There is an inverse relationship between TCR/pMHC affinity and the optimal peptide concentration for Foxp3 induction in vitro. (A) EC₅₀ values for the experiment shown were determined as the peptide concentration resulting in 50% of maximal proliferation. LN cells from 5C.C7 TCR transgenic RAG2^−/− mice were stimulated with irradiated splenocytes and the indicated peptide for 60 h. ³H-methyl-thymidine was used to assess proliferation. (B and C) 5C.C7 RAG2^−/− LN cells were cultured with irradiated splenocytes in the presence of IL-2 and the indicated peptide for 4 d before flow cytometry analysis of the frequency (B) or number (C) of Foxp3⁺ cells. (C) Percentage of maximum cell number is shown, with Foxp3⁺ 5C.C7 represented as a solid line and Foxp3⁻ cells as a dashed line. In A and B, error bars show mean ± SD, with n = 3 or n = 2 wells, respectively. Data are representative of at least three independent experiments.