Figure 3. In vivo expanded T_Regs do not convert to Th17 cells and retain their characteristic suppressive function.

(A) IL-17A levels in the sera of nT_Reg reconstituted animals were analyzed by ELISA. Error bars indicate standard deviation for six different animals tested. Supernatants from normal human PBMCs stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) served as positive control. (B) T_Regs obtained from reconstituted mice and in vitro expanded T_Regs were tested for their ability to suppress proliferation of autologous CD4⁺CD25⁻ T cells at different T_Reg to CD4⁺CD25⁻ responder T cell ratios. Responder cells were stimulated with anti-CD3/CD28 beads and CFSE dilution was analyzed after 5 days of culture under the following conditions: autologous T cells cultured in medium without stimulation (unstimulated), stimulated with anti-CD3/anti-CD28 coated micro beads in the absence of T_Regs (stimulated), in the presence of cultured T_Regs (in vitro T_Regs) or in the presence of T_Regs isolated from reconstituted mice 12 days after reconstitution (In vivo T_Regs). Composite data from all the three animals tested are shown. (C) The functional stability of T_Regs obtained 33 days after in vivo reconstitution was also analyzed by CFSE dilution of CD4⁺CD25⁻ responder cells. Representative data from one of two animals tested is shown. Data depicts CD4⁺CD25⁻ responder cells cultured with medium alone (a), stimulated in the absence of in vivo T_Regs (b) and stimulated in the presence of in vivo T_Regs (c).