The Role of CCR5 in the Recruitment of MDSC to the Tumor Microenvironment

The aim of the study was to analyze the recruitment and accumulation of MDSC in the tumor microenvironment via C-C chemokine receptor type 5 (CCR5)/CRR5 ligand interaction, as well as immunosuppressive activity of CCR5-expressing tumor-infiltrating MDSC. We demonstrated that CCR5+ Mo-MDSC and CCR5+ Gr-MDSC accumulated during tumor progression in melanoma lesions of ret transgenic mice, as well as in melanoma patients at different stages. In the mouse model, the frequency of CCR5+ cells was higher among Gr-MDSC than among Mo-MDSC, whereas in melanoma patients, Mo-MDSC were characterized by higher frequency of CCR5+ cells. CCR5+ MDSC demonstrated an increased immunosuppressive phenotype reflected by enhanced NO and ROS production, as well as ARG-1 and PD-L1 expression, as compared to their CCR5- counterpart. Both in mouse model and in melanoma patients, the concentration of CCR5 ligands such as CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) and inflammatory factors such as granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and interferon-γ (IFN-γ) were increased in skin tumors as compared to the serum that allows the migration of these cells to the tumor microenvironment. Furthermore, we demonstrated that CCR5+ MDSC inhibited T cell proliferation in a dose-dependent manner and showed a tendency to stronger inhibition of T cell proliferation than their CCR5- counterparts. In addition, we detected the CCR5 expression on CD4+ and CD8+ T cells in ret transgenic melanoma bearing mice. Interestingly, the frequency of CCR5+ regulatory T cells (Treg) was significantly higher than that among other T cell subsets. Antigen-experienced CCR5+ Treg accumulated in advanced stage melanoma patients as compared to early stage patients and healthy donor (HD). After intraperitoneal injection of mCCR5-Ig, which blocks CCR5-CCR5 ligand interactions into tumor bearing mice, we observed a significantly increase in mouse survival as compared to the control group. Moreover, the frequency of MDSC and Treg decreased in skin tumors after mCCR5-Ig therapy. NO production by MDSC in skin tumors and the frequency of CD69 expression, reduced activated Treg in metastatic lymph nodes (LN), which indicates the therapeutic effect of mCCR5-Ig. In summary, we demonstrated that during melanoma progression, CCR5 expressing MDSC accumulated in the tumor microenvironment and exerted strong immunosuppressive activity. This accumulation was mediated by CCR5 ligands and other inflammatory factors. Blockage of CCR5-CCR5 ligand interactions induced the prolongation of the survival of tumor bearing mice mediated by a reduced migration and immunosuppressive activity of MDSC and Treg at the tumor site. Our findings define a critical role for CCR5 in recruiting MDSC and Treg,which can be used for the development of novel immunotherapeutic strategies for melanoma patients

LiCl induces TNF-α and FasL production, thereby stimulating apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours.</p> <p>Results</p> <p>Here we show that LiCl induces apoptosis of tumour cells both <it>in vitro </it>and <it>in vivo</it>. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth.</p> <p>Conclusions</p> <p>Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL.</p> <p>Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL</p

Chemokine receptor patterns in lymphocytes mirror metastatic spreading in melanoma

30siopenMelanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4(+) and CD8(+) T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8(+)CCR9(+) naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8(+) T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches.openJacquelot N.; Enot D.P.; Flament C.; Vimond N.; Blattner C.; Pitt J.M.; Yamazaki T.; Roberti M.P.; Daillere R.; Vetizou M.; Poirier-Colame V.; Semeraro M.; Caignard A.; Slingluff C.L.; Sallusto F.; Rusakiewicz S.; Weide B.; Marabelle A.; Kohrt H.; Dalle S.; Cavalcanti A.; Kroemer G.; DI Giacomo A.M.; Maio M.; Wong P.; Yuan J.; Wolchok J.; Umansky V.; Eggermont A.; Zitvogel L.Jacquelot, N.; Enot, D. P.; Flament, C.; Vimond, N.; Blattner, C.; Pitt, J. M.; Yamazaki, T.; Roberti, M. P.; Daillere, R.; Vetizou, M.; Poirier-Colame, V.; Semeraro, M.; Caignard, A.; Slingluff, C. L.; Sallusto, F.; Rusakiewicz, S.; Weide, B.; Marabelle, A.; Kohrt, H.; Dalle, S.; Cavalcanti, A.; Kroemer, G.; DI Giacomo, A. M.; Maio, M.; Wong, P.; Yuan, J.; Wolchok, J.; Umansky, V.; Eggermont, A.; Zitvogel, L

