Myeloid-derived Suppressor Cells Cells Research Articles

Targeted STAT3 disruption in myeloid cells alters immunosuppressor cell abundance in a murine model of spontaneous medulloblastoma

Although the immune system may provide early protection against cancer, tumors may exploit the healing arm of the immune system to enhance their growth and metastasis. For example, myeloid derived suppressor cells (MDSCs) are thought to promote tumor growth by several mechanisms, including the suppression of T cell activity. It has been suggested that STAT3 activation in myeloid cells modulates multiple aspects of MDSC physiology, including their expansion and activity. Whereas most animal studies investigating tumor immunology have used tumor implants, we used transgenic mice (Smo*) that spontaneously develop medulloblastoma brain tumors to investigate the temporal accumulation of MDSCs within tumors and how myeloid STAT3 disruption affects MDSC and other immune cell types. We found distinct populations of MDSC in medulloblastoma tumors, with a high prevalence of CD11b(+)Ly6G(+)Ly6C(low/-) cells, described previously by others as G-MDSCs. These were found early in tumor development, in premalignant lesions located on the surface of the cerebellum of 28-day-old mice. In fully developed tumors, pSTAT3 was found in the majority of these cells. Conditional STAT3 gene disruption in myeloid cells resulted in an enhanced proinflammatory phenotype of macrophages in Smo* mice. Moreover, a significant reduction in the abundance of G-MDSCs and Tregs was observed within tumors along with an increased presence of CD4(+) and CD8(+) cells. Despite these alterations in immune cells induced by myeloid STAT3 disruption, we found no effect on tumor incidence in Smo* mice with this deletion.

Myeloid-Derived Suppressor Cells as a Vehicle for Tumor-Specific Oncolytic Viral Therapy

One of the several impediments to effective oncolytic virus therapy of cancer remains a lack of tumor-specific targeting. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells induced by tumor factors in tumor-bearing hosts. The biodistribution kinetics of MDSC and other immune cell types in a murine hepatic colon cancer model was investigated through the use of tracking markers and MRI. MDSCs were superior to other immune cell types in preferential migration to tumors in comparison with other tissues. On the basis of this observation, we engineered a strain of vesicular stomatitis virus (VSV), an oncolytic rhabdovirus that bound MDSCs and used them as a delivery vehicle. Improving VSV-binding efficiency to MDSCs extended the long-term survival of mice bearing metastatic colon tumors compared with systemic administration of wild-type VSV alone. Survival was further extended by multiple injections of the engineered virus without significant toxicity. Notably, direct tumor killing was accentuated by promoting MDSC differentiation towards the classically activated M1-like phenotype. Our results offer a preclinical proof-of-concept for using MDSCs to facilitate and enhance the tumor-killing activity of tumor-targeted oncolytic therapeutics.

Tumor-Derived Lactate Modifies Antitumor Immune Response: Effect on Myeloid-Derived Suppressor Cells and NK Cells

In this study, we explore the hypothesis that enhanced production of lactate by tumor cells, because of high glycolytic activity, results in inhibition of host immune response to tumor cells. Lactate dehydrogenase-A (LDH-A), responsible for conversion of pyruvate to lactate, is highly expressed in tumor cells. Lentiviral vector-mediated LDH-A short hairpin RNA knockdown Pan02 pancreatic cancer cells injected in C57BL/6 mice developed smaller tumors than mice injected with Pan02 cells. A decrease occurred in the frequency of myeloid-derived suppressor cells (MDSCs) in the spleens of mice carrying LDH-A-depleted tumors. NK cells from LDH-A-depleted tumors had improved cytolytic function. Exogenous lactate increased the frequency of MDSCs generated from mouse bone marrow cells with GM-CSF and IL-6 in vitro. Lactate pretreatment of NK cells in vitro inhibited cytolytic function of both human and mouse NK cells. This reduction of NK cytotoxic activity was accompanied by lower expression of perforin and granzyme in NK cells. The expression of NKp46 was decreased in lactate-treated NK cells. These studies strongly suggest that tumor-derived lactate inhibits NK cell function via direct inhibition of cytolytic function as well as indirectly by increasing the numbers of MDSCs that inhibit NK cytotoxicity. Depletion of glucose levels using a ketogenic diet to lower lactate production by glycolytic tumors resulted in smaller tumors, decreased MDSC frequency, and improved antitumor immune response. These studies provide evidence for an immunosuppressive role of tumor-derived lactate in inhibiting innate immune response against developing tumors via regulation of MDSC and NK cell activity.

Inflammation, immune suppression, and tumor progression.

