4B). ELISA confirmed IFN-γ production in cocultures, albeit lower than in cultures of T cells stimulated

alone (Fig. 4C). Because substantial IFN-γ was produced in cocultures of T cells and Tgfb1+/− liver CD11b+Gr1+ cells Antiinfection Compound Library cell line (Fig. 4C), IFN-γ appears insufficient to confer MDSC activity on liver-resident CD11b+Gr1+ cells. Indeed, when IFN-γ was added exogenously, Tgfb1+/− liver CD11b+Gr1+ cells were unable to inhibit T cell proliferation (Fig. 4D), and NO production was not augmented (Fig. 4B). Thus, IFN-γ is necessary but not sufficient for MDSC activity. Anti-Gr1 recognizes two highly related cell surface proteins, Ly6C and Ly6G.22 Expression patterns of these cell surface proteins distinguish two major MDSC subsets, with the Ly6G−Ly6Chi phenotype characteristic of monocyte-like MDSCs and the Ly6GhiLy6Clo phenotype characteristic of granulocyte-like MDSCs.23 Most CD11b+ cells from Tgfb1−/− livers coexpressed Ly6C ( Fig. 5A). Among CD11b+Ly6C+ cells, approximately two-thirds were Ly6GhiLy6Clo, whereas the rest were Ly6G−Ly6Chi (Fig. 5A). After isolation of these subsets (Fig. 5B), we observed that suppressor activity resides exclusively in the “monocytic” CD11b+Ly6G-Ly6Chi cell population, with no activity found in the “granulocytic” CD11b+Ly6GhiLy6Clo cell population (Fig. 5C). NO production tracked

with suppressor function (Fig. 5D). The rapid accumulation of MDSCs parallels that of CD4+ T cells (Fig. 1). Therefore, we asked whether one cell type influences the accumulation of the other in vivo, by examining CD11b+Gr1+ cell accumulation at day Selleck Forskolin 11 in livers of Tgfb1−/− mice rendered deficient either in all adaptive lymphocytes (Rag1−/−) or specifically in CD4+ T cells (anti-CD4 mAb). Neither Rag1−/−Tgfb1−/−

mice nor anti-CD4–treated Tgfb1−/− mice exhibited an increase in liver CD11b+Gr1+ cells (Fig. 6A). Conversely, CD4+ T cells accumulated to high levels in Tgfb1−/− mice whether CD11b+Gr1+ cells had been depleted (anti-Gr1; Fig. 6B). Thus, CD4+ T cells are required for MDSC accumulation in Tgfb1−/− liver, whereas CD4+ T cells accumulate despite MDSC depletion. see more We examined the role of IFN-γ in MDSC accumulation. Circulating plasma IFN-γ levels are highly elevated in Tgfb1−/− mice,21 IFN-γ is necessary for hepatocellular damage,9 and CD4+ T cells are the only significant source of IFN-γ in this model.21 The Ifng−/−Tgfb1−/− mice exhibited normal liver CD11b+Gr1+ cell numbers ( Fig. 6C). Conversely, depletion of CD11b+Gr1+ cells had no effect on plasma IFN-γ levels in Tgfb1−/− mice (Fig. 6D), which remained elevated. In addition, Ifng−/−Tgfb1−/− liver CD11b+ cells failed to suppress T cell proliferation in vitro (Fig. 6E). Thus, IFN-γ is essential both for the in vivo accumulation of CD11b+Gr1+ cells and for their in vitro suppressor function.