β-hydroxybutyrate potentiates anti-tumor immunity by modulating cytotoxic CD8+ T cell responses

β-hydroxybutyrate potentiates anti-tumor immunity by modulating cytotoxic CD8+ T cell responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-hydroxybutyrate potentiates anti-tumor immunity by modulating cytotoxic CD8 + T cell responses Yupan Bai, Han Xue, Yujie Bao, Yue Pan, Jiayin Tang, Mengna Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8428666/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Ketogenic diets (KDs) have been reported to influence tumor progression through metabolic and immunological modulation of the tumor microenvironment. β-hydroxybutyrate (βOHB), the predominant ketone body elevated by KD, functions not only as an energy substrate but also as a potent signaling metabolite. Despite its role in modulating the tumor microenvironment, the direct impact of βOHB on the function of CD8⁺ T cell, a key mediator of anti-tumor immunity, remains incompletely understood. Here, we demonstrate that βOHB suppresses tumor growth in multiple mouse tumor models by enhancing the accumulation, survival, and effector function of tumor-infiltrating CD8⁺ T cells. In contrast, acetoacetate does not exert comparable immunomodulatory effects. Mechanistically, βOHB upregulates the Tcf7–Lck signaling pathway by engaging with the cell surface receptor Hcar2, rather than through its role as an HDAC inhibitor. Knockdown of either Tcf7 or Hcar2 in CD8 + T cells abolishes the promoting effect of βOHB on CD8 + T function. Our findings elucidate a metabolite-immune axis that directly regulates the functional state of tumor-infiltrating CD8⁺ T cells and provide experimental evidence linking ketone metabolism to anti-tumor immune regulation. β-hydroxybutyrate CD8 + T cell anti-tumor therapy Tcf7 Lck Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ketone bodies generated during ketogenic diets (KD) exert pleiotropic functions beyond energy supply by modulating inflammation, oxidative stress, catabolic pathways, and associated gene expressions ( 1 , 2 ). Accumulating studies also supports KDs as promising adjuncts to cancer therapy, with preclinical studies in glioma, pancreatic cancer, and colon cancer showing that KD-induced immunological changes within the tumor microenvironment (TME) consistently suppress tumor growth and prolong survival in tumor-bearing mice ( 3 – 6 ). Moreover, these immune-modulatory effects enable KDs to sensitize otherwise resistant tumors to immune checkpoint blockade (ICB) therapy ( 7 , 8 ). β-hydroxybutyrate (βOHB), the predominant circulating ketone body, acts not only as an energy substrate but also as a potent signaling metabolite. It has been shown to activate Hcar2 signaling and induce transcriptional factor Hopx to suppress colorectal cancer development in mice ( 9 ). It also exerts anti-angiogenic effects in colitis-associated cancer through reducing HIF-1α-dependent VEGFA secretion from cancer cells ( 10 ). In addition, β-OHB functions as an endogenous inhibitor of HDACs, reducing the activities of HDAC1, HDAC3, and HDAC4, which results in hyperacetylation of histone H3 and upregulation of oxidative stress resistance genes including FOXO3a, MT2, SOD2, and catalase ( 11 ). Together, these studies identify βOHB as a bioactive metabolite with multifaceted anti-cancer activities, supporting further investigation into its therapeutic potential. CD8⁺ T cells are central to the anti-tumor efficacy of ICB and adoptive T cell therapy. However, their durable response to immunotherapy is often compromised by T cell exhaustion (Tex), a dysfunctional state arising during chronic infections and malignancies, characterized by impaired T cell proliferation, reduced cytokine secretion, and sustained expression of multiple inhibitory receptors ( 12 , 13 ). Tex represents an alternative CD8⁺ T cell fate in which extensive transcriptional reprogramming constrains their effector functions and limits their transition into long-lived memory subsets ( 14 , 15 ). A network of transcriptional factors, including Tbx21, Eomes, Id2, Blimp-1, Bcl6, Tcf7 has been identified as central to this process ( 16 ). Specifically, Tbx21 and Eomes coordinate the differentiation and balance of effector and memory CD8⁺ T cells ( 17 ), Id2 promotes the survival of effector T cells through IL-12–dependent signaling pathways ( 18 ), Blimp-1 is a master regulator of terminal effector T cell differentiation ( 19 ), whereas Bcl-6 promotes the development of KLRG1 lo IL-7Rʰⁱ memory T cells ( 20 ). Tcf7/TCF1 is essential for generation and maintenance of stem-like CD8⁺ T cells, which are required for sustained responses under chronic antigen exposure and responsiveness to ICB therapies ( 21 , 22 ). These transcriptional programs are dynamically shaped by extracellular cues within the TME, and their modulation enhances the recruitment, persistence, and functional activity of TME-infiltrated CD8⁺ T cells ( 23 , 24 ), offering a promising strategy to improve the efficacy of cancer immunotherapies. Despite evidence that KDs and βOHB modulate the TME and immune responses, the direct impact of βOHB on CD8⁺ T cell differentiation, activation, and exhaustion, as well as the underlying molecular mechanisms, remain poorly understood. Here, we investigated the regulatory effects of KDs and βOHB on CD8⁺ T cells within the TME and found that βOHB promotes the expansion of CD8⁺ T cells with high Tcf7 expression, which sustain stemness, self-renewal, and effector functions in response to tumor antigens via the HCAR2–Tcf7–Lck signaling axis. These findings provide new insights into the regulation of TME-infiltrated CD8⁺ T cell fate and function, and highlight βOHB as a promising therapeutic candidate to potentiate immune responses and improve clinical outcomes in cancer treatment. Materials and Methods Mice Wild-type female and male C57BL/6 and BALB/c mice, aged 6–8 weeks, were purchased from Jihui Experiment Animal Feeding Co. Ltd. Age-matched littermates of both sexes were used in all experiments, with sexes analyzed separately when indicated. Mice were maintained under specific pathogen-free (SPF) conditions with food and water ad libitum on a 12-h light/dark cycle. All animal procedures were conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (approval number:SH9H-2022-A8-1). Human specimens Tumor-infiltrating T cells were isolated from patients with colon cancer. Plasma samples were collected from patients diagnosed with advanced colorectal carcinoma for this study. All samples were collected with informed consent under procedures approved by the Institutional Review Board (IRB) of Renji Hospital, Shanghai Jiao Tong University School of Medicine (approval number:RA-2022-411). Cell isolation and culture Mouse tumor cell lines (B16F10, CT26) were purchased from National Collection of Authenticated Cell Culture. B16F10 cells were cultured in DMEM (HyClone), and CT26 cells in RPMI 1640 medium (HyClone), both supplemented with 10% FBS. Mouse lymphocytes were isolated from spleen using density gradient centrifugation with Lymphoprep™ (Serumwerk). CD8 + T cells were then purified via positive selection using MicroBeads (Miltenyi Biotec), resuspended at 5×10 5 cells/mL, and activated with anti-CD3 (2 µg/mL) and anti-CD28 (1 µg/mL) mAbs (BD Biosciences) for 24–48 h. Activated CD8 + T cells were maintained in RPMI 1640 medium containing 10% FBS, IL-2 (10 ng/mL), β-mercaptoethanol, L-glutamine (Gibco), MEM (Non-essential amino acid solution, Gibco), HEPES (Sigma), and treated with specific concentrations of βOHB (Sigma) in various experiments. Tumor-infiltrating CD8 + T cells were isolated through mechanical dissociation and filtration through a 70 µm cell strainer, followed by mononuclear cells enrichment using Lymphoprep™ (Serumwerk) via density gradient centrifugation. CD8 + T cells were then further purified using MicroBeads (Miltenyi Biotec) through positive selection. The purified CD8 + T cells were subsequently assessed by FACS. Tumor inoculation and treatment C57BL/6 mice were inoculated subcutaneously in the right flank with 2×10 5 B16F10 melanoma cells, and BALB/c mice were similarly inoculated with 5×10 5 CT26 colon cancer cells. Tumor growth was monitored by measuring tumor volumes every 2 days using digital calipers, and calculated by the formula: tumor volume = (a × b × c)/2. For the ketogenic diet experiment, C57BL/6 mice were randomly assigned to two groups. The ketogenic diet (KD) group received a high-fat diet providing 80% of calories from fat, whereas the control group was fed a standard chow diet (ND group). After two weeks of dietary treatment, mice were subcutaneously