The tumor microenvironment is a complex milieu of tumor and host cells. Host cells can include tumor-reactive T cells capable of killing tumor cells. However, more frequently tumor and host components interact to generate a highly immune suppressive environment that frustrates T cell cytotoxicity and promotes tumor progression through a variety of immune and non-immune mechanisms. Myeloid-derived suppressor cells (MDSC) are a major host component contributing to the immune suppressive environment. MDSC accumulate in most patients and experimental mice with cancer. They inhibit both adaptive and innate immunity through a diverse array of suppressive mechanisms and therefore are a significant obstacle for natural immunity and for active cancer immunotherapies. Their accumulation and suppressive potency are driven by pro-inflammatory mediators. In addition to their inherent immune suppressive function, MDSC amplify the immune suppressive activity of macrophages and dendritic cells via cross-talk which results in the up-regulation of inflammatory mediators. Cross-talk between MDSC and other myeloid cells is itself enhanced by inflammation, resulting in an autocrine tumor microenvironment that sustains and amplifies immune suppression. This talk will describe the cell-cell interactions used by MDSC to inhibit anti-tumor immunity and promote tumor progression, and the role of inflammation in promoting cross-talk between MDSC and other cells in the tumor microenvironment.

Surveillance on the Status of Immune Cells after Echinnococcus granulosus Protoscoleces Infection in Balb/c Mice

BackgroundCystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly.Methodology/Principal FindingsIn this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b+ and CD11c+ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1+ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b+/GR-1+. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4+ and CD8+ T cells following infection. Regulatory T cells expressing CD4+/CD25+/FoxP3 + increased significantly over the course of infection.ConclusionsOur findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.

Granulocytic myeloid-derived suppressor cells are cryosensitive and their frequency does not correlate with serum concentrations of colony-stimulating factors in head and neck cancer

Granulocytic myeloid-derived suppressor cells (MDSC) are a MDSC subset expanded in various cancer types. As many clinical studies rely on the use of stored collections of frozen blood samples, we first tested the influence of freezing/thawing procedures on immunophenotyping and enumeration of granulocytic MDSC (G-MDSC). To identify factors involved in expansion of human G-MDSC, we then analyzed correlations between G-MDSC frequencies, clinical parameters and granulocyte-related factors in the peripheral blood of head and neck cancer patients. HLA-DR, CD14, CD33 and CD66b allowed a clear discrimination of G-MDSC from monocytic MDSC and immature myeloid cells. MDSC subsets were sensitive to cryopreservation with immature G-MDSC showing the highest sensitivity. G-MDSC frequencies were increased in advanced disease stage and associated with the level of CCL4 and CXCL8, but not with colony-stimulating factors, IL-6, S100A8/9, CXCL1 and other cytokines. Our results indicate that the frequency of MDSC, in particular G-MDSC, may be underestimated in retrospective clinical analyses using frozen blood samples. Increased G-MDSC frequencies correlate with advanced disease and increased concentrations of CXCL8, but, unexpectedly, not with growth factors (such as granulocyte colony-stimulating factor), IL-6 and CXCL1. Our data suggest that CXCL8 promotes accumulation of G-MDSC in cancer patients independent of classical colony-stimulating factors.

Liver Stromal Cells Contribute to Liver Transplant Tolerance: Involved Cellular and Molecular Mechanisms

Organ transplantation is currently dependent on life-time administration of immunosuppressive drugs to non-specifically suppress immune response, resulting in severe side effects, including infection and cancer. Induction of specific tolerance is an ideal approach, but not generally successful, which however, actually naturally occurs. Thus, ˜1/4 liver allografts in farm pigs are spontaneously accepted, and induce donor specific tolerance. In humans, immunosuppression can be weaned off until completely stopped in ˜1/4 liver transplant patients. The underlying mechanisms are not completely understood. We and others surprisingly found that liver transplant tolerance is dependent on IFN-g stimulation. Mouse liver allograft, otherwise spontaneously accepted, was acutely rejected in IFN-g-/- recipients. In this study, the liver from IFN-gR1-/- mice (B6, H-2b) was transplanted into C3H (H-2k) recipients (n=6). None of them survived >15 days (WT grafts survived >100 days), suggesting that liver transplant tolerance requires intact IFN-g signaling pathway in liver graft. Both flow and histochemical analyses demonstrated markedly increased CD8+ infiltrates in IFN-gR1-/- grafts associated with lower expression of B7-H1 and less infiltrating T cell apoptosis, suggesting a role of B7-H1, an end product of IFN-g signaling in regulating immune response. This was supported by the result that the liver grafts from B7-H1-/- mice were acutely rejected in WT allogeneic recipients, associated with markedly less apoptosis (annexin V) in the graft infiltrating T cells. Liver grafts deficient in IFN-gR1 did not affect much T cell activation (CD44hiCD62Llo), as well as generation of myeloid-derived suppressor cells (MDSC) and Tregs in early phase. However, Tregs were markedly enhanced in long-term survived WT grafts, suggesting that elimination of effectors in early phase is crucial, while the suppressor cells may largely work to maintain tolerance. To identify the cell components in the liver graft responsible for these changes, the graft non-parenchymal cells were isolated at various time points for flow analysis. Almost all graft CD45+ cells were quickly (in one-two days) become recipient origin (H-2k, IFN-gR1+), while CD45- cells remain donor type (H-2b, IFN-gR1-), even in long-term survived grafts, suggesting that liver non-hematopoietic cells may be responsible for these changes. We have studied isolated hepatic stellate cells (HSC) (liver stromal cells, well known for their storing vitamin A and participating in fibrosis) that were exceptionally immunosuppressive. Cotransplantation with HSC effectively protected islet allografts without requirement of immunosuppression. Interestingly, cotransplantation with HSC isolated from IFN-gR1- mice failed to protect islet grafts, associated with markedly more infiltrating CD8+ T cells and less MDSC and Treg cells, indicating that the immune regulatory activity of HSC depends on intact IFN-g signaling pathway. Expression of B7-H1 was upregulated following exposure to IFN-g in a dose dependent manner, suggesting B7-H1 is an end product of IFN-g signaling. HSC isolated from B7-H1-/- mice largely lost their ability to protect islet allgrafts. This was associated with more CD8 T cells graft infiltration. In conclusion, IFN-g/B7-H1 signaling pathway in liver stromal cells play a critical role in establishment of liver transplant tolerance.

