Regulation of MDSC function by hypoxia. MDSCs were isolated from spleens of CT26 tumor-bearing mice and cultured in medium containing 10 ng/ml GM-CSF and 25% CT26 tumor cell conditioned medium (TCCM) under normoxic and hypoxic (1% O₂) conditions using a hypoxic chamber. (A) Expression of NOX subunits was evaluated in triplicates after 2 d of culture using qRT-PCR. Results are shown as mean ± SD from three experiments performed with similar results. *, statistically significant (P < 0.05) differences between normoxia and hypoxia groups. (B) Cells were collected after 3 d of culture, loaded with DCFDA, and labeled with anti–Gr-1 and anti–CD11b antibodies. Fluorescence intensity of DCFDA was measured within the Gr-1⁺CD11b⁺ population. A typical result of three independently performed experiments is shown. (C) Expression of argI and inos was evaluated in triplicates in MDSC after 24- and 48-h incubations in hypoxic and normoxic conditions using qRT-PCR. Results from three independently performed experiments are shown. *, statistically significant (P < 0.05) differences between normoxia and hypoxia groups. (D) MDSCs were cultured for 48 h under normoxic or hypoxic conditions and their ability to suppress proliferation of anti-CD3/CD28–stimulated splenocytes was evaluated. Cell proliferation was measured in triplicates by ³H-thymidine uptake. A typical result from two performed experiments is shown. (E) BM cells were cultured for 3 d in media containing 10 ng/ml GM-CSF and 25% TCCM. After that time, cells were incubated under normoxic or hypoxic conditions for an additional 2 d. Gr-1⁺ cells were isolated and cultured with splenocytes from BALB/c mice stimulated with anti-CD3/CD28 antibodies. Production of IFN-γ was measured in triplicates by ELISPOT assay. *, statistically significant (P < 0.05) differences from values of splenocytes cultured alone. Two independent experiments were performed. (F) MDSCs were cultured for 5 d with 10 ng/ml GM-CSF and 25% TCCM in normoxia or hypoxia and then fixed and stained with H&E (400× magnification). Bars, 10 µm. A typical example of two independently performed experiments is shown. (G) Phenotype of MDSC cultured for 5 d in hypoxia or normoxia. The proportions of cells with indicated phenotype were evaluated by flow cytometry. Cumulative results of four performed experiments are shown. *, statistical significant differences (P < 0.05) between the groups. Error bars in A, C–E, and G show mean and SD. (H) Cells derived from a 5-d culture of MDSC in hypoxia were tested for nonspecific esterase activity. Assay was repeated using cells isolated from two independent experiments. Bars, 10 µm. (I and J) An association between the presence of MΦ and MDSC in hypoxic areas of the tumor. (I) Number of Gr-1⁺ or F4/80⁺ cells per one 800-µm-×-600-µm field. A total of 8–16 fields was counted for each area and results are shown as mean ± SD. *, statistically significant (P < 0.05) differences between areas with less and more hypoxia. (J) Representative areas of s.c. EL-4 tumors are shown. Brown, staining with antibody detecting area of hypoxia; red; F4/80⁺ or Gr-1⁺ cells. Bars, 100 µm.