Tumor-derived SCF is required for MDSC accumulation in the tumor-bearing mice. (A) Decreased percentage of Gr-1⁺CD115⁺ MDSCs in bone marrow of mice bearing SCF knockdown tumors. BM Percoll fraction II (Fr. II) cells from mice bearing SCF-silenced (4 mice) versus control mock-transfected (5 mice) MCA26 tumors were stained with anti–Gr-1-APC plus anti–CD115-PE or isotype control antibodies followed by flow cytometric analysis. The percentage of MDSCs in mice bearing SCF-silenced tumors is significantly lower than that in mice bearing control tumors (P < .001 by Student t test). (B) Decreased number of Gr-1⁺CD115⁺ cells in mice bearing SCF knockdown tumors. The numbers of Gr-1⁺CD115⁺CD11b⁺ and Gr-1⁺CD115^-CD11b⁺ cells in bone marrow Fr. II derived from mice with large ( > 10 × 10 mm²), medium (7 × 7 mm²), or small ( < 7 × 7 mm²) control versus SCF-silenced tumors were compared. The average numbers of MDSCs in BM are presented. A significantly lower number of MDSCs were observed in mice bearing SCF knockdown tumors with sizes larger than 7 × 7 mm² compared with mice bearing control wild-type (WT) tumors (*P < .05, Student t test). (C) Reduced suppressive activity of Percoll Fr. II cells isolated from mice bearing SCF knockdown tumors. The suppressive activities of Percoll Fr. II, which contains MDSCs, isolated from mice bearing large SCF knockdown or control wild-type tumors were assessed using HA peptide-mediated proliferation at various ratios of CD4 HA TCR transgenic splenocytes/irradiated Fr. II cells. The Fr. III cells were used as negative control. The Fr. II cells from mice with SCF knockdown tumors exhibited lower suppressive activity compared with those from mice bearing wild-type tumors. Con indicates control. (D) Proliferative response of T cells derived from tumor tissues. The anti-CD3/anti-CD28–mediated proliferative responses of the T cells derived from SCF-silenced or control tumor tissues were assessed in a standard [³H]-thymidine incorporation assay. Splenocytes from naive normal mice were used as a positive control. w/o indicates without.