Anti cd3 and anti cd28 antibodies

CD4⁺CD25⁻CD62L^hiCD44^low naïve T cells were isolated from the spleen and peripheral lymph nodes of indicated mice and activated by plate-bound anti-CD3 and anti-CD28 (5 μg/ml). T_H17 cells were induced with the combination of human TGF-β (1 ng/ml), mouse interleukin 6 (mIL-6) (20 ng/ml), mIL-1β (10 ng/ml), and IL-23 (25 ng/ml). To analyze the maintenance of T_H17 cells, 3 days later, the developed T_H17 cells were restimulated with anti-CD3/28 and treated with 2 μM 4-OHT (Sigma-Aldrich) to induce Rorc deletion under T_H17-polarizing condition. About 48 or 96 hours later, the cells were collected for further analysis. To analyze the plasticity, the developed T_H17 cells were treated with 2 μM 4-OHT for 2 days, collected, and restimulated with anti-CD3/28 and cultured under various conditions. T_H1 or T_H1/17 cells were induced by the combination of mIL-12 (20 ng/ml), mouse interferon gamma (mIFN-γ) (10 ng/ml), and IL-23 (25 ng/ml) as indicated. T_H2 cells were induced by IL-4 (20 ng/ml). T_reg cells were induced by mIL-2 (10 U/ml) and human TGF-β (2 ng/ml). Four days later, the cells were collected for further analysis. Anti-CD3 and anti-CD28 antibodies were purchased from Bio X Cell, IL-23 and human TGF-β were from R&D System, and other cytokines were from PeproTech.