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. 2019 Apr 23;9:6374. doi: 10.1038/s41598-019-42932-1

Figure 3.

Figure 3

In vitro–generated G-MDSCs cultured with rapamycin had increased immunosuppressive ability. (A) In vitro–generated G-MDSCs were cultured with DMSO, 1 μM rapamycin (Rapa), 5 μM DON, or 1 μM rapamycin plus 5 μM DON (Rapa + DON), and were then isolated by manual MACS against Ly6G+ and their immunosuppressive ability assessed. CFSE-labeled CD4+ T cells were cultured alone or co-cultured at a 1:1 ratio with in vitro–generated G-MDSCs and stimulated by coating the culture plate with anti-CD3 and CD28 mAbs. After 3 days of culture, the proliferation of CD4+ T cells cultured alone or with G-MDSCs was examined by measuring the CFSE fluorescence using flow cytometry. Results shown are representative of four separate experiments. (B) Real-time PCR analysis of the TGFβ and PD-L1 mRNA levels in isolated in vitro–generated G-MDSCs cultured under the four drug regimens. Each experiment was performed in triplicate. Data are representative of 5 independent experiments. Bars show mean ± SEM. *P < 0.05; **P < 0.01.