Fig 2: 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. If applicable, 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. AMitochondrial ATP production was measured in stimulated CD4+ and CD8+ T cells, n = 14 individual human subjects.B–EExtracellular acidification rate, measured in (B) unstimulated and (C) stimulated CD4+ T cells and (D) unstimulated and (E) stimulated CD8+ T cells, n = 6 individual human subjects.F, GGlycolytic proton efflux rate (glycoPER) and compensatory (maximum) glycoPER measured in stimulated (F) CD4+ T cells and (G) CD8+ T cells, depicted as mean ± 95% CI/mean ± SEM, n = 7/8 individual human subjects.HQuantification of mitochondrial membrane potential using JC1, indicated by MFI PE/FITC in human lymphocytes in vivo. n = 4/5 (unstimulated/stimulated) individual human subjects.I, JQuantification of (I) cellular and (J) mitochondrial ROS using CellROX/MitoSOX, indicated by MFI FITC and MFI PE in native human Pan/CD4+/CD8+ T cells, n = 8 (CellROX), 10/9/10 (MitoSOX) individual human subjects.KQuantification of mitochondrial mass using MitoTracker green, indicated by MFI FITC in native human pan/CD4+/CD8+ T cells, n = 10/10/11 individual human subjects.LTruCulture Interleukin (IL)1a/ß protein quantification of LPS-stimulated whole blood samples, n = 11/12 individual human subjects.MTruCulture IL8 and IL12 subunit p40 protein quantification of unstimulated whole blood samples, n = 5 (IL8: T0 n = 4) individual human subjects. TruCulture IFN?, IL4, IL6, IL23, and TNFa protein quantification of unstimulated whole blood samples were below the lower limit of quantification. Data information: Data depicted as mean ± SEM (ECAR/glycoPER) and box plots (all other) with median, 25th and 75th percentiles and range, dots indicating individual values. *P < 0.05, **P < 0.01, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.

Fig 3: 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.

Fig 4: Beta-hydroxybutyrate enhances human T-cell immune capacity in vitro Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48 h in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T-cell RNA was isolated, and cell culture supernatant was sampled. mRNA expression of CD4+ cytokines IL2, IL4, IL8, and IL22 relative to endogenous controls, n = 13/11/10/8 biological replicates.Protein expression of IL2, IL4, IL6, and IL8, analyzed in the supernatant of stimulated PBMCs, n = 9/12/11/11 biological replicates.Th1/Th2 cell transcription factors Tbet and GATA3 quantified via RT–qPCR and flow cytometric ratio of Th1/Th2 cells following differentiation, n = 6/6/4 biological replicates.mRNA expression of CD8+ cytokines IFN?, PRF1, TNFa, GZMB, and CTLA4 in stimulated human T cells relative to internal controls, n = 15/14/10/14/9 biological replicates.Protein expression of IFN?/TNFa in the supernatant of stimulated PBMCs and cell lysis-dependent calcein fluorescence of isolated T cells, n = 8/12/9 biological replicates.Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n = 7/11 biological replicates.IL10 and TGFß1 mRNA and protein expression of human Treg cells, n = 8/9 (IL10), 5/9 (TGFß1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, 25th and 75th percentiles and range (all other). Dots indicating individual values. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.