HIF-1α regulation of MDSC function. (A) MDSCs isolated from spleens of EL-4 tumor-bearing mice were cultured with 10 ng/ml GM-CSF in hypoxia for 4 or 16 h. The level of HIF1-α was measured by Western blotting. A typical example of three independently performed experiments is shown. (B–F) MDSCs were treated with various concentrations of HIF-1α stabilizer DFO in the presence of 10 ng/ml GM-CSF for 48 h and then washed and used in the experiments. No effect of DFO on MDSC cell viability was observed (not depicted). Each experiment was performed in triplicates and repeated two times with the same result. Results are shown as mean ± SD. (B and C) Effect of MDSC on proliferation (B) and IFN-γ production (C) of splenocytes stimulated with anti-CD3/CD28 antibodies. (D and E) Effect of a 48-h treatment of MDSC with DFO on the expression of arg1, inos, gp91^phox, and p47^phox. Each experiment was performed in triplicates. Three experiments were performed with the same result. (F) Percentage of F4/80⁺CD11b⁺ MΦ differentiated from MDSC treated with DFO for 5 d. Cumulative results (mean ± SD) of three experiments are shown. In D and F, * denotes statistically significant (P < 0.05) differences from untreated (0 µM) samples. (G) Evaluation of BM reconstitution by BM cells from HIF-1α–deficient mice. Expression is shown of hif-1α in HIF-1α^{fl/flCre+/−} and HIF-1α^{fl/flCre−/−} mice after treatment with poly I:C using real-time PCR. Experiments were performed in triplicates in three mice. Error bars show mean and SD. (H) Reconstitution of lethally irradiated CD45.1⁺ congenic mice with CD45.2⁺ BM from HIF-1α–deficient (HIF-1α^flox/flox, Cre^+/−) or WT (HIF-1α ^flox/flox, Cre^−/−) mice. Blood of mice 2 wk after BM transfer was tested. A typical example of three experiments is shown. (I–K) CD45.1⁺ lethally irradiated recipients were reconstituted with BM cells from HIF-1α knockout (KO) or WT CD45.2⁺ mice. 2 wk later, mice were inoculated s.c. with 5 × 10⁵ EL-4 tumor cells. 3 wk after that, CD45.2⁺ HIF-1α WT or KO MDSC were isolated from spleens of tumor-bearing mice and then transferred into ascites of congenic CD45.1⁺ mice. 12 h later, CD45.2⁺ CD11b⁺ donor cells were isolated and used in the subsequent experiments. (I) The MDSCs were cultured with anti-CD3/CD28 antibody-activated T cells (Resp., responder cells) and their proliferation was measured by ³H-thymidine incorporation. Experiments were performed in triplicates. Mean ± SD is shown. Experiments were performed twice with the same result. (J) Expression of arg1 and inos was analyzed in the MDSC before and after adoptive transfer into the tumor milieu. Two independent experiments were performed in triplicates. In J and K, * denotes statistically significant (P < 0.05) differences between the groups. Error bars show mean and SD. (K) ROS level in MDSC after the adoptive transfer was determined with DCFDA. Cumulative results of two experiments are shown. Error bars show mean and SD.