Fig 2: Cytokine secretion of T cells in patients with NSCLC was promoted after rocaltrol treatment. (A) 1α,25(OH)2D3 levels (baseline) in sera of patients with NSCLC, before therapy with docetaxel and rocaltrol (n=17) or docetaxel (n=12) alone. (B) Overview of study design. Patients with NSCLC received one course of intravenous docetaxel (75 mg/m2) followed by treatment with rocaltrol at a dose of 0.5–2.0 µg/day for total of 3 weeks. (C–E) CT scans and waterfall plot showing changes of tumor size in patients with NSCLC who received one course of intravenous docetaxel (75 mg/m2), followed by oral rocaltrol at a dose of 0.5–2.0 µg/day for total of 3 weeks. Waterfall plot of percentage of patients with recurrence and no recurrence after treatment with docetaxel and rocaltrol or docetaxel alone. (F–H) Graphs showed the percentage of IFN-γ+, TNF-α+, perforin+, and GZMB+ cells in CD4+, CD8+, and Vγ9Vδ2+ T cells of patients with NSCLC, before (pre) and after (post) therapy with docetaxel (75 mg/m2) and rocaltrol (0.5–2.0 µg/day) or docetaxel alone at the indicated time points followed by activation of anti-CD3 and anti-CD28 antibodies for 4 hours. Flow cytometry and statistical analysis for the percentage of IFN-γ+, TNF-α+, perforin+, and GZMB+ on T cells (CD4+, CD8+, and Vγ9Vδ2+). (I) Graphs showed the proportion of IFN-γ+, TNF-α+, perforin+, and GZMB+ in CD4+, CD8+, and Vγ9Vδ2+ T cells from patients with NSCLC, after (post) therapy with docetaxel (75 mg/m2) and rocaltrol (0.5–2.0 µg/day) or treatment with docetaxel alone for 3 weeks. n=17 NSCLC donors. (G, H) Paired Student’s t-test. (A, I) Unpaired Student’s t-test. Significance was set to p<0.05 and represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GZMB, granzyme B; IFN-γ, interferon gamma; n.s., not significant; NSCLC, non-small cell lung cancer; TNF-α, tumor necrosis factor alpha.

Fig 3: Ketogenic diet enhances human T-cell immune capacity in vivo Healthy volunteers conducted a 3-week KD with a limited carbohydrate consumption of < 30 g/day. Blood was taken and analyzed prior to (T0) and post-KD (T1). PBMCs were isolated. T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n = 17/18/17 individual human subjects.Tbet and GATA3 mRNA expression in CD4+ T cells, n = 7 individual human subjects.Flow cytometric quantification of Th1/Th2 cells and respective ratio of Th1/Th2 cells, n = 7 individual human subjects.IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n = 19/9 individual human subjects.IFN? GZMB, PRF1, and CTLA4 mRNA expression in CD8+ T cells, n = 16/18/17/17 individual human subjects.Relative CD8+ cell lysis activity as measured by calcein fluorescence of isolated T cells, n = 4 individual experiments. Data information: Data depicted as box plots with median, 25th and 75th percentiles and range. Dots indicating individual values. *P < 0.05, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.

Fig 4: 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 5: Ketone bodies shift T-cell metabolism to oxidative phosphorylationHuman 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. Pan T cells and CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. A–COCR, maximum respiration, spare respiratory capacity, and basal respiration were measured in (A) pan T cells, (B) CD4+ T cells, and (C) CD8+ T cells, n = 9 (pan T), n = 3/4 (CD4 unstimulated), n = 7/8/9 (CD4 stimulated), n = 5 (CD8 unstimulated), and n = 8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, 25th and 75th percentiles and range (all other). Dots indicating individual values. *P < 0.05, **P < 0.01, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.