Characterization of regulatory CD4⁺CD25⁺ cells isolated from AAV-EF1α-ova–transduced DO11.10-tg RAG-2^−/− mice. Expression of FoxP3 in a subpopulation of CD4⁺ splenocytes derived from DO11.10-tg BALB/c (RAG⁺) or DO11.10-tg RAG-2^−/− (RAG⁻) mice. (A) Splenocytes were sorted into CD4⁺ cells and further separated into CD25⁺ or CD25⁻ cells. cDNA from each population was subjected to PCR using FoxP3- or HPRT (hypoxanthine–guanine phosphoribosyl–transferase)–specific primers. (B) Quantification of relative FoxP3 mRNA levels in indicated CD4⁺ T-cell subsets. cDNA samples were subjected to real-time quantitative PCR analyses using primers and an internal fluorescent probe specific for FoxP3 or HPRT. The relative quantity of FoxP3 in each sample was normalized to the relative quantity of HPRT. Shown are average results for 3 independent experiments (± SD). (C) Flow cytometry for FoxP3 expression. Shown are examples for percent CD25⁺FoxP3⁺ of CD4⁺ splenocytes in individual mice (8 weeks after gene transfer with AAV-ova or naive control). (D) Cell dose–dependent suppression of IL-2 expression in DO11.10-tg CD4⁺CD25⁻ cells by CD4⁺CD25⁺ isolated from AAV-EF1α-ova–transduced DO11.10-tg RAG-2^−/− mice upon in vitro coculture (in the presence of APCs) and stimulation with ova. Data represent average (± SD) from n = 4 cultures based on cells pooled from several mice.