Figure 5.

CD39/73 signaling induces MSC mitochondrial transfer to iTregs during IL-2 driven expansion. (a) Mitochondrial quantity was measured from IL-2/media or MSC co-culture expanded iTregs by RT-PCR (n = 6–8). Anti-CD73 antibody (10 μg ml⁻¹) was added in MSC co-culture. Cells were collected at 72 h after mtGFP lentiviral transduced MSC co-culture. (b) Flow analysis of mtGFP⁺ iTregs in anti-CD73 blocking and cytochalasin B treatment. Cells were stained with CD4 antibody in order to identify iTregs at 72 h. (c) Analysis of mtGFP⁺ CD4⁺ iTregs after incubation with CD39 (100 nM) and CD73 (100 nM) inhibitor treated MSC (n = 7–14). Representative flow plot, gated for CD4⁺ iTreg cells after MSC co-culture. (d) Protein and RNA expression of Miro1 in MSC were measured after co-culture with iTreg. (e) After co-culture in the presence of CD39 inhibitor (n = 8–9). The image intensities for western blots were normalized to beta actin. Data are representative of 3–4 different experiments. (f) Effect of CD39 and CD73 inhibitors on suppressive functions of iTreg during MSC co-culture expansion. Inhibitor was added to iTreg and MSC co-culture. Data are representative of n = 3 independent samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 paired t test. See also Supplementary Fig. S6.