inoculated with B16F10 cells as described above. Tumor growth and body weight were monitored throughout the study. For intratumoral treatment, PBS or β-hydroxybutyrate (βOHB) was dissolved in sterile PBS and administered at 100 mg/kg (B16F10) or 200 mg/kg (CT26) in a final injection volume of 100 µL. Injections were performed once daily or every other day starting on day 7 after tumor inoculation. Mice were lightly anesthetized with 2% isoflurane during intratumoral injections to minimize distress. For CD8⁺ T cell depletion, Once B16F10 tumors became palpable (7 days post-inoculation), CD8⁺ T cells were depleted using an anti-CD8a monoclonal antibody (clone 2.43, Bio X Cell, Cat# BE0061). The antibody was diluted in sterile PBS and administered intraperitoneally at 10 mg/kg in a total volume of 100 µL, once weekly. Intratumoral βOHB administration began 24 h after the first CD8⁺ T cell depletion injection and was delivered once daily at 100 mg/kg, 100 µL per injection. Mice were briefly anesthetized with 2% isoflurane during injections to minimize discomfort. Mice were monitored daily for tumor growth, body weight, and general health. Euthanasia was performed when tumors reached 2.5 cm³ or humane endpoints were met, using CO₂ asphyxiation followed by cervical dislocation according to AVMA 2020 guidelines. No animals or data were excluded unless predefined humane endpoints were reached. Animals were not blinded due to visible phenotypic differences and the need to accurately administer treatments and monitor welfare. Flow cytometry For surface staining, cells were pre-incubated with Fc blocking buffer (BD Biosciences, Cat# 553141) for 15 min on ice, and then stained with fluorescence-conjugated monoclonal antibodies in BD™ Stain Buffer(BD Biosciences, Cat# 554656) for 30 min on ice. For intracellular staining, CD8 + T cells were treated with 0 or 5 mM βOHB for 48 h, followed by stimulated with Cell Activation Cocktail (with Brefeldin A) (BioLegend, Cat# 423303) for 6 h. Cells were then fixed in Fixation/Permeabilization solution (BD Biosciences, Cat# 554714) for 40 min at 4℃ in the dark. After washing with Perm/Wash Buffer (BD Biosciences, Cat# 554714), intracellular staining was performed using Alexa Fluor 647 anti-human/mouse Granzyme B antibody, FITC-anti-mouse TNF-α antibody, and PE-anti-mouse IFN-γ antibody for 50 min in the dark. Antibodies were purchased from BD Biosciences or Biolegend. Detailed information was provided in Table S1 . Apoptosis was assessed by staining cells with Annexin V and propidium iodide (PI) in 1× Binding buffer (BD Biosciences) at RT for 15 min in the dark. Flow cytometry was conducted using an LSR-Fortessa (BD Biosciences) instrument, and data were analyzed using FlowJo software (version 10). Cells were gated as singlets, live cells, and specific immune cell populations, including CD8 + T cells (CD45 + CD3 + CD8 + ), CD4 + T cells (CD45 + CD3 + CD4 + ), NK cells (CD45 + CD3 − NK1.1 + ), and B cells (CD45 + CD3 − CD19 + ). Real-time PCR and Western blot CD8 + T cells were incubated with 0 or 5 mM βOHB for 72 h, then washed and harvested. RNA was extracted from these cells using TRIzol reagent (Thermo Fisher Scientific, Cat# 15596026), and reverse transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Cat# RRO47A). Real-time PCR was performed using TB Green Premix Ex Taq (Tli RNase H Plus) kit (TaKaRa, Cat# RR820A) on a LightCycler 480 instrument (Roche). Primers were listed in Table S2. For Western blot analysis, CD8 + T cells, treated as described above, were washed and lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with 1× protease inhibitor cocktail (Proteintech). Total protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Protein samples were then denatured at 95°C for 5 min. Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% fat-free milk for 1 h, followed by overnight incubation at 4°C with primary antibodies, including anti-TCF1/Tcf7, anti-Lck, anti-NFκB, anti-H3K27ac, anti-Notch1, and anti-GAPDH. After incubation with HRP-conjugated secondary antibody for 30 min, protein levels were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). Detailed antibody information was provided in Table S3. Histology and immunofluorescence staining Tumor tissues were fixed in 4% paraformaldehyde (PFA) solution for 24 h, and then embedded in paraffin blocks following standard procedures. For antigen retrieval, tissue sections were immersed in citrate buffer and heated in saturated steam for 20 min. After cooling, the slides were washed with phosphate-buffered saline (PBS) and blocked with 5% bovine serum albumin (BSA) for 30 min to reduce background staining. The sections were then incubated overnight at 4°C with 488-conjugated anti-Mouse CD8a antibody (Proteintech, Cat# CL488-65069). After DAPI staining, fluorescence images were captured to visualize target proteins using a confocal laser scanning microscope (Leica TCS SP8 STED 3X). RNA-seq analysis CD8 + T cells from B16F10 tumor-bearing mice were isolated, sorted, and incubated with 0 or 5 mM βOHB for 72 h. Total RNA was extracted using the TRIzol reagent. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and verified by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was enriched using Oligo(dT) beads, fragmented into short segments using fragmentation buffer, and reverse transcribed into cDNA by using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Cat# 7530). The purified double-stranded cDNA fragments were end-repaired, A-tailed, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1×), followed by size selection through agarose gel electrophoresis and PCR amplification. The cDNA libraries were sequenced on the Illumina NovaSeq 6000 platform. Clean reads were aligned to the mouse reference genome (mm10). Gene-level counts were calculated and normalized as reads per kilobase of transcript per million mapped reads (RPKM) to estimate gene expression levels. The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1354648 . Differentially expressed genes (DEGs) were identified using DESeq2, with genes showing an adjusted p -value < 0.05 and |log₂ (fold change) | ≥ 1 considered significantly differentially expressed. siRNA knockdown assay The siRNAs targeting mouse genes were synthesized as follows: Tcf7-1: 5′- GAAGCCAGUCAUCAAGAAA-3′ Tcf7-2: 5′- GAAAUGCAUUCGGUACUUA-3′ Hcar2-1: 5′-GGCGAGGCAUAUCUGUGUA-3′ Hcar2-2: 5′-CGUUCCUGACGGACAACUA-3′ Scrambled siRNA: 5′-UUCUCCGAACGUGUCACGU-3′ Briefly, 1×10 6 cells were seeded into a six-well plate in 2 mL of RPMI 1640 medium (without FBS or antibiotics) for 24 h. Cells were then transfected with 3 µL of 10 µM siRNA per well, pre-mixed with 9 µL of Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Cat# 13778150) in Opti-MEM medium. After 6 h, the medium was replaced with fresh RPMI 1640 supplemented with 10% FBS. After culturing for 48 h, cells were treated with βOHB for another 48 h before being harvested for further experiments. Cell proliferation assay Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo, Cat# CK04) according to the manufacturer’s instructions. In brief, 90 µL of tumor cells were plated at a concentration of 3,000 cells/well into 96-well plates. Then, 10 µL of CCK-8 solution was added to each well and the cells were incubated at 37°C for 2 h. Absorbance was measured at 450 nm using a microplate reader (VICTOR Nivo). βOHB measurements Each sample from the mouse tumor models was divided in two aliquots for total protein measurement and βOHB quantification, respectively. Tumor samples were washed with PBS and collected by centrifugation at 300×g for 5 min. βOHB levels were determined using the BHB Colorimetric Assay Kit (Cayman). Statistical analysis Statistical analyses were performed using GraphPad Prism 9.0 software, unless otherwise indicated. All experimental data were presented as the mean ± SEM. Statistical significance was determined using a two-tailed Student’s t-test. P-values < 0.05 were considered statistically significant. Results KD retards tumor growth by enhancing CD8 + T cell functions To explore the impact of ketone bodies on tumor growth and anti-tumor immunity, we treated C57BL/6J mice with a ketogenic diet (KD) containing 80% fat under specific pathogen–free (SPF) conditions (Fig. 1 A). Compared with mice receiving a normal diet (ND), KD-fed mice showed a substantial reduction in the growth of subcutaneously implanted B16F10 melanoma tumors, accompanied by elevated βOHB levels in serum but not in tumors, while body weight remained comparable between the two groups (Fig. 1 B–C and Fig. S1 A–C). KD feeding also improved long-term survival of tumor-bearing mice (Fig. S1 D), consistent