Antigen-specific in vivo expansion of CD4⁺CD25⁺ T cells from Marilyn mice. (A) FACS analysis of lymph node cells from female Marilyn mice labeled with anti-CD4, Vβ6, and CD25. (B) FoxP3 mRNA levels of purified CD25⁺ and CD25^– cells from female B6 and Marilyn mice were determined by real-time PCR analysis on fresh splenocytes. (C-D) In vivo expansion of DBY-Tregs: B6- or DBY-Tregs labeled with 5 μM CFSE were transferred together with 10 × 10⁶ female or male 45.1 splenocytes into recipient female 45.1 mice; n = 3 mice per group. At day 6, splenocytes were labeled with CD25, CD4, and CD45.2. Dot plots are gated on CD4⁺CD45.2⁺ cells. Statistical analysis was performed using the Mann-Whitney t test; *P ≤ .05. Panel A is representative of 3 experiments, and panels B-D are each representative of 2 experiments. Error bars in panels B and C show standard error of the mean.