LAP⁺, CD121a⁺, and CD121b⁺ Tregs maintain purity, suppressive function, and phenotype after expansion. (A) Expression and purification based on LAP, CD121a, and CD121b on 48-hour restimulated CD25⁺ cells initially obtained with one-step CD25 selection method and expanded for 21 days. (B) Analysis of FOXP3 during an additional 21 more days of expansion for LAP⁺, CD121a⁺, and CD121b⁺ Tregs with anti-CD3/CD28 Dynabeads and IL-2 and (C) their typical Treg surface markers at the end of the 21-day expansion (42 total days in culture). Data are representative of CD121a⁺ and CD121b⁺ Tregs as well. Numbers in quadrants in panels A and B represent percentage of total population. (D) In vitro suppression assay of day 21 unseparated CD25⁺ cells (CD25+) and postpurified LAP⁺, CD121a⁺, and CD121b⁺ Tregs from panel A. Error bars represent SEM. (E) In vitro suppression assay of fresh Tregs (CD25^hi) and 21-day expanded LAP⁺, CD121a⁺, and CD121b⁺ Tregs from panel B. Error bars represent SEM.