1A and Supporting Information Fig. 1). MDSC expansion was considerably faster in mice bearing 4T1/IL-1β tumors (Fig. 1A), in line with previous results 11. Regarding the distribution of polymorphonuclear (or granulocytic)-MDSC

(PMN-MDSC; identified as Gr-1+CD11b+SSChigh cells) versus monocytic-MDSC (Mono-MDSC; identified as Gr-1+CD11b+SSClow cells) PMN-MDSC expansion was much greater than that of Mono-MDSC in both types of tumor-bearing mice. Thus, when comparing mice with the same tumor diameter, this resulted in much higher numbers of PMN-MDSC in mice bearing established 4T1/IL-1β-tumors than in mice bearing established 4T1-tumors (Fig. 1B; tumor diameter 10–12 mm). At the same time point, Mono-MDSC numbers only increased marginally in the same mice (Fig. 1B). The Matsushima laboratory has recently described PMN-MDSC as Ly6Clow, while Mono-MDSC were click here Ly6Chi20. Analysis of Ly6C

versus Gr-1 expression on gated CD11b+ cells demonstrated that the population of PMN-MDSC (Gr-1high) in 4T1-tumor-bearing mice was not homogeneous, but consisted of 80–90% Ly6Clow (Ly6Clow MDSC) and 10–20% of a buy Deforolimus previously undescribed population of MDSC lacking Ly6C expression (Ly6Cneg MDSC). Interestingly, in 4T1/IL-1β-tumor-bearing mice, the ratio of Lyc6low to Ly6Cneg MDSC was reversed, that is, this newly identified subpopulation of Ly6Cneg MDSC represented 75–90% of polymophonuclear (PMN)-MDSC in those mice (Fig. 1C and D and Supporting Information Fig. 2A). We observed the same pattern of Ly6C expression by Ly6G+ cells when we used antibodies of the clone 1A8 (anti-Ly6G) instead of clone RB6-8C5

(Ly6G/Ly6C) (data not shown). Flow cytometric analyses and Giemsa staining confirmed that Ly6Cneg MDSC were PMN cells (Supporting Information Fig. 2B and C). We detected a similar predominance of Ly6Cneg MDSC in the tumor mass, blood, lymph Methisazone nodes, thymus of 4T1/IL-1β-tumor-bearing mice, while Ly6Clow MDSC predominated in 4T1-tumor-bearing mice at these sites (Supporting Information Fig. 2D). Increasing the availability of IL-1β in 4T1-tumor-bearing mice either via treatment with multiple doses of recombinant IL-1β (rIL-1β; Fig. 2A) or when recipient mice were deficient for IL-1 receptor antagonist (IL-1Ra−/−; Fig. 2B) resulted in significantly more Ly6Cneg MDSC, but not Ly6Clow MDSC. However, the absolute numbers of Ly6Cneg MDSC in these treated or mutant mice were lower than those detected in 4T1/IL-1β-tumor-bearing mice possibly because of different levels of available IL-1. In contrast, reducing the availability of IL-1β via treatment of 4T1/IL-1β-tumor-bearing mice with recombinant IL-1Ra led to a decreased number of Ly6Cneg MDSC and delayed tumor growth compared to untreated mice (Fig. 2C and data not shown). Together, these data supported the importance of IL-1β in the expansion of Ly6Cneg MDSC.