with previous studies supporting the therapeutic potential of KD in cancer ( 8 ). We then evaluated the effects of KD on the tumor immune microenvironment by profiling tumor-infiltrating immune cells via flow cytometry (FACS). KD feeding led to a modest increase in NK cell infiltration and had minimal effects on CD4⁺ T cells, dendritic cells, and B cells. Notably, the accumulation of CD8⁺ T cells within tumors was significantly elevated in KD-fed mice compared with the ND group as shown by FACS and immunofluorescent analysis (Fig. 1 D and Fig. S1 E–I). Further analysis revealed that the tumor-infiltrating CD8⁺ T cells from KD-fed mice exhibited markedly reduced apoptosis, as indicated by decreased Annexin V staining, suggesting improved survival within the tumor microenvironment (Fig. 1 E). Moreover, they also showed elevated expression of key cytotoxic and effector molecules, including Granzyme B, IFNγ, and TNFα (Fig. 1 F–H), implying that KD enhances the cytotoxic potential of CD8⁺ T cells for effective tumor cell killing. The above observations demonstrate that KD suppresses tumor growth by promoting both the accumulation and functional potency of CD8⁺ T cells, underscoring an important role of CD8⁺ T cell-mediated immunity in the antitumor effects of ketogenic diets. βOHB promotes CD8⁺ T cell survival and cytotoxic function To elucidate the molecular basis of KD-mediated enhancement of anti-tumor immunity, we investigated whether βOHB, the predominant ketone body elevated under ketogenic conditions, directly modulates effector CD8⁺ T cell activity in culture. Mouse splenic CD8⁺ T cells were activated with soluble anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) and IL-2, and cultured in complete medium supplemented with increasing concentrations of βOHB. As shown in Fig. 2 A–D, treatment with βOHB reduced apoptosis of activated CD8⁺ T cells and enhanced their cytotoxic effects in a dose-dependent manner, with the strongest effects observed at 5 mM, as indicated by decreased Annexin V staining and elevated Granzyme B, IFN-γ, and TNF-α production. Notably, this effect was attenuated at 10 mM βOHB, suggesting a narrow concentration range in which βOHB optimally supports effector T cell survival and function (Fig. 2 A–D). In contrast, βOHB had minimal impact on naïve CD8⁺ T cells, indicating that it selectively modulates antigen-experienced T cells (Fig. S2A–C). Consistent with its prosurvival effects and enhancement of effector functions, βOHB treatment also promoted proliferation of activated CD8⁺ T cells, as shown by increased EdU incorporation relative to untreated controls (Fig. 2 E). We then examined whether acetoacetate (AcAc), another major ketone body, exerts similar effects. Treatment of activated CD8⁺ T cells with 5 mM AcAc did not affect apoptosis or effector molecule expression, indicating that βOHB, rather than AcAc, is the primary ketone body responsible for enhancing effector CD8⁺ T cell survival and function (Fig. S2D–G). To further evaluate the effects of βOHB in a tumor context, tumor-infiltrating CD8⁺ T cells (CD8⁺ TILs) isolated from human colon cancer specimens were cultured with βOHB at concentrations ranging from 0 to 10 mM. Consistent with observations in activated mouse splenic CD8⁺ T cells, 5 mM βOHB elicited the strongest response, markedly reducing apoptosis and enhancing the production of key effector cytokines in CD8⁺ TILs (Fig. 2 F and Fig. S2H). Together, these results identify βOHB as a selective enhancer of effector CD8⁺ T cell survival, function, and proliferation, providing a direct link between ketogenic conditions and antitumor immunity. βOHB potentiates CD8⁺ TILs to promote anti-tumor immunity To investigate the role of βOHB in CD8⁺ T cell-mediated anti-tumor immunity in vivo , B16F10 melanoma-bearing mice were treated with βOHB intratumorally (Fig. 3 A). βOHB administration elevated βOHB levels within tumor tissue while serum concentrations remained largely unchanged, and markedly suppressed tumor growth without affecting body weight (Fig. 3 B and Fig. S3A–D). We next analyzed immune cell infiltration in tumors by flow cytometry. Compared with PBS-treated controls, βOHB-treated tumors exhibited modest increases in NK cells and dendritic cells, whereas the relative abundance of CD4⁺ T cells and B cells remained unchanged across the examined tumor samples (Fig. S3E–H). Notably, intratumoral CD8⁺ T cells were substantially enriched following βOHB treatment, as confirmed by both flow cytometry and immunofluorescence staining of tumor tissue sections (Fig. 3 C and Fig. S3I). This enrichment aligns with the increased CD8⁺ T cell infiltration observed in tumors from KD-fed mice (Fig. 1 D). Subsequent analysis revealed that CD8⁺ T cells isolated from βOHB-treated tumors exhibited substantially enhanced survival, whereas spleen-derived CD8⁺ T cells showed no significant changes (Fig. 3 D). These tumor-infiltrating CD8⁺ T cells also displayed elevated expression of polyfunctional effector molecules, including Granzyme B, IFN-γ, and TNF-α, indicative of enhanced cytotoxic activity and potentiated antitumor function (Fig. 3 E–H). To determine whether the reduced tumor growth in βOHB-treated mice was mediated by CD8⁺ T cells, B16F10-bearing mice were treated with anti-CD8 antibodies to deplete CD8⁺ T cells. Remarkably, removal of CD8⁺ T cells abrogated the anti-tumor effects of βOHB, confirming that βOHB exerts its anti-tumor efficacy in a CD8⁺ T cell–dependent manner (Fig. 3 I and Fig. S3J, K). We then assessed the reproducibility of these findings in another syngeneic tumor model. In mice bearing CT26 colon carcinoma, βOHB administration similarly suppressed tumor growth, enhanced CD8⁺ T cell survival within the tumor microenvironment, and promoted effector cytokine production (Fig. 4 A–E). These results suggest that the immunopotentiating effects of βOHB on CD8⁺ TILs are conserved across distinct tumor contexts. Besides, when B16F10 and CT26 cancer cells were cultured with βOHB for 48 hours in vitro , only minimal changes were observed in cell viability, apoptosis, or proliferation rates, indicating that βOHB exerts its anti-tumor effects primarily through potentiation of CD8⁺ T cell-driven immune responses rather than through direct cytotoxicity against tumor cells (Fig. S4A–D). βOHB enhances CD8⁺ T cell anti-tumor function though Tcf7-dependent transcriptional regulation To dissect the molecular mechanisms by which βOHB modulates tumor-infiltrating CD8⁺ T cell function, we performed RNA sequencing of CD8⁺ T cells isolated from tumors and cultured in the presence or absence of βOHB. Transcriptomic profiling revealed broad reprogramming of pathways associated with metabolism, effector function, and survival (Fig. S5A). Gene set enrichment analysis (GSEA) showed a strong upregulation of T cell activation–associated genes ( Gzma , Gzmb , Prf1 , Il12rb2 , Ifng , Tnf ) and enrichment of TCR signaling and cytokine pathways (Fig. 5 A and B). Transcription factors (TFs) that orchestrate CD8⁺ T cell differentiation and effector programming, including Tcf7, Tbx21, and Eomes, were also markedly upregulated following βOHB stimulation (Fig. 5 A). Consistently, qPCR validation confirmed that the expression of representative genes ( Tcf7 , Lck , Tbx21 , Eomes , Nfκb2 , and Nfκbiz ) was substantially elevated in βOHB-treated CD8⁺ T cells compared to controls (Fig. 5 C–D and Fig. S5B–D). In agreement with these transcriptional changes, Western blot analysis demonstrated that βOHB treatment also increased Tcf7, Lck, and NF-κB protein levels in both splenic effector and tumor-infiltrating CD8⁺ T cells (Fig. 5 E). Among these transcriptional regulators, Tcf7 stood out due to its well-established role in maintaining CD8⁺ T cell stemness, self-renewal, and effector function in response to viral or tumor antigens ( 25 ). To investigate whether Tcf7 mediates the tumor-suppressive effects of βOHB, CD8 + T cells were isolated from C57BL/6 mice spleens and activated with anti-CD3, anti-CD28 and IL-2. Small interfering RNA (siRNA) targeting Tcf7 was used to knock down Tcf7 expression in activated CD8⁺ T cells (Fig. S5E and F). Compared with control cells transfected with scrambled siRNA, siTcf7 CD8⁺ T cells exhibited markedly reduced expression of Lck and NF-κB, key mediators of antitumor immunity, consistent with the established role of Tcf7 in regulating T cell effector function, and βOHB treatment failed to enhance their expression in si Tcf7 CD8⁺ T cells (Fig. 6 A). Moreover, Tcf7 knockdown significantly attenuated the effects of βOHB on CD8⁺ T cell apoptosis and effector cytokine production (Fig. 6 B–F and Fig. S6A–B). Taken