LAP⁺ and CD121b⁺ Tregs are anergic and manifest potent T-suppressor activity. CD4⁺CD25⁻ T cells were stimulated with anti-CD3 and APCs alone (■) or in the presence of various numbers of unseparated CD25⁺ or purified LAP⁺, LAP⁻, CD121b⁺, and CD121b⁻ cells from 48 hours of restimulated day 14 cultures generated in the absence (A) or presence (B) of rapamycin. Error bars represent SEM. ³H-TdR incorporation was determined after 72 hours of stimulation. CFSE-labeled CD4⁺CD25⁻ T cells were stimulated with anti-CD3 and APCs alone or in the presence of unseparated CD25⁺ or purified LAP⁺, LAP⁻, CD121b⁺, and CD121b⁻ cells from 48-hour restimulated day 14 cultures generated in the absence (C) or presence (D) of rapamycin. Cocultures were performed at responder to suppressor ratios of 4:1 and 8:1. CFSE dilution was measured by FACS analysis after 72 hours of culture. Number in each quadrant represents percentage of dividing cells from the total population. Data are from 1 donor representative of 6.