Phenotype of CD4⁺FoxP3⁺ T cells in the peripheral blood of ACT patients. (A) Cell-surface expression of CD25, CD27, and CD127, as well as intracellular CTLA-4 and FoxP3 expression, were evaluated on CD4-gated T cells from 1 of 29 patient PBMC samples analyzed before, 1 week, and 4 weeks after treatment. Numbers in the top left quadrants indicate the mean fluorescence intensity of CD25/FoxP3 or CTLA-4/FoxP3 expression. (B) Expression of CD45RO on gated CD4⁺FoxP3⁺ T cells from 1 of 8 representative patient PBMC samples obtained before, 1 week, and 4 weeks after treatment. (C) Expression of Ki-67 on gated CD4⁺FoxP3⁺ T cells from 1 of 29 representative patients before, 1 week, and 4 weeks after treatment. (D) CFSE dilution of labeled CD4⁺CD25⁻ effector T cells stimulated with anti-CD3 Ab was assessed after a 96-hour coculture with CD4⁺CD25^high T cells containing > 60% FoxP3⁺ T cells at varying effector T cell-to-Treg ratios. The percentages of T cells undergoing at least 1 division are shown, and data are representative of 2 experiments carried out with week-4 PBMC samples from 2 patients in the CD8⁺ young tumor-infiltrating lymphocyte (TIL) trial.