Resistance to metastatic disease and MDSC regression require FasL. (A) Resistance to metastatic disease requires host cell expression of FasL. BALB/c, FasL^−/−, STAT6^−/−, CD1^−/−, CD1^−/−FasL^−/−, and STAT6^−/−FasL^−/− mice were inoculated on day 0 in the mammary gland with 7000 4T1 tumor cells. Primary tumors were removed 3-4 weeks later when metastatic disease was established, and mice were followed after surgery for survival. Numbers indicate the number of mice surviving > 247 days/total number of mice/group. Data are the pooled results of 2 independent experiments. (B) FasL^−/− mice contain elevated levels of MDSCs. Data are the average + SD of 5 mice/group. (C) MDSC regression requires host cell expression of FasL. The BALB/c, FasL^−/−, STAT6^−/−, CD1^−/−, CD1^−/−FasL^−/−, and STAT6^−/−FasL^−/− mice of Figure 5A were bled 10-12 days after removal of their primary tumors, and the percentage of Gr1⁺CD11b⁺ MDSCs in the blood was determined by flow cytometry. Dotted line represents the average percentage of Gr1⁺CD11b⁺ cells in the blood of tumor-free mice; solid lines represent the average percentage of Gr1⁺CD11b⁺ cells in each group. Each ○ represents an individual mouse. Data are pooled from 2 independent experiments. (D) Gr1⁺CD11b⁺ cells from postsurgery BALB/c, FasL^−/−, STAT6^−/−FasL^−/−, and CD1^−/−FasL^−/− mice are equally suppressive. Gr1⁺CD11b⁺ cells from the peripheral blood of 4T1 tumor–bearing mice were cocultured with DO11.10 splenocytes and cognate (OVA_323-339) peptide. T-cell activation was assessed by ³H-thymidine incorporation. Average +SD of 4 independent experiments for BALB/c and 2 independent experiments for FasL^−/−, CD1^−/−FasL^−/−, and STAT6^−/−FasL^−/−mice. (E) Postsurgery resistance to 4T1-induced metastatic disease is perforin dependent. BALB/c, FasL^−/−, and Pfp^−/− mice were inoculated on day 0 in the abdominal mammary gland with 7000 4T1 tumor cells. Primary tumors were removed 3-4 weeks later when metastatic disease was established, and mice were followed after surgery for survival. Data are pooled from 5 independent experiments for BALB/c (n = 27 mice) and from 3 independent experiments for Pfp^−/− and FasL^−/− mice (n = 10 mice for each group). Pfp^−/− mice survived significantly shorter than wild-type and FasL^−/− mice (P < .01 by log-rank test). (F) FasL is required for MDSC reduction. BALB/c, Pfp^−/−, FasL^−/−, STAT6^−/−Pfp^−/−, and STAT6^−/−FasL^−/− mice were inoculated in the abdominal mammary gland with 7000 4T1 tumor cells. When primary tumors were 4-5 mm in diameter, the mice were bled, and the percentage of Gr1⁺CD11b⁺ MDSCs in the peripheral blood was determined by flow cytometry. *Statistically significantly different groups (P < .05); Average +SD of 3 mice/group. (G) MDSCs apoptose in vivo in response to activated T cells. DO11.10 transgenic mice were injected subcutaneously on day 0 with cognate peptide (OVA_323-339) or irrelevant peptide (HA_110-119). Splenocytes were collected on day 3, depleted for RBCs, and stained for Gr1, CD11b, and cleaved caspase3 or for CD4, DO11.10 TcR, and CD69. (Top) Gated Gr1⁺CD11b⁺ and CD4⁺DO11.10⁺ cells from representative individual mice; (bottom) percentage of CD11b⁺Gr1⁺cleaved caspase3⁺ and percentage of activated (CD69⁺) CD4⁺DO11.10⁺ cells. Average +SD of 5 mice/group. Data are from 1 of 2 independent experiments. (H) Fas^+/+ but not Fas^−/− MDSCs apoptose in vivo in response to activated T cells. C57BL/6 Fas^+/+ and Fas^−/− mice were adoptively transferred with CD8⁺ OT-1 T cells and challenged subcutaneously with cognate peptide. Splenocytes were harvested on day 3 and analyzed according to Figure 5G for percentage of caspase3⁺ MDSCs and for percentage of activated (CD69⁺) OT-1 T cells. Average +SD of 4 mice/group.