Extended Data Fig. 1. Chronic T cell stimulation induces T cell exhaustion.

All experimental analyses were conducted eight days after initial stimulation unless otherwise specified. (a-c) Expression of inhibitory immunoreceptors (PD-1, LAG-3, PD-L1) and intracellular cytokine production (IFN-γ and TNF) in acutely and chronically stimulated T cells following re-stimulation with PMA and ionomycin. (d) Expression of Glut1 in acutely or chronically stimulated OT-I T cells with or without restimulation using bead-bound anti-CD3. Actin is used as a loading control. Experiment was repeated three times with similar results. Uncropped blot can be found within Source Data. (e) Gene set enrichment plot showing that genes associated with chronically stimulated polyclonal T cells in vitro are enriched for genes upregulated in exhausted CD8+ T cells (Texh) but not anergic T cells¹⁵. (f) Killing of peptide-pulsed B16 cells. Luciferase-expressing B16 cells pulsed with Ova peptide at the indicated doses for 4 h were co-cultured with acutely or chronically stimulated T cells for 24 h. The following day, cells were lysed and luciferase expression was assessed using a luminometer. (g) Normalized isotopologue abundance of intracellular lactate in acutely and chronically stimulated T cells following 6 h of re-stimulation by plate-bound anti-CD3 in the presence of U-¹³C-Glucose. Abundance was normalized to cell number at the time of harvest. (h) Median lactate excreted per molecule of glucose consumed in acutely and chronically stimulated T cells following initial stimulation. P values were calculated by unpaired, two-sided Student’s t-test (f-h) relative to acutely stimulated T cells or based on 1,000 permutations by the GSEA algorithm and not adjusted for multiple comparisons (e). Data are presented as the mean ± s.d. of n=3 biologically independent samples from a representative experiment. **P<0.01.