MDSC in peripheral blood and tumor tissues of cancer patients. (A–C) Peripheral blood and tumor tissues were collected from patients with HNC during surgical resection. Single cell suspensions were prepared as described in Materials and methods and cells were stained with anti-CD11b, anti-CD14, and anti-CD33 antibodies. (A) A typical example of gating of CD11b⁺CD14⁻CD33⁺ MDSC from the same patient in flow cytometry. (B) Cells were stained with DCFDA to detect ROS level within the population of CD11b⁺CD14⁻CD33⁺ cells from the same patient. Cumulative results from six patients are shown. **, statistically significant difference between MDSC in the tumor site and peripheral blood (P = 0.007). MFI, mean fluorescence intensity. (C) Cells were labeled with anti-iNOS antibody and the protein level was measured within the population of CD11b⁺CD14⁻CD33⁺ cells. Cumulative results from six patients are shown. *, statistically significant difference between MDSC in the tumor site and peripheral blood (P = 0.04). (D) Proliferation of PHA-stimulated mononuclear cells in the presence of MDSC isolated from tumor and peripheral blood of the same patient. PBMCs (10⁵/well) were stimulated with 5 µg/ml PHA in the presence of indicated amount of MDSC. Proliferation was measured in triplicate on day 3 by uptake of ³H-thymidine. Proliferation of PBMC in the absence of MDSC was considered 100%. ***, statistically significant difference between groups (P = 0.0008). Samples from three patients were tested and cumulative results are shown. Error bars show mean and SD.