. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. miR-34a has been implicated in regulating the expansion of MDSCs. In the present study we demonstrated that silencing of MUC1 expression via lentiviral transduction with a MUC1 specific shRNA resulted in a significant increase in miR-34a expression, as quantified by q-PCR. Consistent with the MUC1 mediated regulation of MDSC expansion by modulation of miR-34a levels, MDSCs induced by co-culture of healthy donor PBMCs with MUC1 silenced AML cells contained 4-fold higher levels of miR-34a, as compared to controls. AML cells were pre-incubated with SYTO® RNASelect™ Green Fluorescent cell stain. After 4 hours, co-cultures of PBMCs and AML cells were analyzed via flow cytometry. AML cells were excluded and cells positive for MDSC markers and containing green fluorescing exosome dye were quantified. AML derived exosomes were found in 18% of MDSCs expanded from AML cells (n=3), demonstrating exosome trafficking from tumor to MDSCs. Finally, miR-34a was over-expressed in MOLM-14 using lentiviral transduction. Over-expression of miR-34a in MUC1 expressing MOLM-14 cells resulted in a 30% reduction in MDSC expansion in co-cultured PBMCs (n=3).In conclusion, MUC1 regulates MDSC expansion in AML, via its effects on miR34a, acting as a critical mediator of tumor mediated immune suppression. Incorporating strategies to reverse the expansion of MDSCs in AML, potentially by targeting MUC1 and increasing miR-34a expression offers a novel therapeutic approach for cancer immunotherapy. DisclosuresKüfe:Genus Oncology, LLC: Equity Ownership.

The thyroid status reprograms T cell lymphoma growth and modulates immune cell frequencies.

In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8(+) T cells, and higher percentage of CD19(+) B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas. T cell lymphoma phenotype is paradoxically influenced by thyroid status. Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination. Thyroid status affects the distribution of immune cell types in the tumor milieu. Thyroid status also modifies the nature of local and systemic immune responses.

Interleukin-17A deficiency ameliorates streptozotocin-induced diabetes.

Interleukin-17 (IL-17) is a cytokine with critical functions in multiple autoimmune diseases. However, its roles in type I diabetes and the underlying mechanisms remain to be fully elucidated. In the current study, we investigated the impact of IL-17 deficiency on streptozotocin (STZ) -induced diabetes. Il-17(-/-) mice exhibited attenuated hyperglycaemia and insulitis after STZ treatment compared with control mice. The Il-17(-/-) mice had fewer CD8(+) cells infiltrating the pancreas than wild-type controls after STZ injection. Wild-type mice showed increased percentage and number of splenic CD8(+) cells and decreased Gr1(+) CD11b(+) myeloid-derived suppressor cells (MDSC) after STZ treatment, but Il-17(-/-) mice maintained the percentages and numbers of splenic CD8(+) cells and MDSC, suggesting that IL-17 is implicated in STZ-induced cellular immune responses in the spleen. We further purified the MDSC from spleens of STZ-treated mice. Il-17(-/-) MDSC showed increased ability to suppress CD8(+) cell proliferation invitro compared with wild-type MDSC. Transfer of MDSC to diabetic mice showed that MDSC from Il-17(-/-) mice could ameliorate hyperglycaemia. Moreover, recipients with MDSC from Il-17(-/-) mice had a decreased percentage of CD8(+) cell in the spleen compared with recipients with MDSC from wild-type mice. These data suggest that IL-17 is required in splenic MDSC function after STZ delivery. In summary, our study has revealed a pathogenic role of IL-17 in an STZ-induced diabetes model with important implications for our understanding of IL-17 function in autoimmune diseases.

Obesity triggers enhanced MDSC accumulation in murine renal tumors via elevated local production of CCL2.

