Activity and redirection of transgenic CD4⁺CD25⁺ T cells. (A) 5 × 10⁴ flow-cytometrically purified CD4⁺CD25^- SJL T cells were cultured in the presence or absence of concanavalin A (conA) with or without an equal number of nontransgenic or MBP89-101-IA^s-ζ receptor transgenic CD4⁺CD25⁺ T cells, and with 2.5 × 10⁵ irradiated splenocyte feeders. Cultures were pulsed with ³H-thymidine 72 hours later and proliferation was measured by scintillation counting. (B) Tg or non-Tg CD4⁺CD25⁺ or CD4⁺CD25^- T cells were purified by flow cytometric sorting and stimulated for 4 days with anti-CD3 and anti-CD28 antibodies. The cells were then washed and recultured in the absence of stimulation, or restimulated with anti-CD3 antibody or 200 Gy irradiated 6F11 MBP89-101–specific T-cell hybridoma cells for 48 hours prior to measuring cytokine production by Bio-Plex assay or ELISA. Coculture of the 6F11 T cells with RMTCs, which express the MBP89-101-IA^s ligand on their chimeric receptor, would be expected to stimulate both the hybridoma and the RMTCs. To control for 6F11-produced cytokines, irradiated 6F11 cells were also stimulated with anti-CD3 antibody. No IFN-γ, IL-10, or TGF-β was produced, while IL-4 levels were approximately 10% of that produced when RMTCs were cocultured with 6F11 cells (data not shown). Error bars indicate 1 SD.