Fig. 8. CD4⁺CD25⁺ Treg cells from TS1×HACII mice suppress responder CD4⁺ T cells in vitro.

(A) CFSE-labeled MACS-purified CD4⁺ T cells from TS1 mice were cultured with 0.1 μg/ml anti-CD3 and irradiated BALB/c splenocytes in the presence or absence of FACS-purified CD4⁺CD25⁺ cells from TS1×HACII mice. Cells were cultured at a Treg:responder cell ratio of 1:2. Responder CD4⁺ T-cell proliferation was analyzed via flow cytometry at day three of culture. (B) 6.5⁺CD4⁺CD25⁺ T cells were FACS-purified from TS1 (black circles) or TS1×HACII (gray circles) mice. Graded numbers of Treg cells were cultured with lymph nodes cells from TS1 mice and S1 peptide. Proliferation was assessed at day three of culture by [³H]-thymidine incorporation. (C) MACS-purified CD4⁺ T cells from TS1 mice were cultured with 0.1 μg/ml anti-CD3 and irradiated BALB/c splenocytes in the presence or absence of FACS-purified CD4⁺CD25⁺ cells from TS1×HACII mice. At day four of culture, IFN-γ production by the responder CD4⁺ T cells was determined by intracellular cytokine staining.