Expansion rates and phenotypic characteristics of CD45RA⁺ and CD45RA^– CD4⁺CD25^high T cells in long-term cultures. (A) Expansion rates (left panel) and coexpression of CD62L and CCR7 (right panel) on in vitro–expanded CD4⁺CD25^high T cells and sorted CD45RA⁺ and CD45RA^– subsets thereof, respectively. Unseparated CD4⁺CD25^high T cells (diamonds), or corresponding CD45RA⁺ (triangles) and CD45RA^– (inverted triangles) subpopulations were FACS-sorted from leukapheresis products and expanded for up to 18 days. Open symbols in the right panel represent values for gated subpopulations in freshly isolated PBMCs (n = 6). Phenotypic analyses of cultured cells were performed at indicated time points during expansion (filled symbols). Phenotype of CD4⁺CD25^– T cells within PBMCs or upon in vitro culture is shown for comparison (circles). Combined data from 10 independent cultures with cells from 8 different donors. Error bars represent SEM. (B) Intracellular CTLA-4 expression of CD45RA⁺ (shaded histogram) and CD45RA^– CD4⁺CD25^high T cells (bold solid line) or CD4⁺CD25^– T cells (dashed line) after 2 weeks of in vitro expansion. Long dashed histogram represents isotype control. (C-D) FOXP3 mRNA (C) and protein expression (D) by sorted CD45RA⁺ (RA+) and CD45RA^– (RA–) subpopulations or total CD4⁺CD25^high (CD25^high) and CD4⁺CD25^– T cells (CD25^neg) during in vitro expansion. (C) Total RNA was isolated from expanded cells on the days indicated. FOXP3 mRNA expression was determined by qRT-PCR and normalized to 18S rRNA. FOXP3 mRNA expression by sorted and 7-day expanded CD4⁺CD25^– T cells was arbitrarily set to 1. One representative example (left panel) and combined data (right panel) for expanded CD45RA⁺ (filled symbols) and CD45RA^– cells (open symbols) from 4 independent cultures are shown. (D) Cells were isolated on the days indicated, rested, and then stained for CD4, CD25, and FOXP3. Representative results from 1 of 3 independent experiments.