Fig. 3

The immunosuppressive effect of T-MSCs on T cell proliferation is partially dependent on NO. (A) The expression levels of Cxcl9, Cxcl10, Cxcl11, iNos, Cox2 and Pd-l1 were detected by real-time PCR at 12 h, 24 h and 48 h after MSCs were stimulated with 20 ng/mL IFN-γ and TNF-α alone or synergically. (B) The expression of iNOS in T-MSCs was detected by flow cytometry after 24 h synergistic stimulation with different concentrations of IFN-γ and TNF-α. (C) The expression of iNOS in T-MSCs was detected by flow cytometry after different time synergistic stimulation with IFN-γ and TNF-α (20 ng/mL each). (D) Cell culture supernatant was obtained from different treatment groups in Fig. 3B, total nitrates level was assayed by a modified Griess reagent. (E) Cell culture supernatant was obtained from different treatment groups in Fig. 3C, total nitrates level was assayed by a modified Griess reagent. (F) T-MSCs and CFSE-labeled splenocytes were co-cultured for 48 to 72 h at the ratio of 1:20, 1:60, 1:180 and 1:540, respectively (anti-CD3/CD28 (1 µg/mL each) was added into the medium to stimulate the proliferation of splenocytes), and then fluorescence intensity of CFSE was detected by flow cytometry. (G) T-MSCs from Nos2^−/− or WT C57BL/6 mice were cocultured with fresh C57BL/6 splenocytes plus anti-CD3/CD28, with or without the iNOS inhibitor, L-NMMA (1 mM). Cell proliferation was assayed after 48 to 72 h. (H) The groups was consistent with Fig. 3G (expect for the splenocytes were not stained with CFSE), and after 48 h of culture, CD3 and Ki-67 were stained, then the proportion of CD3⁺Ki-67⁺ was detected by flow cytometry. Data are represented as mean ± SEM. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001