Obesity is one of the leading risk factors for developing renal cell carcinoma, an immunogenic tumor that is treated clinically with immunostimulatory therapies. Currently, however, the mechanisms linking obesity with renal cancer incidence are unclear. Using a model of diet-induced obesity, we found that obese BALB/c mice with orthotopic renal tumors had increased total frequencies of myeloid-derived suppressor cells (MDSC) in renal tumors and spleens by d14 post-tumor challenge, relative to lean counterparts. Renal tumors from obese mice had elevated concentrations of the known myeloid cell chemoattractant CCL2, which was produced locally by increased percentages of dendritic cells, macrophages, B cells, and CD45- cells in tumors. MDSC expression of the CCL2 receptor, CCR2, was unaltered by obesity but greater percentages of CCR2+ MDSCs were present in renal tumors from obese mice. Of note, the intracellular arginase levels and per-cell suppressive capacities of tumor-infiltrating and splenic MDSCs were unchanged in obese mice relative to lean controls. Thus, our findings suggest that obesity promotes renal tumor progression via development of a robust immunosuppressive environment that is characterized by heightened local and systemic MDSC prevalence. Targeted intervention of the CCL2/CCR2 pathway may facilitate immune-mediated renal tumor clearance in the obese.

The addition of recombinant vaccinia HER2/neu to oncolytic vaccinia-GMCSF given into the tumor microenvironment overcomes MDSC-mediated immune escape and systemic anergy.

Effective immunotherapeutic strategies require the ability to generate a systemic antigen-specific response capable of impacting both primary and metastatic disease. We have built on our oncolytic vaccinia GM-CSF strategy by adding recombinant tumor antigen to increase the response in the tumor microenvironment and systemically. In the present study, orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) was associated with increased Gr1+CD11b+ myeloid derived suppressor cells (MDSCs) both systemically and in the tumor microenvironment. This MDSC population had inhibitory effects on the HER2/neu specific Th1 immune response. VVneu and VVGMCSF are recombinant oncolytic vaccinia viruses that encode HER2/neu and GM-CSF, respectively. Naïve FVB mice vaccinated with combined VVneu and VVGMCSF given systemically developed systemic HER2/neu-specific immunity. NBT1 bearing mice became anergic to systemic immunization with combined VVneu and VVGMCSF. Intratumoral VVGMCSF failed to result in systemic antitumor immunity until combined with intratumoral VVneu. Infection/transfection of the tumor microenvironment with combined VVGMCSF and VVneu resulted in development of systemic tumor-specific immunity, reduction in splenic and tumor MDSC, and therapeutic efficacy against tumor. These studies demonstrate the enhanced efficacy of oncolytic vaccinia virus recombinants encoding combined tumor antigen and GM-CSF in modulating the microenvironment of MDSC-rich tumors.

Increasing glioma-associated monocytes leads to increased intratumoral and systemic myeloid-derived suppressor cells in a murine model.

Patients with glioblastoma multiforme (GBM) exhibit marked intratumoral and systemic immunosuppression. GBM is heavily infiltrated with monocytic cells. Monocytes contacting GBM cells develop features of immunosuppressive myeloid-derived suppressor cells (MDSCs), which are elevated in GBM patients. Therefore, we hypothesized that circulating MDSC levels could be raised in vivo by increasing glioma-associated macrophages. GL261-luciferase glioma was implanted intracranially in C57BL/6 mice with or without additional normal syngeneic CD11b+ monocytes. Tumor growth and intratumoral and systemic MDSC (CD11b+/Gr-1+) levels were determined. Green fluorescent protein (GFP)-transgenic monocytes were coinjected intracranially with GL261-luciferase cells. GFP+ cell frequency among splenic and bone marrow MDSCs was determined. Impact of increased MDSC's on spontaneous immune responses to tumor cells expressing a model antigen (ovalbumin [OVA]) was determined. Tumors grew faster and MDSC's were increased in tumor, spleen, and bone marrow in mice receiving GL261-Luc plus monocytes. Many (30%-50%) systemic MDSC's were GFP+ in mice receiving intracranial tumor plus GFP-transgenic monocytes, suggesting that they originated from glioma-associated monocytes. Tumor-infiltrating OVA-specific CD8+ T cells were markedly reduced in mice receiving GL261-OVA and monocytes compared with mice receiving GL261-OVA alone. Increasing glioma-associated macrophages in intracranial GL261 glioma decreases survival and markedly increases intratumoral and systemic MDSC's, many of which originate directly from glioma-associated macrophages. This is associated with decreased spontaneous immune responses to a model antigen. To our knowledge, this is the first evidence in cancer that systemic MDSC's can arise directly from normal monocytes that have undergone intratumoral immunosuppressive education.