Mast cells activate MDSC to promote inflammation (177.2)

Abstract Mast cells play an important role in promoting inflammation, controlling parasitic infection, and potentiating allergic disease. Conversely, myeloid-derived suppressor cells (MDSC) are found in tumor-bearing individuals and in models of septic shock, where they are known to have regulatory effect. We aimed to determine a possible role for interactions between MDSC and mast cells and the effect of these interactions in a mouse model of parasitic infection. Surprisingly, co-culture of these cells resulted in a drastic increase in pro-inflammatory mediator release, which was attributed entirely to MDSC. Depletion of MDSC from mice infected with Nippostrongylus brasiliensis resulted in delayed worm expulsion and a drastically increased intestinal worm burden. Conversely, this inflammatory role of MDSC seems detrimental when inflammation is chronic, as MDSC depletion in an airway hyperresponsiveness model decreased the pathology of this disease. Interestingly, we have found that mast cell conditioned-media activates both the STAT3 and ERK pathways in MDSC, which could represent a novel means of pro-inflammatory cytokine induction in MDSC. We therefore conclude that mast cells can reprogram MDSC to a pro-inflammatory phenotype, suggesting that the evolutionary purpose of these suppressive cells may be to potentiate inflammatory Th2 responses necessary for parasite expulsion.

Abstract LB-495: Myeloid-derived suppressor cells promote cancer-associated fibroblast migration and carcinoma cell invasion

Abstract Metastasis the most devastating part of cancer, yet we know very little about the earliest steps of metastasis. Myeloid-Derived Suppressor Cells (MDSC) are CD11b+, Gr-1+ bone marrow-derived cells that have been shown to promote tumor development by suppressing T cells. In addition to immune suppression, MDSC can promote metastasis. MDSC localize to the invasive edges of primary tumors and promote breast cancer lung metastasis. Localized invasion of cancer cells is the earliest step of metastasis and fibroblasts are required for breast cancer cell invasion. Fibroblasts remodel the ECM and leave tracks for collective migration of carcinoma cells. These studies seek to identify the mechanism of MDSC promotion of metastasis by determining if MDSC can stimulate fibroblast migration and lead localized carcinoma invasion. Freshly isolated MDSC from a mammary tumor-bearing mouse increase fibroblast migration. TGFβRII-/- fibroblasts demonstrate a further increase in migration in response to MDSC, suggesting that TGFβ signaling is not required for MDSC promotion of fibroblast migration. MDSC secretions act directly on fibroblasts since conditioned medium from MDSC increases fibroblast migration to the same extent as live MDSC. Antibody array analysis identified several MDSC secreted proteins, including CXCL11, CXCL15, FGF2, IGF-I, IL-4 and Shh. MDSC increase carcinoma cell invasion through Matrigel, but only when fibroblasts are present. MDSC treatment of fibroblasts stimulates Akt and ERK1/2 phosphorylation and loss of secreted proteins, including SDF-1, TIMP-1, TNFα and VEGF. Analysis of subsets of MDSC identified that monocytic MDSC (CD11b+, Ly6Chi, Ly6G-) increase fibroblast migration more than other myeloid cell populations. Tumor-derived MDSC promote fibroblast migration more that splenic MDSC. These studies show that MDSC can lead fibroblasts to stimulate carcinoma invasion and suggest that perivascular MDSC may be the earliest stimulus for localized invasion during metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-495. doi:1538-7445.AM2012-LB-495

