Function Of Myeloid-derived Cells Research Articles

Hypoxia-inducible factor controls immunoregulatory properties of myeloid cells in mouse cardiac allografts - an experimental study.

Hypoxia-inducible factors (HIFs) play a critical role in inflammatory properties of myeloid-derived cells. The effect of HIFs on myeloid-derived cell functions in organ transplantation remains unknown, however. We transplanted hearts into transgenic mice with myeloid cell-targeted deletions of HIF-1α or its negative regulator von Hippel-Lindau (VHL) to investigate the effects of HIF-1α inactivation or HIF pathway activation, respectively, on ischemia-reperfusion injury (IRI) and acute rejection. Deletion of VHL in myeloid cells enhanced mRNA expression of anti-inflammatory genes IDO, Arg-1, and HO-1 in vitro. In vivo, VHL-/- myeloid-derived cells of allograft recipients alleviated IRI and acute rejection, evidenced by reduced cardiomyocyte damage, decreased proinflammatory cytokine mRNA levels, and absence of inflammatory infiltrate at 5 days after transplantation. Ultimately, allograft survival was significantly prolonged. In vitro, VHL-/- myeloid-derived cells dose-dependently inhibited T-cell proliferation. Myeloid cells with HIF-1α-deletion retained proinflammatory qualities in vitro and in vivo. Deletion of VHL in myeloid cells of nonimmunosuppressed cardiac allograft recipients reduced myocardial injury and acute rejection. We suggest that HIF transcription factors induce a regulatory phenotype in myeloid-derived cells, which may be harnessed as a novel therapeutic strategy to regulate immune responses after heart transplantation.

Elucidation of Tumor-Promoting Effects of Novel Adherent Immature Myeloid Cells in Tumor

IntroductionSolid tumors are infiltrated by a variety of myeloid-derived cells (MDCs), such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Extensive studies have revealed that they play pivotal roles in tumor progression, such as immunosuppression, angiogenesis, and enhancing tumor cell invasion, and metastasis. It has been suggested, however, that undefined MDCs exist in tumors and play key roles in tumor progression. In order to develop a novel anti-tumor strategy, we have been searching undefined tumor-infiltrating MDCs that associate with tumor progression. Recently we have isolated adherent MDCs (AMCs), which strongly adhere to culture dishes, from subcutaneous tumors of Lewis lung carcinoma (LLC). AMCs contained CD45(+) CD11b(+) F4/80(-) undefined cell population (named F4/80(-)AMC) that exhibit protumoral functions. In this research, we characterized F4/80(-)AMC and explored its protumoral functions.MethodsF4/80(-)AMC were subcutaneously transplanted into syngenic C57BL/6J mice with firefly luciferase-expressing LLC (LLC/luc) cells and bioluminescence (BL) signals corresponding to tumor burden were monitored every four days. The direct effect of F4/80(-)AMC on cancer cell growth was examined in vitro by culturing LLC/luc cells with (co-culture) or without (mono-culture) F4/80(-)AMC. In addition, conditioned medium (CM) collected from co-culture and mono-culture were analyzed by using mouse cytokine protein array. Receptors for markedly increased cytokines in co-culture CM were knocked down in LLC/luc cells to examine their involvement in LLC growth, and the candidate cytokines were further investigated for their direct effect on LLC growth by adding their recombinant proteins to the culture medium and monitoring the growth of LLC/luc cells. Furthermore, the possibility that F4/80(-)AMC could have a role to recruit other MDCs to tumor site was examined by in vitro chemotaxis assays using transwell chambers, and the impact of the neutralizing antibody against candidate cytokines on MDCs chemotaxis was evaluated.ResultsInvestigation of surface makers on F4/80(-)AMC revealed that F4/80(-)AMC is distinct from any other already-known myeloid cells [Figure 1]. BL signal from tumors co-transplanted with F4/80(-)AMC was significantly increased compared to the signal from LLC/luc tumors, indicating that F4/80(-)AMC promotes tumor growth in subcutaneous tumor models [Figure 2]. Since F4/80(-) AMC was able to enhance proliferation of LLC cells in an in vitro co-culture without cell-cell contact [Figure 3], we hypothesized that F4/80(-)AMC may directly enhance tumor cell growth through cytokine release. Cytokine array showed that 6 cytokines (lipocalin-2, CXCL1, CXCL2, adiponectin, CCL2, and CCL5) were markedly increased in co-cultured CM compared to mono-cultured CM [Figure 4]. Knocking down (KD) of receptors for these cytokines indicate that only KD of CXCR2, a receptor for CXCL1/CXCL2, significantly abrogated F4/80(-)AMC-induced LLC growth [Figure 5]. CXCL1 and CXCL2 dose-dependently promoted LLC proliferation [Figure 6], demonstrating that F4/80(-)AMC directly enhance cancer cell proliferation via CXCL1 and CXCL2. Furthermore, in tumors co-transplanted with F4/80(-) AMC, monocytic MDSC (Mo-MDSC) and TAM were elevated, while activated CTLs were reduced [Figure 7]. It has been known that Mo-MDSC can suppress CTLs activities and that TAM is differentiated from Mo-MDSC in tumors. Therefore, above results may reflect F4/80(-)AMC-mediated recruitment of Mo-MDSC, which subsequently suppress CTLs and differentiate into TAM. Moreover, antibodies against CCL2 and CCL5 significantly suppressed the migration of Mo-MDSC toward CM of F4/80(-) AMC, suggesting that F4/80(-)AMC recruits Mo-MDSC via CCL2 and CCL5 secretion [Figure 8]. Taken together, these results strongly suggest that F4/80(-)AMC contributes to tumor progression by creating an immunosuppressive microenvironment.ConclusionsOur study sheds light on the protumoral functions of novel MDCs: F4/80(−)AMC. Further characterization of F4/80(−)AMC and elucidation of its relationship with known MDCs are required to understand overall roles of MDCs in malignant progression. Our goal in this work is to identify the cell surface markers of F4/80(−)AMC and develop a novel treatment strategy for advanced cancers based on the knowledge of F4/80(−)AMC. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Abstract Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with >58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Abstract A192: The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Abstract Introduction The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is expressed in immune cells and has limited expression in epithelial cancer cells. Genetic inactivation of PI3K-γ highlights its role in the development and function of myeloid-derived cells that constitute a key component of the suppressive tumor microenvironment (Schmid 2011). Targeting PI3K-γ in tumor-associated myeloid cells could potentially relieve tumor immune tolerance, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD of 0.29 nM for PI3K-γ and enzymatic assays confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over the other Class I PI3K isoforms. Comparison of IPI-549 to previously reported PI3K-γ inhibitors in this enzymatic confirmed its unique potency and selectivity for PI3K-γ. Cellular assays for individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). In kinase screens, IPI-549 is selective for PI3K-γ over other kinases, receptors, ion channels, and transporters. In vitro assays demonstrated that IPI-549 blocked immune suppressive M2 murine macrophage polarization in response to IL-4 and MCSF1. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of PI3K-γ relative to other Class I PI3K isoforms. To characterize IPI-549's ability to inhibit PI3K-γ in vivo, mice with air pouches treated with IPI-549 showed dose responsive inhibition of PI3K-γ-dependent neutrophil migration. The effect of IPI-549 on tumor growth was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell numbers and activity, as assessed by marker studies. Nude or CD8 T-cell depleted mice studies demonstrated a T-cell dependence of IPI-549-mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors or following chemotherapy showed increased tumor growth inhibition compared to monotherapies. Conclusions IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. Citation Format: Karen McGovern, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Kerry White, David Winkler, Vito Palombella, Jeffery Kutok. The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A192.

