IL-2 release as a function of time after in vitro culture of CD4⁺CD25⁺ and CD4⁺CD25^– cells. Cells were isolated by magnetic cell sorting from DO11.10 mice 60 days after gene transfer with AAV-EF1α-ova (A) or AAV-EF1α-GFP (B). All cultured cells were stimulated in vitro with ova (100 μg/mL). APC indicates antigen-presenting cells. Note that neither APCs alone nor CD25^– cells nor CD25⁺ and CD25^– cells cocultured at 1:1 ratio yielded a response to ova antigen for both experimental groups. Panel C shows CD4⁺CD25⁺ (from AAV-EF1α-ova–transduced DO11.10 mice) dose-dependent suppression of IL-2 release from CD4⁺CD25^– cells (isolated from AAV-EF1α-GFP DO11.10 mice). Data represent average from n = 4 cultures based on cells pooled from several mice.