Figure 6

mTOR inhibition with rapamycin promotes immunosuppressive activity of MDSCs in vitro. (a) CD11b⁺Ly-6G⁺Ly-6C^low G-MDSCs and CD11b⁺Ly-6G⁻Ly-6C^high M-MDSCs with DMSO or rapamycin treatment were co-cultured with CFSE-stained CD4⁺ T cells at the ratio of 1:10 and 1:3 for 5 days, respectively, then the proliferation of T cells was assayed by flow cytometry. The decreased percentage of CFSE MFI after DMSO or rapamycin treatment indicated the ability of suppression of T-cell proliferation. Rapamycin treatment promoted immunosuppressive function of both M-MDSCs and G-MDSCs compared with control. (b) Arginase-1 and iNOS mRNA expression in CD11b⁺Ly-6G⁻Ly-6C^high M-MDSCs and CD11b⁺Ly-6G⁺Ly-6C^low G-MDSCs were examined by RT-PCR. The expression of Arginase-1 and iNOS mRNA were upregulated in M-MDSCs, whereas in G-MDSCs only Arginase-1 mRNA was upregulated after rapamycin treatment. (c) Arginase-1 and iNOS protein levels in CD11b⁺Ly-6G⁺Ly-6C^low G-MDSCs and CD11b⁺Ly-6G⁻Ly-6C^high M-MDSCs were examined by western blot. For both G-MDSCs and M-MDSCs, the protein levels of Arginase-1 and iNOS were increased after rapamycin treatment when compared with the control group. (d)The protein levels of Runx1 in G-MDSCs and M-MDSCs with or without rapamycin treatment were examined by western blot. The levels of Runx1 protein in both G-MDSCs and M-MDSCs were significantly reduced after rapamycin treatment in comparison with the control groups. There was almost no Runx1 expression in G-MDSCs after rapamycin treatment. (e) The levels of runx1 mRNA in G-MDSCs and M-MDSCs were examined by RT-PCR. Both G-MDSCs and M-MDSCs presented downregulated expression of runx1 mRNA after rapamycin treatment, however, the decrease of runx1 mRNA in M-MDSCs was not significant. (data were shown as mean±S.D.; n=5 duplicate tests; Arg-1, Arginase-1; CFSE, carboxyfluorescein succinimidylester; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity)