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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Cytotherapy. 2021 Apr 20;23(8):704–714. doi: 10.1016/j.jcyt.2021.02.118

Figure 1: Sort-purified naïve tTreg maintain Foxp3 and suppressive function after multiple stimulations. In vitro expansion of naïve human tTreg and naive CD4 T cells.

Figure 1:

Naïve PB CD4+ T cells and tTreg were sort-purified (CD4+25–127+45RA+ and CD4+25hi127−45RA+, respectively) and expanded with anti-CD3 mAb-loaded KT64/86 using two rounds of stimulation. tTreg were also stimulated 5 times in the presence of rapamycin. (A) Average expansion for CD4 T cells (Teff) and tTreg-No Rapa. (B) Representative example (left) and average percent CD127−Foxp3+ (CD4-gated). (C) Average (n=3) %TSDR demethylation of naïve tTreg stimulated once or twice in the absence of rapamycin compared with Foxp3 expression. (D) Average expansion for naïve tTreg stimulated 1–5 times in the presence of rapamycin. (E) Representative example (above) and individual data points along with average percent CD127−Foxp3+ (CD4-gated) for tTreg+Rapa stimulated 1–5 times as indicated. Mean ± 1 SEM is shown. (F) Average relative Foxp3 expression in naïve tTreg expanded in rapamycin with the indicated number of stimulations normalized to 1 stimulation. (G) Percent suppression of in vitro CD8 T cell proliferation tTreg/PBMNC ratios of 1:2, 1:4 and 1:8 for tTreg cultures stimulated the indicated number of times in the presence or absence of rapamycin. Data shown are mean ± SEM. No significant differences were observed in suppression. All results are from 3–4 independent experiments.