Ionomycin

Cells were stained with surface antibodies and Fixable Viability Dye (Zombie UV, BioLegend) in PBS containing 2% fetal calf serum (FCS) (FACS buffer) for 30 min at 4 °C in the presence of human (BioLegend) or mouse (BioXcell) Fc block. For experiments involving intracellular staining of cytokines, T cells were stimulated for 3 h (mouse) and 6 h (human) with phorbol 12-myristate 13-acetate (PMA; 50 ng ml⁻¹) (BioGems) and ionomycin (1 µg ml⁻¹) (BioGems) in the presence of brefeldin A (1 µg ml⁻¹) (GolgiPlug, BD Biosciences). Cells were washed with FACS buffer followed by fixation with the Foxp3 fixation/permeabilization kit (eBioscience) in accordance with the manufacturer's instructions, and stained with intracellular antibodies for 60 min at 4 °C.

CD4⁺ T cells or CD8⁺ T cells were purified from PBMCs using magnetic positive selection with biotin human anti-CD4 or anti- CD8 antibodies, respectively, followed by incubation with streptavidin-coupled magnetic microbeads (BioLegend) and positive selection with magnets. Purity was assessed by flow cytometry on a BD LSRFortessa using anti-CD4 (RPA-T4, 1:400) antibodies and anti-CD8 (RPA-T8, 1:400) antibodies. Cells were plated on 48-well plates, coated with anti-CD3 (1 μg ml⁻¹) and anti-CD28 (10 μg ml ) antibodies, in RPMI medium or DMEM, supplemented with 10% FCS, penicillin, streptomycin, HEPES, glutamine, pyruvate, nonessential amino acids and β-mercaptoethanol diluted with PBS (1:1), in the presence of IL-2 (10 ng ml⁻¹, Peprotech) and IL-12 (10 ng ml⁻¹, Peprotech) (T_H1 conditions), with or without amino acids. Human CD4⁺ T cell cultures were supplemented on days 0, 1 and 2 with 5 mM NaCl or 5 mM BHB and collected after 6 days of culture. Cultured cells were stimulated for 6 h with PMA (50 ng ml⁻¹) (BioGems) and ionomycin (1 µg ml⁻¹) (BioGems) in the presence of brefeldin A (1 μg ml⁻¹) (GolgiPlug, BD Biosciences), stained for viability (Zombie UV Fixable Viability Dye (BioLegend)) and surface markers, fixed and permeabilized with 3.7% formaldehyde (Sigma) and 0.1% NP-40, respectively, and stained for intracellular cytokines.

Naive CD4⁺ T cells or CD8⁺ T cells were purified from mouse spleens using the magnetic negative selection kit (Mojosort mouse CD4 naive T cell isolation kit, BioLegend; CD8⁺ T cell isolation kit, Miltenyi) according to the manufacturer’s instructions. Purity was assessed by flow cytometry on a BD LSRFortessa Flow Cytometer using anti-mouse CD4 (RM4-5, 1:400) and anti-mouse CD8 (Ly-3, 1:400) antibodies. Cells were plated on 48-well plates, coated with anti-CD3 (1 μg ml⁻¹) and anti-CD28 (10 μg ml⁻¹) antibodies, in RPMI medium supplemented with 10% FCS, penicillin, streptomycin, HEPES, glutamine, pyruvate, nonessential amino acids and β-mercaptoethanol diluted with PBS (1:1) in the presence of IL-12 (3 ng ml⁻¹, Peprotech) (T_H1 conditions). Cultures were supplemented on days 0, 1 and 2 with 5 mM NaCl or 5 mM BHB. Mouse CD4⁺ T cells were collected at days 3 and 6 of culture. Viability was assessed by Zombie UV Fixable Viability Dye (BioLegend) and cytokine production was measured by intracellular cytokine staining after 3 h stimulation with PMA (50 ng ml⁻¹) (BioGems) and ionomycin (1 µg ml⁻¹) (BioGems) in the presence of brefeldin A (1 μg ml⁻¹) (GolgiPlug, BD Biosciences). Cells were stained for viability and surface markers, fixed with 3.7% formaldehyde (Sigma), permeabilized with 0.1% NP-40 and stained for intracellular cytokines.