Vasoactive intestinal peptide induces CD14+HLA-DR‑/low myeloid-derived suppressor cells in gastric cancer.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of cells, which have been revealed to inhibit T-cell responses in tumor-bearing mice. In addition, a number of immune suppressive mechanisms have linked MDSCs and the development of human cancer. However, the role of MDSCs in human gastric cancer tissue remains to be elucidated as specific markers are lacking. Therefore, the aim of the present study was to investigate the frequency and immune suppressive function of MDSCs denoted in the present study as cluster of differentiation 14 (CD14)+human leukocyte antigen (HLA)-DR-/low in gastric cancer patients. In the present study, MDSCs were directly isolated and characterized from the tumor and adjacent normal tissue of gastric cancer patients. Functional analysis of the CD14+HLA-DR-/low MDSCs co-cultured with allogeneic CD4+ T cells were performed and compared with controls. In addition, the interferon-γ (IFN-γ) and interleukin (IL)-2 production was compared in order to investigate the capacity of vasoactive intestinal peptide (VIP) to induce CD14+HLA-DR(-/low) MDSC-mediated CD4+ T-cell dysfunction and whether IL-10 secretion is involved in this mechanism. As a result, the quantity of CD14+HLA-DR(-/low) cells in tumor tissue from gastric cancer patients was significantly higher than that in the adjacent normal tissue. In addition, CD14+HLA-DR-/low MDSCs isolated from tumor tissue were observed to inhibit the CD4+ T-cells' immune responses in comparison with those from the adjacent normal tissue. Furthermore, VIP was able to induce the differentiation of CD14+ mononuclear cells isolated from healthy donor peripheral blood mononuclear cells into activated MDSC cells. Of note, the immunosuppressive effect of VIP-induced CD14+HLA-DR(-/low) MDSCs on CD4+ T cells was mediated by IL-10 secretion, which was demonstrated in the subsequent decrease of IFN-γ and IL-2 production. In conclusion, CD14+HLA-DR(-/low) cells were significantly increased in gastric cancer tissue and were shown to have a critical role in CD4+T-cell immunosuppression. In addition, VIP as a novel cytokine may induce the differentiation of CD14+ mononuclear cells towards CD14+HLA-DR(-/low) MDSCs. An improved understanding of phenotypic heterogeneity and the mechanism of generation of MDSCs in gastric cancer patients is important in the design of effective immunotherapeutic strategies.

Microenvironment Induced Myelodysplastic Syndrome (MDS) in S100A9 Transgenic Mice Caused by Myeloid-Derived Suppressor Cells (MDSC)

Abstract 788Understanding the pathophysiology of myelodysplastic syndrome (MDS) is limited by a complex molecular mechanism and lack of an adequate animal model that recapitulates the role of inflammation in the abnormal hematopoiesis. We recently showed that patients with MDS have expansion of inflammation-related hematopoietic suppressive cells called immature myeloid-derived suppressor cells (MDSC) that display direct cytotoxic and suppressive effects on autologous hematopoietic progenitor cells (HPCs). Expansion of bone marrow (BM) MDSCs contributed to the production of inflammatory cytokines and reduced HPC survival underlying BM failure in lower risk patients. Here we provide evidence that MDSC activation, expansion and development is driven by overexpression of inflammatory-related signaling molecules, myeloid-related protein 8 (MRP8, encoded by S100A8) and MRP14 (encoded by S100A9). Both MRP proteins serve as the native endogenous ligands for Toll-like receptor 4 (TLR4), which is an important damage-associated molecular pattern (DAMP) mediating inflammatory response. We found higher expression of MRP8 and MRP14 in BM mononuclear cells from MDS patients compared to healthy donors, in whom these proteins were not detectable. High surface expression of both TLR2 and TLR4 in MDS MDSCs compared to healthy donor MDSCs confirmed that this signaling pathway is activated in MDS. Inhibition of MRP8/MRP14 proteins in MDSCs using specific shRNAs dramatically attenuated IL-10 and TGF-β production and rescued BFU-E and CFU-GM colony formation of autologous bone marrow progenitors. These data show that inflammation-associated MRP8/MRP14 expression plays a critical role in the suppressive activities of MDS MDSCs. We therefore generated S100A9 transgenic mice (S100A9Tg) overexpressing the murine MRP14 homologue and investigated the role of this protein in bone marrow failure. Significant MDSC accumulation was evident in the BM of S100A9Tg mice by 6 weeks, but not in S100 knockout (KO) or wild type (WT) mice. Similar to human MDS, MDSCs from S100A9Tg mice, but not S100KO or WT mice, significantly inhibited BFU-E colony formation. Depletion of MDSCs in vitro rescued BM colony formation in the S100A9Tg mice indicating that the BM suppression is mediated by MDSC cells. TGF-β and IL-10 secretion was significantly increased in S100A9Tg mice, substantiating the role of S100A9 as an essential inflammatory factor that regulating MDSC suppressive activity. Analogous to human MDS, 6-month old S100A9Tg mice developed ineffective hematopoiesis with severe anemia, leukopenia, and thrombocytopenia accompanied by MDS-like morphological features. BM aspirates and core biopsies from S100A9Tg mice were hypercellular with trilineage cytological dysplasia characteristic of MDS. Treatment with ATRA, which induced the differentiation of MDSCs rescued hematopoiesis in S100A9Tg mice. Our findings indicate that primary BM expansion of MDSC is sufficient to perturb hematopoiesis and result in the development of MDS, supporting the notion of microenvironment-conducive oncogenesis. S100A9Tg transgenic mice provide a novel in vivo model of human MDS for target discovery and testing of novel therapeutics. Disclosures:No relevant conflicts of interest to declare.

