For analysis of the OCR (in pmol/min) and ECAR (in mpH/min), the Seahorse XFe96 metabolic extracellular flux analyzer was used (Seahorse Bioscience). Total or sorted CD4+ T cells were resuspended in serum-free, unbuffered RPMI 1640 (Sigma-Aldrich) and were plated onto Seahorse cell plates (2.5 × 105 cells per well) coated with Cell-Tak (BD Biosciences) to enhance T cell attachment. Perturbation profiling of the use of metabolic pathways was done by the addition of oligomycin (1 μM), FCCP (2 μM), and rotenone (1 μM; all are given as final concentrations, all from Sigma-Aldrich). For a description of how the various metabolic parameters were calculated, see fig. S1. The contribution of FAO to OCR was assessed by the addition of etomoxir (40 μM; Sigma-Aldrich). Assessment of the OCR of isolated mitochondria was also performed with the Seahorse XFe96 metabolic extracellular flux analyzer and followed a protocol described previously (38). Sorted effector memory CD4+ T cells were plated onto Seahorse cell plates, as described earlier, in RPMI/10% AB containing TGF-β (5 ng/ml) where indicated in the figure legends and cultured at 37°C and 5% CO2 for 5 hours. The medium was then exchanged for mannitol and sucrose buffer [70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM Hepes, and 1 mM EGTA (pH 7.2); all reagents from Sigma-Aldrich] containing fatty acid–free bovine serum albumin (4 mg/ml; Sigma-Aldrich). During the assay, first plasma membrane permeabilizer (1 nM; Seahorse Bioscience) and then the following substrates (final concentrations given) together with adenosine diphosphate (1 mM) and FCCP (2 μM; both from Sigma-Aldrich) were added: complex I: pyruvate (5 mM)/malate (2.5 mM; both from Sigma-Aldrich); complex II: succinate (10 mM; Sigma-Aldrich); complex III: duroquinol (0.5 mM; TCI America); and complex IV: tetramethyl-p-phenylenediamine (0.5 mM)/ascorbate (2 mM; both from Sigma-Aldrich). Last, the following specific inhibitors were added: complex I: rotenone (1 μM); complex II: malonate (40 μM); complex III: antimycin A (20 μM); and complex IV: sodium azide (20 mM; all from Sigma-Aldrich).

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