O6.04 * GRANULOCYTIC MYELOID-DERIVED CELLS OBTAINED FROM BLOOD MAINLY SUPPRESS T-CELL PROLIFERATION IN PATIENTS WITH GLIOBLASTOMA

OBJECTIVE: To study the importance of myeloid-derived suppressor cells (MDSC) in primary glioblastomas, we investigated the frequency, phenotype, and immune-suppressive function of different myeloid-derived cells (MDC) in the blood and freshly resected tumors of 40 patients. RESULTS: Multicolour flow cytometric analysis showed that the frequency of CD14lowCD15+ granulocytic CD11bhighCD33b+ MDC was higher in peripheral blood and tumor samples of glioblastoma patients compared to healthy controls and positively correlated with the tumor volume before resection. Granulocytic MDC expressed CD16, CD66b, CD114, CD195 and could be further divided into CD14lowCD15high neutrophilic and CD14lowCD15int eosinophilic MDC. Moreover, we could detect an intratumoral accumulation of CD14highCD15+ monocytic CD11bhighCD33b+ MDC expressing CD115, CD124, CD184, CD206 and CX3CR1. All MDC subsets stained positively for reactive oxygen species, but Arginase 1 was specifically expressed in tumor-infiltrating MDC. T cell suppression assays with FACS-sorted MDC revealed that granulocytic and most prominently eosinophilic MDC obtained from the blood, but not the tumor could efficiently suppress non-specific T cell proliferative responses. Contrary to blood-derived MDC, HLA-DR expression was significantly increased on tumor-infiltrating MDC. Upon LPS or R848 stimulation, blood-derived MDC showed an increased production of IL-10, in contrast to tumor-infiltrating MDC that were resistant to Toll-like-receptor (TLR) stimulation. Although the frequency of granulocytic MDC inversely correlated with CD4+ and CD8+ T memory cells in the blood, no association with time to tumor progression in glioblastoma patients could be found. CONCLUSION: Our findings provide a detailed characterization of the nature and suppressive function of MDC in glioblastoma patients and indicate that granulocytic MDC obtained from the blood but not from the tumor mainly suppress T-cell proliferation.