Immunomodulatory Effects of Histone Deacetylase 6 Inhibition in Suppressor Immune Cells in Multiple Myeloma

Abstract 128 The interaction of myeloma (MM) cells with bone marrow accessory cells and/or the extracellular matrix induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most efficient anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC), regulatory T cells (Treg) and IL-17 secreting Th (Th17) cells act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. Since, we and others have reported increased numbers of Treg and Th17 cells in MM, we here assessed MDSCs in both peripheral blood (PBMC) and bone marrow (BMMC) of patients with MM compared to healthy donors. Phenotypic analysis by flow cytometry showed a significant increase in CD14−CD11b+HLA-DRlowCD15+ MDSCs in both PBMC and BMMC from MM patients compared to healthy donors (p Disclosures: Hideshima:Acetylon: Consultancy. Raje:Celgene: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees; Millenium: Membership on an entity9s Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity9s Board of Directors or advisory committees; Acetylon: Research Funding. Bradner:Acetylon: Scientific Founder. Richardson:Bristol Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees; Millennium: Membership on an entity9s Board of Directors or advisory committees. Munshi:Celgene: Membership on an entity9s Board of Directors or advisory committees; Millennium: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees; Onyx: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Anderson:Novartis: Membership on an entity9s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Merck: Membership on an entity9s Board of Directors or advisory committees; Onyx: Membership on an entity9s Board of Directors or advisory committees; Millennium: Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees; Acetylon: Membership on an entity9s Board of Directors or advisory committees.

Abstract LB-159: Bisphosphonates are internalised by myeloid derived suppressor cells (MDSCs) in vivo in a mouse model of breast cancer

Abstract Zoledronic acid (Zometa; ZOL), a nitrogen-containing bisphosphonate (N-BP), has become the standard treatment for metastatic bone disease due to its ability to inhibit osteoclast-mediated resorption. The molecular target of ZOL in osteoclasts is FPP synthase, an enzyme of the mevalonate pathway. Inhibition of this enzyme prevents the synthesis of isoprenoid lipids necessary for the prenylation of small GTPases such as Rho, Rac and Rap, thereby causing the accumulation of the unprenylated form of these proteins and fundamentally affecting cell function and survival. ZOL and other N-BPs have anti-tumour effects in some preclinical models of skeletal and non-skeletal tumours, and, more recently, have tantalising effects on patient survival in some clinical trials. However, the exact mechanisms responsible for these anti-tumour effects in vivo are still unknown, and the extent to which N-BPs are internalised by cells other than osteoclasts (such as immune cells) is unclear. We have sought to address this question in the 4T1 murine breast cancer model using a novel, fluorescently-labeled N-BP (AF647-RIS). 24 hours after a subcutaneous injection, using flow cytometry we have demonstrated for the first time the cellular uptake of this N-BP by Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs), as well as by CD14+F4/80+ monocytes/macrophages (TAMs) in mammary tumours, whole blood, liver, spleen and bone marrow. Uptake of N-BP was not detectable in the tumour cells or other cell types. Furthermore, 7 days after a single injection of a clinically relevant dose of ZOL into tumour-bearing mice, we detected a significant reduction in MDSC number in the spleen. It remains unknown whether ZOL affects protein prenylation in MDSCs or TAMs in vivo. Currently available methods to detect changes in protein prenylation, using western blotting to detect unprenylated small GTPases, are too insensitive to detect subtle effects of N-BPs on prenylation in cells in vivo. Importantly, we have now optimised a new, highly sensitive enzyme-based approach to detect the accumulation of unprenylated proteins in cell and tissue extracts from N-BP-treated animals. Additional studies are now in the very final stages to determine conclusively whether a clinically-relevant dose of ZOL inhibits protein prenylation in MDSCs and TAMs in 4T1 mice. In summary, we provide the first conclusive evidence that N-BPs can be internalized in vivo by MDSC and other myeloid cells such as TAMs. Given the important role of these cells in the progression of tumour growth and metastasis, our studies will finally clarify the mechanism underlying the preclinical, and potentially clinical, anti-tumour activity of these agents. 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 LB-159. doi:10.1158/1538-7445.AM2011-LB-159

