Irrapc

Memory CD4⁺ T cells were cultured in complete RPMI (RPMI (Labtech) supplemented with 10% FCS (Sigma Aldrich), 2 mM L-glutamine with 1% penicillin/streptomycin (Sigma Aldrich) with irradiated antigen-presenting cells (irrAPC), anti-CD3 (eBioscience) and, where indicated, in the presence of dichloroacetate (DCA) (10 mM, Sigma Aldrich), rapamycin (20 nM, Sigma Aldrich), 5-(Tetradecyloxy)-2-furoic acid (TOFA) (1.2 μg/ml, Sigma Aldrich), C-75 (0.6 μg/ml, Sigma Aldrich), cerulenin (3.1 μM, Sigma Aldrich), or palmitate (25 μM, Seahorse Biosciences) for 5 days. Isolated CD161⁺, CD161⁻, and Tconv cells were cultured with irradiated antigen presenting cells (irrAPC), anti-CD3 and IL-2 (20 u/ml, eBioscience) for 6 days or in some cases with PMA/ionomycin for 18 h prior to Seahorse analysis. Isolated Treg cells were cultured using the Treg cell expansion kit (Miltenyi Biotec) in the presence of IL-2 (500 u/ml) for 6 days prior to Seahorse analysis. During glucose deprivation, memory CD4⁺ T cells were cultured in glucose-free complete RPMI (RPMI (Biosciences) supplemented with 10% dialysed FCS (Sigma Aldrich), 1% vitamin cocktail (InvivoGen), and 1% selenium/insulin cocktail (InvivoGen) supplemented with either 10 mM glucose (Sigma Aldrich) or 10 mM galactose (Sigma Aldrich) for 5 days.