together, these data suggest that βOHB enhances CD8⁺ T cell anti-tumor activity through Tcf7-dependent transcriptional reprogramming and activation of the Lck–NFκB signaling axis. Hcar2 is required for βOHB-mediated Tcf7 upregulation To elucidate the molecular mechanisms by which βOHB upregulates Tcf7 , we comprehensively evaluated potential regulatory pathways in activated mouse splenic CD8⁺ T cells. We first assessed whether HDAC inhibition by βOHB contributes to Tcf7 induction. Although βOHB treatment modestly increased global H3K27ac levels, the extent of acetylation was much lower than that induced by the potent HDAC inhibitor Trichostatin A (TSA). Notably, TSA could not upregulate Tcf7 expression as observed with βOHB treatment instead it led to a reduction in Tcf7 protein levels (Fig. S6C and D). These results suggest that βOHB-induced Tcf7 expression is independent of its HDAC-inhibitory activity. NOTCH signaling has been reported to induce Tcf7 and its downstream targets such as Bcl11b , Gata3 , Lck , and Lat ( 26 – 28 ). However, the levels of Notch1 responsible for downstream transcriptional activation, remained unchanged following βOHB treatment, suggesting that NOTCH signaling is not involved in βOHB-mediated upregulation of Tcf7 (Fig. S6E). We next explored whether βOHB regulates Tcf7 expression through receptor-mediated signaling pathways. βOHB functions as a signaling metabolite by engaging its cell-surface receptor Hcar2 (Gpr109a), which has been implicated in immune regulation and transcriptional control ( 29 , 30 ). To determine whether Hcar2 mediates βOHB-induced Tcf7 expression, CD8⁺ T cells were transiently transfected with siRNA targeting Hcar2 or with scrambled control siRNA and subsequently treated with βOHB or PBS for 48 hours. qPCR analysis showed that βOHB markedly upregulated Tcf7 expression in control cells, whereas this induction was nearly abolished upon Hcar2 knockdown (Fig. 6 G and Fig. S6F). These results identify Hcar2 as a key receptor mediating βOHB-induced activation of Tcf7 transcription and CD8⁺ T cell-dependent antitumor immunity. Discussion β-hydroxybutyrate (βOHB), the major ketone body elevated during fasting or ketogenic states, has been shown to suppress tumor growth, in part by modulating the tumor immune microenvironment( 31 ); however, its impact on CD8⁺ T cell-mediated antitumor immunity remains poorly understood. In this study, we revealed that βOHB inhibits tumor progression by enhancing CD8⁺ T cell accumulation, survival, and effector function within the tumor microenvironment. Mechanistically, βOHB upregulates the transcription factor Tcf7 and its downstream Lck–NFκB signaling cascade via the βOHB receptor Hcar2, independently of its canonical HDAC-inhibitory activity or Notch signaling. The identified βOHB–Hcar2–Tcf7 axis establishes a direct molecular link between metabolic signaling and transcriptional regulation in effector T cells, highlighting βOHB as a key immunometabolic cue that sustains CD8⁺ T cell persistence and effector potency within the tumor microenvironment. Ketogenic diets can inhibit tumor growth and metastasis by restricting energy availability to cancer cells, inhibiting angiogenesis, alleviating oxidative stress in normal cells, and modulating oncogenic signaling pathways ( 32 ). KDs have also been shown to enhance T cell-mediated antitumor immunity by inhibiting immunosuppressive signals in TME, such as KLF5-dependent CXCL12 expression decreased intratumoral accumulation of immunosuppressive cells, increased infiltration of NK and cytotoxic T cells ( 6 ). CD8⁺ T cells, the principal cytotoxic effectors of antitumor immunity, are essential for tumor clearance and immune surveillance. Previous work reported the ketogenic diet enhances immune function in healthy individuals by inducing ketone body-driven metabolic reprogramming toward mitochondrial oxidation, thereby strengthening CD4⁺, CD8⁺, and regulatory T-cell activity and memory formation ( 33 ). However, whether βOHB directly regulates cytotoxic CD8⁺ T cell activity in tumors remained unclear. Our study indicates that KD and βOHB treatment in mouse tumor models change the tumor immune microenvironment by markedly enhancing CD8⁺ T cell infiltration and effector functions, resulting in increased antigen-specific cytotoxicity and improved tumor suppression. These observations align with recent studies showing that ketogenic diets and ketone bodies can augment the efficacy of PD-1 blockade in mouse tumor models ( 8 ), supporting the hypothesis that ketogenic interventions may potentiate CD8⁺ T cell-mediated antitumor immunity and help overcome resistance to immune checkpoint blockade. Recent single-cell RNA sequencing analyses in melanoma patients have shown that CD8⁺ TILs consist of progenitor-like (Tex-stem) and terminally differentiated (Tex-term) subsets ( 34 ). Tex-stem cells display self-renewal, durability, and the capacity to differentiate into terminal effector cells, with these properties critically dependent on TCF1, a transcription factor encoded by Tcf7 that maintains the stemness and long-term persistence of antigen-responsive CD8⁺ T cells ( 27 ). In our study, βOHB treatment significantly upregulated Tcf7 expression in mouse CD8⁺ TILs, along with its downstream targets Lck and NFκB. Silencing Tcf7 abrogated βOHB-mediated enhancement of CD8⁺ TIL functions, suggesting that βOHB may promote the stem-like properties and functional capabilities of CD8⁺ T cells within the tumor microenvironment through Tcf7 signaling. The presence of TCF1/Tcf7⁺ stem-like CD8⁺ TILs within tumors is associated with enhanced therapeutic efficacy in immune checkpoint blockade ( 35 , 36 ). Our findings that βOHB increases Tcf7 expression and enhances CD8⁺ T cell–mediated antitumor immunity suggest that dietary or pharmacological βOHB supplementation could serve as an adjuvant to checkpoint inhibitors to promote durable immune responses in cancer patients. Moreover, βOHB supplementation during ex vivo T cell expansion may enhance the generation and persistence of therapeutic T cells, such as CAR-T cells, thereby augmenting their antitumor potency and long-term survival in patients. Our study further reveals that βOHB upregulates Tcf7 expression in CD8⁺ T cells through receptor-mediated signaling, rather than through its traditional role in HDAC inhibition. Knockdown of Hcar2, a known cell surface receptor for βOHB, abolished this effect, establishing Hcar2 as an essential mediator of βOHB-induced Tcf7 expression. βOHB binding to Hcar2 activates a G-protein coupled receptor signaling cascade, but the specific intracellular signaling pathways downstream of Hcar2 that regulate Tcf7 expression remain to be further elucidated. In colorectal cancers, previous studies indicate that βOHB activates the anti-tumorigenic transcription regulator Hopx via Hcar2 signaling, independently of HDAC inhibition, suggesting that Hcar2 signaling plays an important role in the anti-tumor effects of βOHB ( 9 ). These results also underscore the potential of targeting the βOHB–HCAR2 axis, such as by combining PD-1 blockade with selective HCAR2 agonists, to enhance the efficacy of immune checkpoint therapy and advance immunometabolic interventions. Beyond cancer therapy, these mechanisms may also be relevant to chronic infections and immune-mediated disorders characterized by T cell exhaustion and metabolic stress, providing a framework for restoring immune function through metabolite-driven transcriptional regulation. In summary, our work demonstrates that βOHB reinforces antitumor immunity by enhancing CD8⁺ T cell persistence and effector function, which is mediated through activation of the Hcar2–Tcf7 axis and the downstream Lck–NFκB pathway (Fig. 7 ). These findings provide a mechanistic basis for metabolite-driven immunomodulation of CD8⁺ T cell, offering a rationale for integrating ketogenic or ketone-based therapies with current immunotherapeutic strategies to enhance cancer treatment outcomes. Data and materials availability All data is present in the manuscript and supplemental figures. RNA-seq data reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA1354648 . Declarations Competing interests The authors declared that they have no competing interests. Ethics approval All the animal experiments were conducted in accordance with the ARRIVE guidelines and approved by the Laboratory Animal Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (SH9H-2022-A8-1). Human samples were obtained after written informed consent from all participants. All experiments involving human specimens were conducted in accordance with the ethical principles of the Declaration of