Natriuretic peptide receptor A knock out restricts tumorigenesis through ablation of myeloid derived suppressor cells expansion (66.3)

Abstract Recent data from our group and a number of others have demonstrated that expansion and activation of Myeloid Derived Suppressor Cells(MDSC) play a pivotal role in tumorigenesis. The receptor for atrial natriuretic peptide, natriuretic peptide receptor A (NPRA) has been implicated in cancers. We have shown that NPRA expression and signaling is important for tumor growth. We hypothesize a novel link between NPRA expression, altered differentiation of myeloid cells and tumor formation. MDSC population consists of two major subsets of Ly6G+ granulocytic and Ly6C+ monocytic cells. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. MDSC isolated from tumor bearing mice expressed high levels of NPRA. Next we hypothesized that NPRA knock out would result in restricted tumor growth. Using NPRA deficient mice, we report that these mice have altered differentiation of myeloid cells. As a consequence tumor cells injected subcutaneously to NPRA KO mice failed to grow. We investigated the nature of cells responsible for tumorigenesis and proved that ablation of a subset of MDSC cells restricts tumor formation. This work may indicate key roles for NPRA in myeloid cell differentiation and cancer formation.

Small Molecule SHIP Agonist AQX-MN100 Inhibits Multiple Myeloma (MM) Cell Growth In NOD/SCID Mice and Reduces Immune Suppressive Myeloid Derived Suppressor Cells (MDSC) and Regulatory T Cells (Tregs) In Mouse Model

AbstractAbstract 792We recently described a novel anti-MM drug (AQX-MN100) which is a small molecule agonist of SHIP (Src homology-2 (SH2) containing inositol-5¢-phosphatase) a signaling molecule found only in hemopoietic cells.(Ong et al, Blood; 110:1942, 2007) The molecule was developed using a high-throughput SHIP enzyme assay to screen an invertebrate marine natural product library and isolate the Pelorol.(Yang et al Org Lett; 7:1073, 2005) SHIP normally functions to negatively regulate the PI3K pathway important to normal hemopoietic cells growth and function. Inappropriate activation of the phosphoinositide 3- kinase (PI3K) pathway has been shown to be involved in the pathogenesis of MM and tumour aggressiveness correlates with the degree of activation. The critical role the PI3K/Akt signaling pathway plays in regulating MM cell survival, has stimulated efforts in designing therapeutics that target this pathway. Pan PI3K inhibitors have limited utility in a clinical setting because of their inhibitory effects on all isoforms of the PI3K family as well as non-PI3K targets. SHIP is an exceptionally good target for MM and other hematopoietic disorders that display elevated PI3K/Akt signaling because its expression is restricted to hemopoietic cells. We have shown that an analogue of Pelorol, AQX-MN100 is able to inhibit PI3K signaling and prevent phosphorylation of Akt. AQX-MN100 induced MM cell line apoptosis mediated by caspase and was specific for SHIP expressing cells which are exclusively hematopoietic. AQX-MN100 also enhances the growth inhibition effects of current myeloma drugs Dexamethasone and Bortezomib on human MM tumour cell lines in vitro. (Kennah et al Expt Hematol; 37:1274, 2009)In this study we have extended these finding to further evaluate the role of this compound in the treatment of myeloma. NOD-SCID mice were injected in the lateral flanks with 2 million luciferase tagged MM1.S multiple myeloma cells in Matrigel. Tumors were allowed to establish for two weeks and then either AQX-MN100 or vehicle was administered in an oil deposit subcutaneously in the lower flank at a dose of 50 mg/kg every three days. Tumor volume was quantified by imaging on a Xenogen IVIS 200 after 6 and 11 days. These studies demonstrate a significant reduction of tumor volume at 6 days p<0.05 and a highly significant reduction at 11 days p<0.01 in the mice receiving AQX-MN100 as compared to vehicle.We have shown that AQX-MN100 can directly kill MM cells in in vitro and in vivo. However, based on the known functions of SHIP, we predict that SHIP agonists will additionally target critical steps in MM pathogenesis in vivo, including the ability of MM cells to interact with stromal elements and to subvert the immune system. In order to evaluate this later feature we evaluated the ability of SHIP agonists to reverse the tumor associated immune suppression in MM patients. Tumor and host cell/tumor microenvironment secreted factors promote the production and activation of cells associated with cancer progression: the immune suppressive myeloid derived suppressor cells (MDSC) and regulatory T cells (Tregs). These cells normally regulate immune responses by inhibiting the activation of immune effector cells. The involvement of SHIP in the regulation of these cells is predicted by the observation that MDSC and Treg numbers are elevated in SHIP deficient mice. In this study Balb/C mice, 6 mice/group in duplicate were given either AQX-MN100 3 mg/kg and 10 mg/kg or vehicle once daily orally. At the end of three weeks mesenteric lymph nodes were harvested and subjected to FACS analysis to determine the proportion of MDSC (CD11b+Gr1+) and Treg (CD4+CD25+FoxP3+) cells. Spleen cells were also analysed for B cells, NK cells and granulocytes. In both of the AQX-MN100 treated groups the numbers of MDSC and Tregs were significantly lower than controls while Total CD11b, Total CD3, and spleen B, NK and granulocytes were not different from vehicle treated controls.The known role of SHIP in regulating hemopoietic cell function and the role of SHIP agonists in MM cell killing as well as additional actions on other aspects of MM pathophysiology may make them a powerful treatment option for MM, either alone or in synergy with other known MM therapies. Further development of this agent for the treatment of MM is ongoing. Disclosures:Mui:Aquinox: Equity Ownership, Patents & Royalties. Ong:Aquinox: Equity Ownership, Patents & Royalties. Anderson:Aquinox: Equity Ownership, Patents & Royalties. Sutherland:Celgene: Honoraria; Orthobiotech: Honoraria.

