Other Calcium Channels

TLR2 expression in G-MDSCs or M-MDSCs (right)

TLR2 expression in G-MDSCs or M-MDSCs (right). macrophages, and iNOS expression required interferon- (IFN-) production by CD8+ T cells that had been transiently stimulated by M-MDSC-derived macrophages in an antigen/TLR2-dependent manner. Although Pam2CSK4 triggered DC maturation and tumor regression via induction of tumor antigen-specific cytotoxic T lymphocyte (CTL) responses in tumor-bearing mice, Pam2CSK4 plus antigen increased the frequency Mouse monoclonal to CD16.COC16 reacts with human CD16, a 50-65 kDa Fcg receptor IIIa (FcgRIII), expressed on NK cells, monocytes/macrophages and granulocytes. It is a human NK cell associated antigen. CD16 is a low affinity receptor for IgG which functions in phagocytosis and ADCC, as well as in signal transduction and NK cell activation. The CD16 blocks the binding of soluble immune complexes to granulocytes of iNOS+ macrophages in the tumor. Treatment with iNOS inhibitor enhanced the therapeutic efficacy of Pam2CSK4. Hence, the results suggest that TLR2 ligand and T cell-derived IFN- enhance M-MDSC-mediated immunosuppression, which may negatively regulate anti-tumor CTL response. and (Fig.?1F). Thus, activation of TLR2 signaling enhanced the survival of both MDSC subsets, which may be responsible, at least in part, for their accumulation in tumor-bearing mice treated with Pam2CSK4. Open in a separate window Figure 1. Pam2CSK4 sustains the survival of CD11b+Gr1+ MDSCs. (A) EG7 tumor-bearing mice were subcutaneously injected twice with PBS or 50?nmol Pam2CSK4 and OVA protein every 4?days. After 24?hours from last injection, the proportion of CD11b+Gr1+ cells in the spleen was analyzed by flow cytometry. Numbers adjacent to outlined GDC-0449 (Vismodegib) areas indicate the percentage of relevant population. (B) CD11b+Gr1+ cells were isolated from EG7 tumor-bearing B6 WT, TLR2?/?, or IL-6?/? mice, and cultured in the presence of PBS or Pam2CSK4. After 24?h, cell viability was measured by WST-1 assay. (C) CD11b+Gr1+ cells treated with PBS (thin line histogram) or Pam2CSK4 (bold line histogram) for 24?hours were analyzed by flow cytometry after staining with PI. (D) Real-time PCR analysis of transcripts for in CD11b+Gr1+ cells isolated from tumor-bearing B6 WT or TLR2?/? mice after in vitro GDC-0449 (Vismodegib) treatment with Pam2CSK4 or PBS for 4?h. (E) Flow cytometric analysis of Ly6G and Ly6C expression in CD11b+Gr1+ cells (left). TLR2 expression in G-MDSCs or M-MDSCs (right). (F) G-MDSCs and M-MDSCs were isolated from tumor-bearing mice and incubated with Pam2CSK4 or PBS for 24?h. Cell viability was measured by WST-1 assay. Data represent means standard deviation (SD) in graph. n = 3. **P 0.005. *P 0.05. All data shown are representative of more than 2 independent experiments. Pam2CSK4 promotes differentiation of M-MDSCs into CD11b+F4/80+CD115+ macrophages We tested whether the frequency of MDSC differentiation into macrophages was GDC-0449 (Vismodegib) affected by Pam2CSK4 treatment, given that MDSCs have the potential to differentiate GDC-0449 (Vismodegib) into macrophages and TLR2 ligands induce macrophage differentiation from monocytes.20 CD11b+Gr1+ cells isolated from tumor-bearing mice were labeled with fluorescent dye to trace their fate in vivo, then adoptively transferred into tumor-bearing mice that were injected with PBS or Pam2CSK4. A small GDC-0449 (Vismodegib) proportion of the CD11b+Gr1+ MDSCs up-regulated macrophage markers, F4/80 and CD115 (M-CSFR), and decreased Gr1 expression in PBS-treated mice (Fig.?2A). Interestingly, Pam2CSK4 treatment increased the frequency of F4/80+ and CD115+ cells derived from adoptively transferred CD11b+Gr1+ cells (Fig.?2A). To determine which MDSC subset had the potential to differentiate into macrophages, we isolated each subset and cultured the cells in the presence of Pam2CSK4. F4/80+ and CD115+ cells were generated from M-MDSCs, but not G-MDSCs, and this response was enhanced by Pam2CSK4 (Fig.?2B). These results suggested that Pam2CSK4 promoted macrophage differentiation of M-MDSCs, in addition to prolonging their survival. Open in a separate window Figure 2. CD11b+Gr1+ cells differentiate into F4/80+/CD115+ macrophages and promoter activity in M-MDSC-derived macrophages through STAT125 may be absent from CD11c+ DCs. Alternatively, IFN–induced STAT1 signaling may be negatively regulated by PIAS1 and STAT3, as observed in IL-15-induced DCs.28 Analyzing the differential responses of M-MDSC-derived macrophages and CD11c+ DCs to IFN- could help us identify a critical molecule for the regulation of immunosuppression by M-MDSCs. M-MDSC-mediated T cell suppression is reportedly dependent on the production of NO and Arg1, as well as immunosuppressive cytokines including IL-10 and TGF-.29.