Anti cd3ε cd28

Naive CD4⁺CD25⁻ T cells were purified from LNs and spleen of WT C57BL/6, CD4^CrePkm2^fl/fl or control littermate (CD4^Cre and Pkm2^fl/fl) mice with the untouched CD4 T cell isolation kit (Miltenyi Biotec) and a biotinylated CD25 monoclonal antibody (eBioscience) by using an AutoMACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer’s protocol. Purified cells were activated with soluble anti-CD3ε:CD28 (both 1 µg/ml; BD Biosciences) on U-bottomed plates (10⁵/well). Skewing conditions were as follows: Th17, 2.5 ng/ml rhTGF-β1 (eBioscience) plus 20 ng/ml rmIL-6 (R&D Systems) with or without 20 ng/ml rmIL-23 (R&D Systems); Th1, rmIL-12, and rmIL-2 (both 20 ng/ml; R&D Systems); Th2, anti-IFN-γ (10 µg/ml), rmIL-4, and rmIL-2 (both 20 ng/ml; R&D Systems). For iT reg cell polarization, naive T cells were cultured with plate-bound CD3ε:CD28 (both 1 µg/ml; BD Biosciences) in the presence of 1 ng/ml rhTGF-β1 (eBioscience). When indicated, 0.1 µM rapamycin (Cayman Chemical), 100 µM TEPP-46 (Millipore), or 2 µM Stattic (Tocris) was used.

Naive CD4⁺CD25⁻ T cells were labeled with Cell Proliferation Dye eFluor 670 or CellTrace Violet (both 5 µM; Invitrogen) following the manufacturer’s protocol. Cells were then resuspended in culture medium and activated with anti-CD3ε:CD28 (both 1 µg/ml; BD Biosciences) in the presence or absence of rmIL-2 (20 ng/ml; R&D Systems) or cultured under Th17 cell–skewing conditions for 3 d. The stepwise dilution of the fluorescence in daughter cells as indicative of cell proliferation was assessed by flow cytometry.