Helsinki and were approved by the Institutional Review Board (IRB) of Renji Hospital, Shanghai Jiao Tong University School of Medicine (RA-2022-411). Funding information This work was supported by the National Key R&D Program of China (2022YFA1302800), the National Natural Science Foundation of China (32022036), The Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212300 and JWAIZD-2). Author Contribution Y.B. performed all ex vivo experiments and analyzed the data; Y.B., H.X., and Y.P. performed the animal experiments; J.T. performed the collection of operative samples; Y.B, Yuj. B, and M.W. prepared the figures and helped with manuscript preparation; J.X., and Y.W. provided valuable advice on the project and the manuscript; J.H. and J.X. designed and supervised the research; J.H., Y.B., and J.X. wrote the manuscript with help from all other authors. Acknowledgement We are grateful to the staff of the core facilities at Shanghai Institute of Precision Medicine for their instrument support and technical assistance, as well as to the patients who generously donated tissue samples for this study. We sincerely thank F.X. and X.C. for insightful discussions related to the animal studies, and Q.D., Y.C., and S.L. for constructive suggestions on the flow cytometry experiments. Data Availability All data is present in the manuscript and supplemental figures. RNA-seq data reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number **PRJNA1354648** . References Puchalska P, Crawford PA (2017) Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics. Cell Metab 25:262–284. 10.1016/j.cmet.2016.12.022 Matsuura TR, Puchalska P, Crawford PA, Kelly DP (2023) Ketones and the Heart: Metabolic Principles and Therapeutic Implications. Circ Res 132:882–898. 10.1161/CIRCRESAHA.123.321872 Weber DD, Aminzadeh-Gohari S, Tulipan J, Catalano L, Feichtinger RG, Kofler B (2020) Ketogenic diet in the treatment of cancer - Where do we stand? Mol Metab 33:102–121. 10.1016/j.molmet.2019.06.026 Kesarwani P, Kant S, Zhao Y, Miller CR, Chinnaiyan P (2022) The Influence of the Ketogenic Diet on the Immune Tolerant Microenvironment in Glioblastoma. Cancers (Basel). 14. 10.3390/cancers14225550 Husain Z, Huang Y, Seth P, Sukhatme VP (2013) Tumor-derived lactate modifies antitumor immune response: effect on myeloid-derived suppressor cells and NK cells. J Immunol 191:1486–1495. 10.4049/jimmunol.1202702 Wei R, Zhou Y, Li C et al (2022) Ketogenesis Attenuates KLF5-Dependent Production of CXCL12 to Overcome the Immunosuppressive Tumor Microenvironment in Colorectal Cancer. Cancer Res 82:1575–1588. 10.1158/0008-5472.CAN-21-2778 Dai X, Bu X, Gao Y et al (2021) Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade. Mol Cell. 81: 2317-31 e6. 10.1016/j.molcel.2021.03.037 Ferrere G, Tidjani Alou M, Liu P et al (2021) Ketogenic diet and ketone bodies enhance the anticancer effects of PD-1 blockade. JCI Insight. 6. 10.1172/jci.insight.145207 Dmitrieva-Posocco O, Wong AC, Lundgren P et al (2022) beta-Hydroxybutyrate suppresses colorectal cancer. Nature 605:160–165. 10.1038/s41586-022-04649-6 Huang C, Tan H, Wang J et al (2024) beta-hydroxybutyrate restrains colitis-associated tumorigenesis by inhibiting HIF-1alpha-mediated angiogenesis. Cancer Lett 593:216940. 10.1016/j.canlet.2024.216940 Shimazu T, Hirschey MD, Newman J et al (2013) Suppression of oxidative stress by beta-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339:211–214. 10.1126/science.1227166 Philip M, Schietinger A (2022) CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol 22:209–223. 10.1038/s41577-021-00574-3 Wherry EJ (2011) T cell exhaustion. Nat Immunol 12:492–499. 10.1038/ni.2035 McLane LM, Abdel-Hakeem MS, Wherry EJ (2019) CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol 37:457–495. 10.1146/annurev-immunol-041015-055318 Beltra JC, Manne S, Abdel-Hakeem MS et al (2020) Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms. Immunity. 52: 825 – 41 e8. 10.1016/j.immuni.2020.04.014 Chang JT, Wherry EJ, Goldrath AW (2014) Molecular regulation of effector and memory T cell differentiation. Nat Immunol 15:1104–1115. 10.1038/ni.3031 Intlekofer AM, Takemoto N, Wherry EJ et al (2005) Effector and memory CD8 + T cell fate coupled by T-bet and eomesodermin. Nat Immunol 6:1236–1244. 10.1038/ni1268 He Y, Fu L, Li Y et al (2021) Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8(+) T cell immunity. Cell Metab. 33: 988–1000 e7. 10.1016/j.cmet.2021.03.002 Kallies A, Xin A, Belz GT, Nutt SL (2009) Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity 31:283–295. 10.1016/j.immuni.2009.06.021 Cui W, Liu Y, Weinstein JS, Craft J, Kaech SM (2011) An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8 + T cells. Immunity 35:792–805. 10.1016/j.immuni.2011.09.017 Shan Q, Hu S, Chen X, Danahy DB, Badovinac VP, Zang C, Xue HH (2021) Ectopic Tcf1 expression instills a stem-like program in exhausted CD8(+) T cells to enhance viral and tumor immunity. Cell Mol Immunol 18:1262–1277. 10.1038/s41423-020-0436-5 Zhou X, Yu S, Zhao DM, Harty JT, Badovinac VP, Xue HH (2010) Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity 33:229–240. 10.1016/j.immuni.2010.08.002 Lotscher J, Marti ILAA, Kirchhammer N et al (2022) Magnesium sensing via LFA-1 regulates CD8(+) T cell effector function. Cell. 185: 585–602 e29. 10.1016/j.cell.2021.12.039 Gnanaprakasam JNR, Kushwaha B, Liu L et al (2023) Asparagine restriction enhances CD8(+) T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response. Nat Metab 5:1423–1439. 10.1038/s42255-023-00856-1 Gounari F, Khazaie K (2022) TCF-1: a maverick in T cell development and function. Nat Immunol 23:671–678. 10.1038/s41590-022-01194-2 Germar K, Dose M, Konstantinou T et al (2011) T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc Natl Acad Sci U S A 108:20060–20065. 10.1073/pnas.1110230108 Zhao X, Shan Q, Xue HH (2022) TCF1 in T cell immunity: a broadened frontier. Nat Rev Immunol 22:147–157. 10.1038/s41577-021-00563-6 Weber BN, Chi AW, Chavez A, Yashiro-Ohtani Y, Yang Q, Shestova O, Bhandoola A (2011) A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476:63–68. 10.1038/nature10279 Taggart AK, Kero J, Gan X et al (2005) (D)-beta-Hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. J Biol Chem 280:26649–26652. 10.1074/jbc.C500213200 Yang Y, Pei T, Hu X et al (2024) Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell. Cell Rep Med 5:101718. 10.1016/j.xcrm.2024.101718 Feng S, Wang H, Liu J, Aa J, Zhou F, Wang G (2019) Multi-dimensional roles of ketone bodies in cancer biology: Opportunities for cancer therapy. Pharmacol Res 150:104500. 10.1016/j.phrs.2019.104500 Wan S, Zhou X, Xie F, Zhou F, Zhang L (2025) Ketogenic diet and cancer: multidimensional exploration and research. Sci China Life Sci 68:1010–1024. 10.1007/s11427-023-2637-2 Hirschberger S, Strauss G, Effinger D et al (2021) Very-low-carbohydrate diet enhances human T-cell immunity through immunometabolic reprogramming. EMBO Mol Med 13:e14323. 10.15252/emmm.202114323 Deng W, Ma Y, Su Z et al (2021) Single-cell RNA-sequencing analyses identify heterogeneity of CD8(+) T cell subpopulations and novel therapy targets in melanoma. Mol Ther Oncolytics 20:105–118. 10.1016/j.omto.2020.12.003 Sade-Feldman M, Yizhak K, Bjorgaard SL et al (2018) Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell. 175: 998–1013 e20. 10.1016/j.cell.2018.10.038 Krishna S, Lowery FJ, Copeland AR et al (2020) Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 370:1328–1334. 10.1126/science.abb9847 Additional Declarations No competing interests reported. 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19:25:21","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128836,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/cef48737312866162aa2b1a9.html"},{"id":99836995,"identity":"a6ab69b5-f396-446d-b219-0536be2fb203","added_by":"auto","created_at":"2026-01-08 19:25:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":426982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKD suppressed tumor growth by enhancing CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell-mediated antitumor immune responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of the dietary experiment design: C57BL/6 mice (n=40) were randomly divided into ketogenic diet (KD) and normal diet (ND) groups, followed by subcutaneous inoculation with B16F10 melanoma cells. Tumor growth and tumor-infiltrating CD8+ T cells were subsequently monitored and analyzed.