2D), suggesting that tumor-derived IL-1β could substitute for the absence of host IL-1β. The discovery of a novel subpopulation of MDSC prevailing in 4T1/IL-1β-tumor-bearing mice may explain the reported phenotypic differences of MDSC from these mice compared to those from 4T1-tumor-bearing mice 11. It has been reported that splenic MDSC derived from 4T1/IL-1β-tumor-bearing

mice expressed more ROS and were more effective T-cell suppressors 11. We hypothesized that these differences may be attributable to the presence (and predominance) of the Ly6Cneg Palbociclib datasheet MDSC subset. Indeed, we found that Ly6Cneg MDSC expressed higher levels of inducible nitric oxide synthase (iNOS or NOS2) and ROS than Ly6Clow MDSC (Supporting Information Fig. 3A). In line with these observations, we observed that Ly6Cneg MDSC on a per cell basis were significantly more potent ATR inhibitor inhibitors of the proliferation of antigen-activated T cells than Ly6Clow MDSC (Supporting Information Fig. 3B). To study the ability of Ly6Cneg MDSC versus Ly6Clow MDSC to inhibit innate immunosurveillance, we assessed the capacity of 4T1/IL-1β versus 4T1 cells to form solid tumors upon injection into the footpad of BALB/c, BALB/c Rag2−/− (T- and B-cell

deficient) and BALB/c Rag2−/−IL-2Rβ−/− mice (lacking NK cells in addition to T and B cells). While 4T1 cells induced local tumor growth in all mice (Fig. 3A), the kinetics of tumor growth varied in the different recipients. Notably, in BALB/c Rag2−/−IL-2Rβ−/− mice, tumor development was significantly faster than in BALB/c Rag2−/− mice (Fig. 3A), indicating the involvement of NK cells in the delayed tumor growth in the latter.

In contrast, there was no difference in the kinetics of tumor growth upon inoculation of 4T1/IL-1β tumor cells in the various recipients; however, the IL-1β-secreting tumors grew consistently faster than 4T1 tumors in NK-proficient BALB/c Rag2−/− mice (Fig. 3B). Depletion of MDSC using either anti-Gr-1 monoclonal antibodies or Gemcitabine (GEMZAR, 30) resulted in a significant delay of tumor growth in Rag2−/− mice transplanted with 4T1/IL-1β cells (Fig. 3C and data not shown; p<0.05). Together, these data suggested the involvement Niclosamide of NK cells in the host anti-tumor response and that Gr-1+ cells were involved in the inhibition of the Rag2-independent anti-tumor activity in 4T1/IL-1β-tumor-bearing mice. We analyzed the NK cell compartment in mice bearing established tumors and detected a reduced number (p<0.05) of CD122+NKp46+ NK cells in the bone marrow of 4T1- (30% of control cell numbers) and 4T1/IL-1β-tumor-bearing mice (15% of control cell numbers) (Fig. 4A, left, and Supporting Information Fig. 4). We then analyzed the development of NK cells in the different mice. CD27 is a marker of immature NK cells, while sequential upregulation of CD11b and KLRG-1 expression is associated with NK cell maturation 25.