Suppression of CD8+ T Cell Proliferation by Myeloid Cells

Ly6G⁺ cells were purified from spleen cells or tumor cells by positive selection using biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). The purity of the cell populations was >95%. CD11b⁺Ly6C^highLy6G⁻ cells were isolated from spleen cells by cell sorting on a FACSAria cell sorter (BD Biosciences). CD8⁺ T cells from PMEL mice that recognize the gp100-derived peptide, were used as responders. Splenocytes from PMEL mice were mixed with splenocytes from naïve mice at 1:4 ratio in complete RPMI media and plated into 96-well U-bottom plates at 10⁵ cells/well. Ly6G⁺ or CD11b⁺Ly6C^highLy6G⁻ cells were added to the wells at 1:16–1:1 ratios. Murine gp100 peptide (25 (link)–33 (link)) EGSRNQDWL (AnaSpec, Inc.) was added into the wells at the final concentration of 0.1 μg/mL. After 48 hours, cells were pulsed with ³H-thymidine (1 μCi/well; GE healthcare) for 16 hours. ³H-thymidine uptake was counted using a liquid scintillation counter as counts per minute (cpm) and calculated the percentage of proliferation to the positive control (the wells with responder cells and peptide but without suppressive cells).