\u003cstrong\u003e (B) \u003c/strong\u003eTumor volume of B16F10 melanoma-bearing mice on a KD or ND diet (n=6/group). \u003cstrong\u003e(C)\u003c/strong\u003e Serum βOHB levels in B16F10 melanoma-bearing mice fed a KD (n=5) or ND (n=7) diet. \u003cstrong\u003e(D)\u003c/strong\u003e Bar graphs showing the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells in CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e cells isolated from mouse tumor tissues, as determined by flow cytometry (n=5/group). (\u003cstrong\u003eE\u003c/strong\u003e) FITC-Annexin V/propidium iodide (PI) flow cytometry analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from tumor tissues of mouse fed a KD or ND diet. Bar graphs show the quantitative analysis of apoptotic cell ratios (n=5/group). (\u003cstrong\u003eF–H\u003c/strong\u003e) Flow cytometry analysis of Granzyme B, TNFα, and IFN-γ expression in tumor-infiltrating CD8⁺ T cells from KD and ND groups. Bar graphs show the percentages of Granzyme B⁺, IFN-γ⁺, and TNFα⁺ tumor-infiltrating CD8⁺ T cells (n = 5/group). Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/76b213b3e86b5318d5dd6915.png"},{"id":99836998,"identity":"d1a50271-6b8a-4fb4-a95c-67a21a92f84a","added_by":"auto","created_at":"2026-01-08 19:25:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":717514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβOHB enhances CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell function by suppressing apoptosis and promoting proliferation and cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eApoptosis was assessed by\u003cstrong\u003e \u003c/strong\u003eFITC-Annexin V/propidium iodide (PI) flow cytometry analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from mouse spleen, activated with anti-CD3 and anti-CD28 antibodies, and treated with varying concentrations of βOHB (0-10mM). Bar graphs showing the quantitative analysis of apoptotic cell ratios (n=6/group)\u003cstrong\u003e (B–D)\u003c/strong\u003e Bar graphs showing the percentage of Granzyme B, IFN-γ, or TNFα\u003csup\u003e \u003c/sup\u003eexpression in activated mouse splenic CD8\u003csup\u003e+\u003c/sup\u003e T cells treated with varying concentrations of βOHB (0-10 mM), as analyzed by flow cytometry (n=6/group). \u003cstrong\u003e(E)\u003c/strong\u003e Flow cytometry analysis of Edu incorporation in activated mouse splenic CD8\u003csup\u003e+\u003c/sup\u003e T cells treated with 0 and 5 mM βOHB. Bar graphs showing the percentage of Edu\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e (n=6/group).\u003cstrong\u003e (F)\u003c/strong\u003e Flow cytometry analysis of TNFα and IFN-γ expression in tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from human colon cancer and treated with 0 and 5 mM βOHB. Bar graphs showing the percentage of TNFα\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (n=5/group). Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/07678acd6f18ed312c83141f.png"},{"id":99836996,"identity":"1fdf37e6-677d-41bf-98fe-7e9602e2b83e","added_by":"auto","created_at":"2026-01-08 19:25:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":680019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβOHB slows tumor progression through CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell-dependent anti-tumor immunity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of the B16F10 melanoma mouse model experiment: C57BL/6 mice (n=40) were subcutaneously inoculated with B16F10 melanoma cells. After 7 days, the mice were randomly divided into PBS control and βOHB treatment groups. \u003cstrong\u003e(B) \u003c/strong\u003eTumor growth curves of B16F10 melanoma-bearing mice treated with PBS or βOHB (n=6/group). \u003cstrong\u003e(C)\u003c/strong\u003e Bar graphs showing the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells in CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e cells isolated from mouse tumor tissues, as analyzed by flow cytometry (n=6/group). \u003cstrong\u003e(D)\u003c/strong\u003e FITC-Annexin V/propidium iodide (PI) flow cytometry analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from tumor and spleen tissues under PBS or βOHB treatment conditions. Bar graphs show the quantitative analysis of apoptotic cell ratios for tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=4/group) and spleen naïve CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=6/group). \u003cstrong\u003e(E) \u003c/strong\u003eFlow cytometry analysis of Granzyme B, TNFα and IFN-γ expression in tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from B16F10 melanoma-bearing mice under PBS or βOHB treatment conditions. \u003cstrong\u003e(F–H)\u003c/strong\u003e Bar graphs showing the percentage of Granzyme B\u003csup\u003e+\u003c/sup\u003e, IFN-γ\u003csup\u003e+\u003c/sup\u003e and TNFα\u003csup\u003e+\u003c/sup\u003e tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells in the PBS and βOHB treatment groups, as analyzed by flow cytometry (n=5/group).\u003cstrong\u003e (I) \u003c/strong\u003eTumor growth curves of B16F10 melanoma-bearing mice with or without CD8\u003csup\u003e+\u003c/sup\u003e T cell depletion, followed by βOHB treatment (n = 6/group). Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/f175f1d4c1e55f05a0c094f9.png"},{"id":99837000,"identity":"dbf5b75c-06bc-4b42-84ac-3a964ac9627c","added_by":"auto","created_at":"2026-01-08 19:25:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":493690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβOHB inhibits CT26 tumor growth by modulating CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of the CT26 tumor model: BALB/c mice (n=30) were subcutaneously inoculated with CT26 colon cancer cells. After 7 days, the mice were randomly divided into PBS control and βOHB treatment groups. \u003cstrong\u003e(B) \u003c/strong\u003eTumor growth curves of CT26 tumor-bearing mice treated with PBS or βOHB (n=6/group). \u003cstrong\u003e(C)\u003c/strong\u003e Tumor size of CT26 tumor mice under PBS or βOHB treatment conditions (n=6/group).\u003cstrong\u003e (D)\u003c/strong\u003e Annexin V-FITC/propidium iodide (PI) flow cytometry analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from tumor and spleen tissues under PBS or βOHB treatment conditions. Bar graphs show the quantitative analysis of apoptotic cell ratios for tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=6/group) and spleen naïve CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=6/group). \u003cstrong\u003e(E)\u003c/strong\u003e Flow cytometry analysis of TNFα and IFN-γ expression in tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from CT26-bearing mice in the PBS and βOHB treatment groups.\u003cstrong\u003e \u003c/strong\u003eBar graphs showing the percentage of IFN-γ\u003csup\u003e+\u003c/sup\u003e and TNFα\u003csup\u003e+\u003c/sup\u003e tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells analyzed by flow cytometry in the two groups (n=6/group). Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/1b64d349db173d5cd264f679.png"},{"id":100356739,"identity":"90e9e85f-73c5-4e3a-a9ed-f2d2bbc14052","added_by":"auto","created_at":"2026-01-16 07:17:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":468503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβOHB upregulates the Tcf7-Lck dependent pathway in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Heat map showing differentially expressed genes in tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells under PBS or βOHB treatment conditions. \u003cstrong\u003e(B) \u003c/strong\u003eGene set enrichment analysis (GSEA) of functional pathways and signaling networks in βOHB-treated tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003e(C–D)\u003c/strong\u003e qPCR analysis of Tcf7 and Lck mRNA expression in βOHB-treated activated splenic CD8\u003csup\u003e+\u003c/sup\u003e T cells and tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=3/group).\u003cstrong\u003e (E)\u003c/strong\u003e Western blot analysis of Tcf7, Lck, and NF-κB protein levels in βOHB-treated activated splenic CD8\u003csup\u003e+\u003c/sup\u003e T cells and tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells. Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/6822b8c90d7321e9da313db5.png"},{"id":100356845,"identity":"f71af874-f768-42a1-aea1-ee0386b307c9","added_by":"auto","created_at":"2026-01-16 07:17:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":734530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHcar2 mediates βOHB-induced Tcf7 expression in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Tcf7, Lck, and NF-κB protein expression in wide-type (WT), scrambled siRNA (siNC), and siTcf7 CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without βOHB treatment. \u003cstrong\u003e(B)\u003c/strong\u003e Annexin V-FITC/propidium iodide (PI) flow cytometry analysis of WT, siNC, and siTcf7 CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without βOHB treatment.