Enhancement of T cell responses after Treg depletion. (A and B) CD4⁺CD25^high cells were isolated by FACS sorting and incubated for 6 hours in the presence of increasing concentrations of DAB₃₈₉IL-2 (left panel). In order to determine DAB₃₈₉IL-2–mediated toxicity, PBMCs and PBMCs admixed with CD4⁺/CD25^high cells at a 1:1 ratio were incubated with or without DAB₃₈₉IL-2 (5 nM) for 6 hours. In all experiments, cell viability was determined through MTT assays (A) or 7-AAD staining (B). Representative results from 3 evaluable subjects are presented. (C) Treg-depleted PBMCs (PBMC+DAB) or nondepleted PBMCs (PBMC–DAB) from an RCC patient were analyzed in allogeneic MLRs using DCs at a responder to stimulator ratio of 20:1. Cell proliferation was significantly inhibited when isolated CD4⁺/CD25^high cells were added to PBMCs at a 1:1 PBMC/CD4⁺CD25^high cell ratio (DC+Treg). This inhibition was reversible when the added CD4⁺/CD25^high cells were pretreated with DAB₃₈₉IL-2 (5 nM) for 48 hours (DC+Treg+DAB). Exposure of PBMCs to DAB₃₈₉IL-2 during the T cell–priming phase (day 2) led to complete inhibition of T cell proliferation (DC+DAB). (D) DCs transfected with mRNA encoding hTERT or MART-1 were used to stimulate CTL from Treg-depleted (filled symbols) or nondepleted (open symbols) human PBMCs.In addition, DCs loaded with MART-1–derived peptide 26-35 ELAGIGILTV (MART-1 pep) were used as stimulators. Following 2 stimulation cycles, CTLs were analyzed for their capacity to lyse their cognate (squares) or control targets (circles). As control targets, DCs loaded with GFP mRNA (mock transfected) or irrelevant peptide were used.