Figure 4. STAT3 inhibition expanded potent allostimulated iTregs.

(A) Naive CD4⁺, CD45RO⁻/CD25⁻ T cells were purified through magnetic bead selection and depleted of nTregs before allogeneic coculture. nTregs are shown as CD127⁻, CD25^Bright gating on the CD4⁺ T cells before and after depletion. (B) Representative histogram demonstrates the degree of Foxp3 expression among CD4⁺, CD127⁻, CD25^Bright T cells after allostimulation of naive CD4⁺ T cells with mature moDCs (DC:T cell ratio 1:30 for 5 days) treated with either S3I-201 (STAT3 inhibitor), CAS 285986-31-4 (STAT5 inhibitor), or DMSO-diluent control. (C) Expansion of iTregs vs. Tconvs in absolute number (triplicate mean ± sd) from 5 day allogeneic cocultures treated with S3I-201, CAS 285986-31-4, or DMSO. (D) The effects of STAT3 vs. -5 inhibition on the ratio (triplicate means ± sd) of iTregs to Tconvs, calculated according to absolute number of T cells. Results are from 1 representative experiment of 5. (E) The suppressive capacity of sorted iTregs expanded from STAT3-treated allogeneic cocultures vs. purified untreated nTregs was tested at different ratios to self CD4⁺CD25⁻ responders stimulated by original allogeneic moDCs (DC:T cell ratio 1:30). Graphs show the percentage of proliferation based on ³H-thymidine incorporation and triplicate means ± sd of counts per minute at day 6. (F) Triplicate means ± sd of percentage of demethylated Foxp3 among purified iTregs expanded in allogeneic cocultures treated with S3I-201, CAS 285986-31-4, or DMSO. Results shown are from 1 representative experiment of 2. *P < 0.05, paired t test.