Figure 2 (See previous page).

CTL-induced M-MDSCs produce NO. (A) C57BL/6 mice were injected with B16 melanoma cells and 9 days later, tumor-bearing mice (n = 3) were treated as described in Fig. 1. Tumor-infiltrating cells were analyzed on days 0, 5, and 10 after CTL transfer. The absolute number of eFluor450⁻CD45⁺ cells at the indicated time points is shown. (B) The eFluor450⁻CD45⁺ cells were stained with anti-CD11b and -Gr1 mAbs to detect MDSCs. (C) The absolute number of CD11b⁺Gr1⁺ cells at the indicated time points is shown, numbers of cells in each population were calculated as described in the Materials and Methods section and adjusted by the tumor weight (cells/g). (D) Monocytic MDSC were gated as Gr1^intLy6C⁺ cells in the CD11b⁺Gr1⁺ population. (E) The absolute numbers of Gr1^intLy6C⁺ monocytic MDSC are shown. (F) CD11b⁺Gr1⁺ cells were stained with Diff-quick as described in Materials and Methods; their morphology was assessed using an OLYMPUS BX41 microscope (magnification ×400 left, ×1000 right). (G) Sorted CD11b⁺Gr1⁺ cells in F were incubated for 30 min at 37°C with 5 μM DAF-FM (Diaminofluorescein-FM) (SEKISUI MEDICAL) and then stained with biotin-conjugated anti-Gr1, followed by staining with PE-conjugated anti-CD11b, Streptavidin-APC and analyzed by FLUOVIEW FV10 i (OLYMPUS, Tokyo, Japan). The experiments were performed independently at least three times with similar results.