\u003cstrong\u003e \u003c/strong\u003eBar graphs show the quantitative analysis of apoptotic cell ratios (n=3/group).\u003cstrong\u003e (C)\u003c/strong\u003e Flow cytometry analysis of Granzyme B expression in WT, siNC, and siTcf7 CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without βOHB treatment. \u003cstrong\u003e(D–F) \u003c/strong\u003eBar graphs showing the percentage of Granzyme B\u003csup\u003e+\u003c/sup\u003e, TNFα\u003csup\u003e+\u003c/sup\u003e, and IFN-γ\u003csup\u003e+ \u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in WT, siNC, and siTcf7 CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without βOHB treatment (n=3/group). \u003cstrong\u003e(G)\u003c/strong\u003e Tcf7 mRNA expression in wide-type (WT), scrambled siRNA (siNC), and siHcar2 CD8\u003csup\u003e+\u003c/sup\u003e T cells with or without βOHB treatment (n=6/group). Data are presented as the mean ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/1d364c1f89aca16da984438a.png"},{"id":100357185,"identity":"9ff96e76-0551-4cf0-ac40-07d2d98f4d39","added_by":"auto","created_at":"2026-01-16 07:19:13","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":140188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic model of Ketogenic Diets/βOHB-mediated CD8⁺ T cell activation. \u003c/strong\u003eketogenic dietsand βOHB enhance anti-tumor CD8⁺ T cell responses via the Hcar2–Tcf7–Lck axis, suggesting dietary or metabolite-based interventions as potential cancer immunotherapy strategies.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/5625a75cb238ca3e360670f7.jpeg"},{"id":100406144,"identity":"e0d2d6c0-ce50-4692-95cf-da9addd3d987","added_by":"auto","created_at":"2026-01-16 12:44:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4774732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/4cb168a6-204f-48df-be37-f6f5c2f20d86.pdf"},{"id":100356755,"identity":"78cb181e-f115-4bc5-8d35-39eb48afe26f","added_by":"auto","created_at":"2026-01-16 07:17:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2564229,"visible":true,"origin":"","legend":"","description":"","filename":"Bai20251222SMHJ.docx","url":"https://assets-eu.researchsquare.com/files/rs-8428666/v1/18e47d328f8e735fc78da2b7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eβ-hydroxybutyrate potentiates anti-tumor immunity by modulating cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cell responses\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKetone bodies generated during ketogenic diets (KD) exert pleiotropic functions beyond energy supply by modulating inflammation, oxidative stress, catabolic pathways, and associated gene expressions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Accumulating studies also supports KDs as promising adjuncts to cancer therapy, with preclinical studies in glioma, pancreatic cancer, and colon cancer showing that KD-induced immunological changes within the tumor microenvironment (TME) consistently suppress tumor growth and prolong survival in tumor-bearing mice (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Moreover, these immune-modulatory effects enable KDs to sensitize otherwise resistant tumors to immune checkpoint blockade (ICB) therapy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). β-hydroxybutyrate (βOHB), the predominant circulating ketone body, acts not only as an energy substrate but also as a potent signaling metabolite. It has been shown to activate Hcar2 signaling and induce transcriptional factor Hopx to suppress colorectal cancer development in mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It also exerts anti-angiogenic effects in colitis-associated cancer through reducing HIF-1α-dependent VEGFA secretion from cancer cells (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In addition, β-OHB functions as an endogenous inhibitor of HDACs, reducing the activities of HDAC1, HDAC3, and HDAC4, which results in hyperacetylation of histone H3 and upregulation of oxidative stress resistance genes including FOXO3a, MT2, SOD2, and catalase (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Together, these studies identify βOHB as a bioactive metabolite with multifaceted anti-cancer activities, supporting further investigation into its therapeutic potential.\u003c/p\u003e \u003cp\u003eCD8⁺ T cells are central to the anti-tumor efficacy of ICB and adoptive T cell therapy. However, their durable response to immunotherapy is often compromised by T cell exhaustion (Tex), a dysfunctional state arising during chronic infections and malignancies, characterized by impaired T cell proliferation, reduced cytokine secretion, and sustained expression of multiple inhibitory receptors (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Tex represents an alternative CD8⁺ T cell fate in which extensive transcriptional reprogramming constrains their effector functions and limits their transition into long-lived memory subsets (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A network of transcriptional factors, including Tbx21, Eomes, Id2, Blimp-1, Bcl6, Tcf7 has been identified as central to this process (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Specifically, Tbx21 and Eomes coordinate the differentiation and balance of effector and memory CD8⁺ T cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), Id2 promotes the survival of effector T cells through IL-12\u0026ndash;dependent signaling pathways (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), Blimp-1 is a master regulator of terminal effector T cell differentiation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), whereas Bcl-6 promotes the development of KLRG1\u003csup\u003elo\u003c/sup\u003e IL-7Rʰⁱ memory T cells (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Tcf7/TCF1 is essential for generation and maintenance of stem-like CD8⁺ T cells, which are required for sustained responses under chronic antigen exposure and responsiveness to ICB therapies (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These transcriptional programs are dynamically shaped by extracellular cues within the TME, and their modulation enhances the recruitment, persistence, and functional activity of TME-infiltrated CD8⁺ T cells (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), offering a promising strategy to improve the efficacy of cancer immunotherapies.\u003c/p\u003e \u003cp\u003eDespite evidence that KDs and βOHB modulate the TME and immune responses, the direct impact of βOHB on CD8⁺ T cell differentiation, activation, and exhaustion, as well as the underlying molecular mechanisms, remain poorly understood. Here, we investigated the regulatory effects of KDs and βOHB on CD8⁺ T cells within the TME and found that βOHB promotes the expansion of CD8⁺ T cells with high Tcf7 expression, which sustain stemness, self-renewal, and effector functions in response to tumor antigens via the HCAR2\u0026ndash;Tcf7\u0026ndash;Lck signaling axis. These findings provide new insights into the regulation of TME-infiltrated CD8⁺ T cell fate and function, and highlight βOHB as a promising therapeutic candidate to potentiate immune responses and improve clinical outcomes in cancer treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eWild-type female and male C57BL/6 and BALB/c mice, aged 6\u0026ndash;8 weeks, were purchased from Jihui Experiment Animal Feeding Co. Ltd. Age-matched littermates of both sexes were used in all experiments, with sexes analyzed separately when indicated. Mice were maintained under specific pathogen-free (SPF) conditions with food and water ad libitum on a 12-h light/dark cycle. All animal procedures were conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee of the Ninth People\u0026rsquo;s Hospital, Shanghai Jiao Tong University School of Medicine (approval number:SH9H-2022-A8-1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHuman specimens\u003c/h3\u003e\n\u003cp\u003eTumor-infiltrating T cells were isolated from patients with colon cancer. Plasma samples were collected from patients diagnosed with advanced colorectal carcinoma for this study. All samples were collected with informed consent under procedures approved by the Institutional Review Board (IRB) of Renji Hospital, Shanghai Jiao Tong University School of Medicine (approval number:RA-2022-411).\u003c/p\u003e\n\u003ch3\u003eCell isolation and culture\u003c/h3\u003e\n\u003cp\u003eMouse tumor cell lines (B16F10, CT26) were purchased from National Collection of Authenticated Cell Culture. B16F10 cells were cultured in DMEM (HyClone), and CT26 cells in RPMI 1640 medium (HyClone), both supplemented with 10% FBS.