β‑hydroxybutyrate metabolism enhances CAR T cell function via transcriptional and epigenetic reprogramming

Abstract Introduction. The impact of patient (pt) lifestyle, particularly diet, on chimeric antigen receptor (CAR) T cell therapy remains poorly understood. In our previous work (ASH 2024, Plenary Session), we identified the ketogenic diet-derived metabolite, β-hydroxybutyrate (BHB), as a potentiator of CAR T cell antitumor function across multiple preclinical tumor models. We also found that BHB boosted T cell oxidative phosphorylation and citric acid (TCA) cycle, shifting metabolism toward aerobic respiration. Here, we investigate how BHB shapes CAR T cells through transcriptional, epigenetic, and metabolic changes. Methods and Results. To study the effect of BHB on CAR T cell function, we cultured anti-CD19 CAR T cells on recombinant CD19-coated wells with 5 mM BHB or vehicle. After 48 hours, we collected the cells and performed bulk RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to assess transcriptional and chromatin accessibility changes. BHB treatment induced strong transcriptional reprogramming, namely upregulation of inflammatory mediators (e.g., IL22, ADGRG1, CXCL8) and downregulation of interferon-stimulated genes (e.g., ISG15, OAS1, IFI6, USP18). Gene set enrichment analysis revealed signatures of enhanced oxidative phosphorylation. Chromatin profiling by ATAC-seq showed increased accessibility at promoter regions of genes essential for T cell activation, co-stimulation, and survival (e.g., LCK, CD2, CD28, RELA), suggesting that BHB may epigenetically prime CAR T cells for heightened responsiveness. Notably, regions with enhanced chromatin accessibility were associated with immunoregulatory pathways central to T cell function, including checkpoint regulation, TCR signaling, and NF-κB activation. Integrated analysis of RNA-seq and ATAC-seq data revealed coordinated regulation of genes involved in immune activation and tissue remodeling. Lastly, to further explore epigenetic changes, CUT&RUN-seq profiling of H3K27ac demonstrated increased histone acetylation at loci relevant to T cell function (e.g., STAT2, KDM1A), supporting a role for BHB in promoting a transcriptionally and epigenetically active state in CAR T cells. Next, to confirm these observations in an in vivo model, we characterized circulating CAR T cells in BHB-treated tumor-bearing mice. First, we implanted diffuse large B-cell lymphoma (DLBCL) tumors (CD19⁺ OCI-Ly18, 4×10⁶ cells, subcutaneous) into immunodeficient (NOD SCID gamma) mice and gave daily oral BHB or vehicle. Ten days post-implantation, a curative dose of CART19 (5×10⁶ CAR⁺ cells) was infused. Seventeen days post CAR T cell infusion, we collected peripheral blood from vehicle- and BHB-treated mice (n = 7 mice per arm), isolated peripheral T cells, and performed 5' single-cell RNA sequencing (scRNA-seq, 10X Genomics). ScRNA-seq of the circulating CAR T cells revealed six major T cell subsets (activated CD4⁺, cytotoxic CD8⁺, proliferating CD4/CD8, T central memory, and gamma-delta). We observed that BHB-treated CAR T cells represented a higher proportion of cytotoxic T cells. Moreover, when performing differentially expressed genes analysis, we observed upregulation of critical genes involved in mitochondrial metabolism (MT-CO2 or cytochrome c; ATP5MC3 or ATP synthase), effector function (GZMB, GZMH), and a downregulation of exhaustion/dysfunction-related genes (RGS16, TGFB1) in BHB-treated CAR T cells. Finally, unbiased pathway analysis (KEGG) of BHB-induced genes confirmed that BHB indeed enriched the oxidative phosphorylation pathway and cell cycling. Conclusions. Our results show that BHB supplementation metabolically reprograms antigen-activated CAR T cells, driving transcriptional and epigenetic changes that enhance aerobic mitochondrial respiration and antitumor function. These findings will be validated in patient-derived CAR T cells in an ongoing first-in-human trial of BHB supplementation in relapsed/refractory DLBCL patients receiving commercial CART19 (NCT06610344). *S.L. and P.G. contributed equally. **M.R. and M.L. jointly supervised this work.