Fig 2: 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.

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. 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 4: TNFα was the main mediator released by engaged CAR-T cells in inducing endothelial pro-inflammatory response. (A) The cytokine in the co-cultured supernatant of CAR-T/Nalm6 (sCAR-T) was analyzed by Luminex. (B) HUVEC were stimulated with TNFα (10 μg/ml), IFNγ (50 μg/ml), IL8 (25 μg/ml), and GM-CSF (50 μg/ml) respectively for 4h. The mRNA expression of endothelial activation-related markers was determined by quantitative RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. (C) The protein expression of adhesion molecules E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. (D) The protein expression of TF in HUVEC was determined by western blot. (E) The venn analysis of HUVEC stimulated with sCAR-T and TNFα. * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.

Fig 5: FAK inhibition effectively abolished endothelial activation induced by CAR-T/Nalm6/PBMC co-cultured supernatant. (A) The protein level of phosphorylated FAK at tyrosine 397 as well as TF was determined by western blot. (B) The mRNA levels of endothelial activation-associated markers were determined by RT-PCR. GAPDH was taken as the housekeeping gene and data was expressed as fold changes relative to control. n = 3. (C) The protein expression of E-selectin, VCAM1, and ICAM1 was determined by flow cytometry. n = 3. (D) The concentration of IL6 and IL8 in the supernatant was determined by ELISA. (E) The concentration of Ang2 secreted by HUVEC was assessed by ELISA. n = 3. (F) Confluent HUVEC cultured in Transwell were incubated with sCAR-T supplemented with or without adalimumab for 12h. The permeability of endothelial monolayer was determined by Evans blue-BSA assay. (G) The expression levels of indicated proteins were determined by western blot. * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.0001. ns represents not significant. All data were representative of at least three independent experiments.