Fig 1: Ketogenic diet primes human T cells to mitochondrial metabolism and memory cell development in vivo Forty-four healthy volunteers conducted a 3-week KD with a limited carbohydrate consumption of < 30 g/day. Blood was taken and analyzed prior to starting the diet (T0) and again after 3 weeks of strict adherence to the diet (T1). PBMCs were isolated. T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. A, BOCR, maximum respiration, spare respiratory capacity, and basal respiration were measured in (A) CD4+ and (B) CD8+ T cells, n = 14 individual experiments, each performed in three technical replicates.CQuantification of cellular and mitochondrial ROS using CellROX/MitoSOX, indicated by MFI FITC/MFI PE in human Pan/CD4+/CD8+ T cells, n = 9/10/10 (CellROX), 12/11/12 (MitoSOX) individual human subjects.DQuantification of mitochondrial mass using MitoTracker green, indicated by MFI FITC in human CD4+/CD8+ T cells, n = 11/8 individual human subjects.EWestern blot of mitochondrial oxidative phosphorylation proteins in CD4+/CD8+ T cells as indicated, representative of five individual experiments.FConfocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.GFlow cytometric quantification of memory T cells in vivo: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RA- (PerCP-) and CD45RO+ (Pacific Blue+) staining, representative histogram plots (top), T0 = gray, T1 = purple. Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (bottom), n = 8/7 (CM/EM) individual human subjects.HTruCulture IFN?, IL4, IL6, IL8, IL12 subunit p40, IL23, and TNFa protein quantification of LPS-stimulated whole blood samples, n = 11/12/11/10/11/10/11 individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, 25th and 75th percentiles and range (all others), crosses indicating mean (A and B). Dots indicating individual values. *P < 0.05, **P < 0.01, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate. Source data are available online for this figure.