Chemokine receptor patterns in lymphocytes mirror metastatic spreading in melanoma

Melanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4+ and CD8+ T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8+CCR9+ naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8+ T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches

CCR5 Directs the Mobilization of CD11b+Gr1+Ly6Clow Polymorphonuclear Myeloid Cells from the Bone Marrow to the Blood to Support Tumor Development

Cells of hematopoietic origin can be subdivided into cells of the lymphoid lineage and those of the myeloid lineage, among which are myeloid-derived suppressor cells (MDSCs). The MDSCs can be further divided into CD11b+Ly6G−Ly6Chi monocytic (Mo) MDSCs and CD11b+Ly6G+Ly6Clow polymorphonuclear (PMN) MDSCs. Both subtypes support tumor growth and suppress anti-tumor immunity. Their accumulation at the tumor site includes mobilization from the bone marrow to the blood followed by colonization at the tumor site. The present study examines the mechanism by which PMN-MDSCs are mobilized from the BM to the blood to later accumulate at the tumor site. We show that the chemokine receptor CCR5 is a key driver of this event. We also show that, beyond chemoattraction, the interaction between CCR5 and its ligands promotes the proliferation of CCR5+ PMN-MDSCs at the BM and, later, potentiates their immune-suppressive activities at the tumor site in part by inducing arginase-1

Chronic ethanol feeding modulates inflammatory mediators, activation of nuclear factor-κB, and responsiveness to endotoxin in murine Kupffer cells and circulating leukocytes

Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κB(EGFP) reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1 β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner

Circulating and Tumor Myeloid-derived Suppressor Cells in Resectable Non–Small Cell Lung Cancer

Rationale: Myeloid-derived suppressor cell (MDSC) expansion has been found to play a role in disease progression in patients with cancer. However, the characteristics of MDSCs in lung cancer are poorly understood. Objectives: We prospectively investigated MDSCs and inflammatory factors in tumor and peripheral blood samples from patients with resectable non-small cell lung cancer and studied their correlations with the disease prognosis. Methods: A complex analysis of MDSC subsets and inflammatory mediators was performed using flow cytometry and a Bio-Plex assay. Measurements and Main Results: A significant increase in the frequency of circulating monocytic (M)-MDSCs was observed in the patients with non-small cell lung cancer compared with the healthy donors (HDs). Moreover, the frequencies of M-and polymorphonuclear (PMN)-MDSCs were higher in tumors than in the peripheral blood of the same patients. This accumulation was associated with elevated concentrations of inflammatory mediators involved in MDSC migration to and activation in the tumor microenvironment. An analysis of the MDSC immunosuppressive pattern showed increased programmed death-ligand 1 expression on circulating cells from patients compared with HDs. Tumor PMN-MDSCs displayed higher programmed death-ligand 1 expression levels than the same cells in the peripheral blood. The frequency of CCR5 (C-C chemokine receptor 5) expression on circulating M-MDSCs was significantly higher in the patients than in the HDs. Clinical data analysis revealed negative correlations between recurrence-free survival and the frequencies of PMN-MDSCs and CCR5(+) M-MDSCs in the circulation but not in tumors. Conclusions: Our findings suggest that the level of MDSCs in the peripheral blood but not in tumor tissues predicts recurrence after surgery

Mouse models of pediatric high-grade gliomas with MYCN amplification reveal intratumoral heterogeneity and lineage signatures

Abstract Pediatric high-grade gliomas of the subclass MYCN (HGG-MYCN) are highly aggressive tumors frequently carrying MYCN amplifications, TP53 mutations, or both alterations. Due to their rarity, such tumors have only recently been identified as a distinct entity, and biological as well as clinical characteristics have not been addressed specifically. To gain insights into tumorigenesis and molecular profiles of these tumors, and to ultimately suggest alternative treatment options, we generated a genetically engineered mouse model by breeding hGFAP-cre::Trp53 Fl/Fl ::lsl-MYCN mice. All mice developed aggressive forebrain tumors early in their lifetime that mimic human HGG-MYCN regarding histology, DNA methylation, and gene expression. Single-cell RNA sequencing revealed a high intratumoral heterogeneity with neuronal and oligodendroglial lineage signatures. High-throughput drug screening using both mouse and human tumor cells finally indicated high efficacy of Doxorubicin, Irinotecan, and Etoposide as possible therapy options that children with HGG-MYCN might benefit from