The Effect of Rhg-CSF on MDSC In Bone Marrow and Peripheral Blood Cells

AbstractAbstract 4709 Objective:As granulocyte colony-stimulating factor, recombinant human granulocyte colony stimulating factor (rhG-CSF) is widely used in neutropenic patients. In addition to stimulating the growth of granulocyte, rhG-CSF can promote hematopoietic stem cells from bone marrow (BM) to peripheral blood (PB) and has the effect of immune regulation. Myeloid-derived suppressor cells (MDSC) are a group of heterogeneous cells, derived from bone marrow progenitor cells and immature myeloid cells. Recently, MDSC is researched in the field of solid tumor, but not in the field of hematopoietic stem cell transplantation. Here, we investigate rhG-CSF's effect on MDSC in healthy donors' BM, PB and the relationship between MDSC and graft-verse-host disease (GVHD). Methods:We obtained the BM and PB samples before mobilization and the BM APB and peripheral blood stem cell collection (PBSC) on the 5th day after the rhG-CSF mobilization from 12 healthy donors, respectively. Then we used the flow cytometry to check the absolute number of MDSC. Finally, we analyzed the relationship between the number of MDSC and the incidence of GVHD. Results:In normal physiological conditions, the MDSC could be detected in healthy donor's PB and BM. In PB, the proportion of MDSC in the mononuclear cells was 1.35 ± 0.35%. In BM, the proportion was 2.44 ± 1.11%. The proportion in BM is higher than that in PB, the difference was statistically significant (P=0.047). On the 5th day after the rhG-CSF mobilization, the MDSC ratio of mononuclear cell in PB were 4.01 ± 1.82%. In BM, the ratio was 4.38 ± 2.19%. The difference between the ratio of MDSC in BM and PB was no significant (P=0.076). The number of mobilized peripheral blood MDSC was significantly higher than that before mobilization (P=0.015), while the difference between the numbers of bone marrow MDSC cells before and after mobilization was not significant (P=0.083). The numbers of MDSC in collection and the incidence of GVHD had a significant negative correlation (P=0.048). Conclusion:MDSC could be detected in the healthy donors' PB and BM, the numbers of MDSC in BM were higher than that in PB. The rhG-CSF could mobilize more MDSC from BM to the peripheral blood, and the increased s of MDSC in PB after rhG-CSF mobilization might be related to the low incidence of GVHD in hematopoietic stem cell transplantation.Supported by National Natural Science Foundation of China (30971300), Science and Technology Planning Project of Guangdong Province of China (2009A030200007) and China Postdoctoral Science Foundation (200902332, 20080440776). Disclosures:No relevant conflicts of interest to declare.

