Fig. 4: CD28 regulates Tpex metabolism.

(A) P14 Tpex (CD73⁺ CD39^neg) and P14 Tex (CD39⁺) control (Cd28^fl/fl CreERT2^neg) or CD28^neg (homozygous Cd28^fl/fl CreERT2) were sorted from life-long LCMV chronically infected mice 2-weeks post tamoxifen for RNA sequencing analysis. Gene set enrichment analysis (GSEA) shows Hallmark Oxidative Phosphorylation gene set in CD28⁺ versus CD28^neg Tpex. (B-H) Tpex control, CD28^int and CD28^neg were sorted as described in Fig. 3A and S7A and used for metabolic analysis by seahorse (B, C, G and H) or electron microscopy (E and F). (B) Extracellular flux analysis showing oxygen consumption rate (OCR) of sorted CD28^hi (control), CD28^int and CD28^neg Tpex 15 days post tamoxifen. (C) Spare respiratory capacity (SRC) calculated as difference between maximal and basal OCR normalized to the mean of control CD28^hi Tpex. (D) MitoSOX (mitochondrial reactive oxygen species) staining in CD28^hi (black, from Cd28^fl/fl CreERT2^neg mice), CD28^int (blue, from Cd28^fl/wt CreERT2⁺ mice) and CD28^neg (red, from Cd28^fl/fl CreERT2⁺) splenic Tpex from life-long LCMV chronically infected animals, 15 days post tamoxifen. (E) Electron microscopy of CD28^hi, CD28^int and CD28^neg sorted Tpex 15 days post tamoxifen. Black arrows highlight healthy mitochondria with organized cristae, red arrows point to disorganized mitochondria. Manification 50000X, scale bar is 500 nm, bottom images are an enlargement (x3) of the field highlighted on top images, scale bar is 167 nm. (F) Maximal cristae width, symbols represent measurements for individual crista. (G) Extracellular acidification rate (ECAR) of sorted CD28^hi (black), CD28^int (blue) and CD28^neg (red) Tpex 15 days post tamoxifen. (H) Maximum ECAR post-stimulation, glycolytic reserve and compensatory glycolysis of CD28^hi (black), CD28^int (blue) and CD28^neg (red) Tpex. (I-J) Splenocytes from life-long LCMV chronically infected P14-chimera mice were stimulated with GP33 peptide with or without αCD28. (I) 2-NBDG uptake by P14 after 6h of stimulation. (J) IRF4 expression on P14 stimulated for 24h. Data in (B and G) are representative of 3 independent experiments with two to three samples per group, symbol represent mean value of all replicates analyzed and error bars indicate SEM. Data in (D, I, and J) are representative of 3 independent experiments with three to five mice per group. Data in (C and H) show combined data from three independent experiments. Symbols show individual replicates (C, H, I and J) or mice (D), bars show mean value of all samples analyzed and error bars indicate SEM. (C, D, F, H, I, and J) Significance was determined using ANOVA with Sidak’s correction for multiple comparisons *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.