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. 2019 Mar 7;116(13):6298–6307. doi: 10.1073/pnas.1812471116

Fig. 7.

Fig. 7.

The tolerogenic microenvironment dictates the ex vivo induction of FOXP3 iTregs by CD4-naive TH0 cells transdetermination. (A) Analysis of FOXP3+ expression in iTregs generated ex vivo from polyclonally stimulated naive CD4+ T cells with different nTreg polarizing media. Naive CD4+ T cells were stimulated for 12 d with plate-bound anti-CD3 (4 µg/mL) in the presence of IL-2 (100 IU/mL). Where indicated, TGFβ (5 ng/mL), rapa (10 nM), and PGE2 (1 µM) were added. (A1 and A2) Frequency (A1) and expression level (evaluated by MFI) (A2) of FOXP3 in CD4+ T cell culture. (B) Ex vivo suppressive capacity of human Tregs generated with the polarizing medium containing TGFβ (5 ng/mL), rapa (10 nM), and PGE2 (1 µM). (B1 and B2) The suppressive capacity of ex vivo-generated Tregs was evaluated in quiescent (B1) and inflammatory (B2) conditions with the standard polyclonal nTreg assay. CFSE-labeled Tconvs were cocultured with ex vivo-generated Tregs at different ratios. The percent inhibition of TconvCFSE proliferation by Tregs is depicted. Fresh nTregs and Tconvs served as controls. (B3) IL-17 production by ex vivo-generated iTregs measured in supernatant culture by ELISA. **P < 0.01; ***P < 0.001; ****P < 0.0001.