Corticosterone

CD8⁺ T cells from splenocytes and lymph nodes were isolated using CD8 microbeads (Miltenyi). Cells were further stained with antibodies against CD8, CD62L and CD44, and CD8⁺CD62L^hiCD44⁻ naive cells were sorted by BD FacsAria (BD Biosciences). Sorted cells were cultured for 9 days as described below in DMEM supplemented with 10% (vol/vol) FCS, 50 mM mercaptoethanol, 1 mM sodium pyruvate, nonessential amino acids, L-glutamine, and 100 U/ml penicillin and 100 g/ml streptomycin. Specifically, naive CD8⁺ cells were stimulated with plate bound anti-CD3 (145–2C11, 1 μg/ml) and anti-CD28 (PV-1, 1μg/ml) in the presence of either 10 nM dexamethasone (Sigma), 100 nM Corticosterone (Fisher Scientific), 25 ng/ml IL-27 (R&D), or both dexamethasone and IL27 for 3 days. Cells were then rested in the presence of 5 ng/ml IL2 (Miltenyi) for 3 days followed by restimulation with plate bound anti-CD3 (145–2C11, 1 μg/ml) and anti-CD28 (PV-1, 1μg/ml) in the presence of either 10nM dexamethasone ( Sigma), 100 nM Corticosterone (Fisher Scientific), 25 ng/ml IL-27 (R&D), or both dexamethasone and IL27 for an additional 3 days.

CD4⁺ T cells or CD8⁺ T cells from splenocytes and lymph nodes were isolated using microbeads (Miltenyi Biotec). CD4⁺CD25⁻CD62L^hi CD44⁻ and CD8⁺CD25⁻CD62L^hiCD44⁻ naïve cells were sorted by BD FACSAria (BD Biosciences) and cultured for 9 days as previously described (22 (link)). Briefly, naïve cells were stimulated with plate-bound anti-CD3 (145–2C11; 1 μg/ml) and anti-CD28 (PV-1; 1 μg/ml) in the presence of 100 nM corticosterone (Thermo Fisher Scientific) for 3 days. Cells were then rested in the presence of IL-2 (5 ng/ml; Miltenyi Biotec) for 3 days, followed by restimulation with plate-bound anti-CD3 (145–2C11; 1 μg/ml) and anti-CD28 (PV-1; 1 μg/ml) in the presence of 100 nM corticosterone (Thermo Fisher Scientific) for an additional 3 days.