Figure 2.

MSC co-culture stabilises FOXP3 expression in iTregs in the presence of inflammatory cytokines. (a) Suppressive effects of iTregs were measured day 21 of ex vivo expansion of iTregs in IL-2/media vs MSC co-culture, CD25^high FACS sorted iTregs were used to test suppressive function. 4 days after stimulation, CFSE dilution was examined by FACS (n = 3). (b) Expression of FOXP3 on iTregs was measured following 72 h restimulation with CD3 and CD28 antibodies (1 μg ml⁻¹) in the presence of inflammatory cytokines IFNγ (10 μg ml⁻¹), IL-6 (10 μg ml⁻¹), and TNF (10 μg ml⁻¹) at concentration of 5 × 10⁵ cells ml⁻¹ and analyzed by FACS. Intracellular IFNγ and IL-17 were detected by flow cytometry analysis. Data are representative of two independent experiments ± SD (n = 6–7) **p < 0.01, ***p < 0.001 paired t test. See also Supplementary Fig. S2.