Changes in MDSC function and differentiation induced by the tumor-microenvironment require HIF-1α. (A) Percentage of MΦ and DCs in the population of CD11b⁺Gr-1⁻CD45.2⁺ donor cells 18 h after the adoptive transfer of HIF-1α^+/+ and HIF-1α^−/− MDSC into ascites tumors of congeneic recipients. Each experiment included two mice per group and was performed twice. *, statistically significant (P < 0.05) differences between groups. (B and C) HIF-1α–deficient and WT MDSCs were generated in vivo by transfer of BM cells into tumor-bearing recipients as described in A. MDSCs were isolated and cultured with GM-CSF for 5 d under hypoxic or normoxic conditions. Error bars show mean and SD. (B) Expression of arg1 and inos was measured in triplicates in RT-PCR. Two independent experiments with the same results were performed. Error bars show mean and SD. (C) The phenotype of cells differentiated from MDSC after a 5-d culture with GM-CSF. Two independent experiments with the same results were performed. (D) Tumor growth of lethally irradiated mice reconstituted with either WT or HIV-1α–deficient BM. Tumor was established by s.c. inoculation of 3 × 10⁵ B16.F10 tumor cells 2 wk after transfer of BM to lethally irradiated recipients. Two groups of mice (control) were left untreated. Mice from two other groups received 3 × 10⁶ splenocytes from Pmel-1 T cell receptor transgenic mice on day 6 after tumor inoculation. 1 d later, the mice were immunized with specific peptide. Tumor growth was monitored every 3–4 d in individually tagged mice by measuring two opposing diameters with a set of calipers. Each group included four to five mice. Results of individual mice are shown.