Abstract 5307: Indomethacin reverts tumor mediated immune suppression in a murine lung adenocarcinoma

Abstract Tumor-induced immune suppression is widespread in patients and experimental animals with malignant tumors and is likely to be a significant impediment to immunotherapy. Multiple mechanisms are thought to facilitate this suppression state, being Myeloid-Derived Suppressor Cells (MDSC) and T regulatory cells (Tregs) major contributors. Our aim was to analyze immune parameters indicative of immune suppression in BALB/C mice bearing LP07 (COX+) lung tumor (TBM) during tumor growth and the effect of a Non Steroidal Anti-inflammatory drug (NSAID), Indomethacin, which is a non-specific COX inhibitor, on these parameters. We determined 1) the effect of Indomethacin on LP07 tumor growth; 2) the content of MDSC and Treg cells in lymphoid organs (spleen, tumor-draining lymph nodes-TDLN-) and in the tumors at different times by FACS; 3) arginase activity in lung, spleen, liver and tumor measured as µg Urea/mg protein; 4) specific Delayed Type Hypersensitivity (DTH) response with formalinized LP07 cells, by the foot pad swelling assay. Results: Indomethacin treatment significantly inhibited tumor growth. The percentage of MDSC (CD11b+ Gr1+) augmented significantly in the spleen of TBM in advanced stages of tumor progression (33 days): Normal: 2,6%; TBM 10 days: 2,4%; TBM 20 days: 5,6%; TBM 33 days: 8,3%, p&amp;lt;0,05 vs control. This rise was prevented by Indomethacin treatment (4,6%). Intratumoral and TDLN CD4+CD25+Foxp3+ Tregs frequency augmented during tumor growth, peaking at day 20. Interestingly, Indomethacin treatment decreased Treg cell and MDSC induction (p&amp;lt;0.05) in TDLN. Arginase activity increased in all analyzed tissues of TBM compared to those of control mice. Furthermore arginase activity increase in the spleen was in line with MDSC induction. Specific DTH response decreased in late stages of tumor progression, while Indomethacin treatment reversed this inhibition (TBM: 0,06 ± 0,045 mm; Indo:0,14 ± 0,038 mm). Conclusion: In our model, the increment in MDSC and Tregs frequency during tumor progression correlates with specific immunesuppression demonstrated by DTH responses and increased arginase activity. We suggest that Indomethacin treatment prevented this immunesuppression by modulating COX activity in the tumor microenvironment, which is known to activate MDSC and differentiate Treg cells. 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 5307.

The Novel Role of Tyrosine Kinase Inhibitor in the Reversal of Immune Suppression and Modulation of Tumor Microenvironment for Immune-Based Cancer Therapies

In tumor-bearing hosts, myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) play important roles in immune suppression, the reversal of which is vitally important for the success of immune therapy. We have shown that ckit ligand is required for MDSC accumulation and Treg development. We hypothesized that sunitinib malate, a receptor tyrosine kinase inhibitor, could reverse MDSC-mediated immune suppression and modulate the tumor microenvironment, thereby improving the efficacy of immune-based therapies. Treatment with sunitinib decreased the number of MDSC and Treg in advanced tumor-bearing animals. Furthermore, it not only reduced the suppressive function of MDSCs but also prevented tumor-specific T-cell anergy and Treg development. Interestingly, sunitinib treatment resulted in reduced expression of interleukin (IL)-10, transforming growth factor-beta, and Foxp3 but enhanced expression of Th1 cytokine IFN-gamma and increased CTL responses in isolated tumor-infiltrating leukocytes. A significantly higher percentage and infiltration of CD8 and CD4 cells was detected in tumors of sunitinib-treated mice when compared with control-treated mice. More importantly, the expression of negative costimulatory molecules CTLA4 and PD-1 in both CD4 and CD8 T cells, and PDL-1 expression on MDSC and plasmacytoid dendritic cells, was also significantly decreased by sunitinib treatment. Finally, sunitinib in combination with our immune therapy protocol (IL-12 and 4-1BB activation) significantly improves the long-term survival rate of large tumor-bearing mice. These data suggest that sunitinib can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy for advanced malignancies.

γδT cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation pathway

Maternal immune system must tolerate semiallogenic fetus to establish and maintain a successful pregnancy. Despite the existence of several strategies of trophoblast to avoid recognition by maternal leukocytes, maternal immune system may react against paternal alloantigenes. Leukocytes are important components in decidua. Not only T helper (Th)1/Th2 balance, but also regulatory T (Treg) cells play an important role in pregnancy. Although the frequency of Tregs is elevated during normal pregnancies, their frequency and function are reduced in reproductive defects such as recurrent miscarriage and preeclampsia. Tregs are not the sole population of suppressive cells in the decidua. It has recently been shown that regulatory B10 (Breg) cells participate in pregnancy through secretion of IL-10 cytokine. Myeloid derived suppressor cells (MDSCs) are immature developing precursors of innate myeloid cells that are increased in pregnant women, implying their possible function in pregnancy. Natural killer T (NKT) cells are also detected in mouse and human decidua. They can also affect the fetomaternal tolerance. In this review, we will discuss on the role of different immune regulatory cells including Treg, γd T cell, Breg, MDSC, and NKT cells in pregnancy outcome.