\u003c/p\u003e \u003cp\u003eMouse lymphocytes were isolated from spleen using density gradient centrifugation with Lymphoprep\u0026trade; (Serumwerk). CD8\u003csup\u003e+\u003c/sup\u003e T cells were then purified via positive selection using MicroBeads (Miltenyi Biotec), resuspended at 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL, and activated with anti-CD3 (2 \u0026micro;g/mL) and anti-CD28 (1 \u0026micro;g/mL) mAbs (BD Biosciences) for 24\u0026ndash;48 h. Activated CD8\u003csup\u003e+\u003c/sup\u003e T cells were maintained in RPMI 1640 medium containing 10% FBS, IL-2 (10 ng/mL), β-mercaptoethanol, L-glutamine (Gibco), MEM (Non-essential amino acid solution, Gibco), HEPES (Sigma), and treated with specific concentrations of βOHB (Sigma) in various experiments.\u003c/p\u003e \u003cp\u003eTumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated through mechanical dissociation and filtration through a 70 \u0026micro;m cell strainer, followed by mononuclear cells enrichment using Lymphoprep\u0026trade; (Serumwerk) via density gradient centrifugation. CD8\u003csup\u003e+\u003c/sup\u003e T cells were then further purified using MicroBeads (Miltenyi Biotec) through positive selection. The purified CD8\u003csup\u003e+\u003c/sup\u003e T cells were subsequently assessed by FACS.\u003c/p\u003e\n\u003ch3\u003eTumor inoculation and treatment\u003c/h3\u003e\n\u003cp\u003eC57BL/6 mice were inoculated subcutaneously in the right flank with 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e B16F10 melanoma cells, and BALB/c mice were similarly inoculated with 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e CT26 colon cancer cells. Tumor growth was monitored by measuring tumor volumes every 2 days using digital calipers, and calculated by the formula: tumor volume = (a \u0026times; b \u0026times; c)/2.\u003c/p\u003e \u003cp\u003eFor the ketogenic diet experiment, C57BL/6 mice were randomly assigned to two groups. The ketogenic diet (KD) group received a high-fat diet providing 80% of calories from fat, whereas the control group was fed a standard chow diet (ND group). After two weeks of dietary treatment, mice were subcutaneously inoculated with B16F10 cells as described above. Tumor growth and body weight were monitored throughout the study.\u003c/p\u003e \u003cp\u003eFor intratumoral treatment, PBS or β-hydroxybutyrate (βOHB) was dissolved in sterile PBS and administered at 100 mg/kg (B16F10) or 200 mg/kg (CT26) in a final injection volume of 100 \u0026micro;L. Injections were performed once daily or every other day starting on day 7 after tumor inoculation. Mice were lightly anesthetized with 2% isoflurane during intratumoral injections to minimize distress.\u003c/p\u003e \u003cp\u003eFor CD8⁺ T cell depletion, Once B16F10 tumors became palpable (7 days post-inoculation), CD8⁺ T cells were depleted using an anti-CD8a monoclonal antibody (clone 2.43, Bio X Cell, Cat# BE0061). The antibody was diluted in sterile PBS and administered intraperitoneally at 10 mg/kg in a total volume of 100 \u0026micro;L, once weekly. Intratumoral βOHB administration began 24 h after the first CD8⁺ T cell depletion injection and was delivered once daily at 100 mg/kg, 100 \u0026micro;L per injection. Mice were briefly anesthetized with 2% isoflurane during injections to minimize discomfort.\u003c/p\u003e \u003cp\u003eMice were monitored daily for tumor growth, body weight, and general health. Euthanasia was performed when tumors reached 2.5 cm\u0026sup3; or humane endpoints were met, using CO₂ asphyxiation followed by cervical dislocation according to AVMA 2020 guidelines. No animals or data were excluded unless predefined humane endpoints were reached. Animals were not blinded due to visible phenotypic differences and the need to accurately administer treatments and monitor welfare.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eFor surface staining, cells were pre-incubated with Fc blocking buffer (BD Biosciences, Cat# 553141) for 15 min on ice, and then stained with fluorescence-conjugated monoclonal antibodies in BD\u0026trade; Stain Buffer(BD Biosciences, Cat# 554656) for 30 min on ice. For intracellular staining, CD8\u003csup\u003e+\u003c/sup\u003e T cells were treated with 0 or 5 mM βOHB for 48 h, followed by stimulated with Cell Activation Cocktail (with Brefeldin A) (BioLegend, Cat# 423303) for 6 h. Cells were then fixed in Fixation/Permeabilization solution (BD Biosciences, Cat# 554714) for 40 min at 4℃ in the dark. After washing with Perm/Wash Buffer (BD Biosciences, Cat# 554714), intracellular staining was performed using Alexa Fluor 647 anti-human/mouse Granzyme B antibody, FITC-anti-mouse TNF-α antibody, and PE-anti-mouse IFN-γ antibody for 50 min in the dark. Antibodies were purchased from BD Biosciences or Biolegend. Detailed information was provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Apoptosis was assessed by staining cells with Annexin V and propidium iodide (PI) in 1\u0026times; Binding buffer (BD Biosciences) at RT for 15 min in the dark. Flow cytometry was conducted using an LSR-Fortessa (BD Biosciences) instrument, and data were analyzed using FlowJo software (version 10). Cells were gated as singlets, live cells, and specific immune cell populations, including CD8\u003csup\u003e+\u003c/sup\u003e T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e), CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), NK cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003eNK1.1\u003csup\u003e+\u003c/sup\u003e), and B cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR and Western blot\u003c/h2\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells were incubated with 0 or 5 mM βOHB for 72 h, then washed and harvested. RNA was extracted from these cells using TRIzol reagent (Thermo Fisher Scientific, Cat# 15596026), and reverse transcribed using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (TaKaRa, Cat# RRO47A). Real-time PCR was performed using TB Green \u003cem\u003ePremix Ex Taq\u003c/em\u003e (Tli RNase H Plus) kit (TaKaRa, Cat# RR820A) on a LightCycler 480 instrument (Roche). Primers were listed in Table S2.\u003c/p\u003e \u003cp\u003eFor Western blot analysis, CD8\u003csup\u003e+\u003c/sup\u003e T cells, treated as described above, were washed and lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with 1\u0026times; protease inhibitor cocktail (Proteintech). Total protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Protein samples were then denatured at 95\u0026deg;C for 5 min. Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% fat-free milk for 1 h, followed by overnight incubation at 4\u0026deg;C with primary antibodies, including anti-TCF1/Tcf7, anti-Lck, anti-NFκB, anti-H3K27ac, anti-Notch1, and anti-GAPDH. After incubation with HRP-conjugated secondary antibody for 30 min, protein levels were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). Detailed antibody information was provided in Table S3.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistology and immunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eTumor tissues were fixed in 4% paraformaldehyde (PFA) solution for 24 h, and then embedded in paraffin blocks following standard procedures. For antigen retrieval, tissue sections were immersed in citrate buffer and heated in saturated steam for 20 min. After cooling, the slides were washed with phosphate-buffered saline (PBS) and blocked with 5% bovine serum albumin (BSA) for 30 min to reduce background staining. The sections were then incubated overnight at 4\u0026deg;C with 488-conjugated anti-Mouse CD8a antibody (Proteintech, Cat# CL488-65069). After DAPI staining, fluorescence images were captured to visualize target proteins using a confocal laser scanning microscope (Leica TCS SP8 STED 3X).\u003c/p\u003e\n\u003ch3\u003eRNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells from B16F10 tumor-bearing mice were isolated, sorted, and incubated with 0 or 5 mM βOHB for 72 h. Total RNA was extracted using the TRIzol reagent. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and verified by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was enriched using Oligo(dT) beads, fragmented into short segments using fragmentation buffer, and reverse transcribed into cDNA by using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Cat# 7530). The purified double-stranded cDNA fragments were end-repaired, A-tailed, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1\u0026times;), followed by size selection through agarose gel electrophoresis and PCR amplification.\u003c/p\u003e \u003cp\u003eThe cDNA libraries were sequenced on the Illumina NovaSeq 6000 platform. Clean reads were aligned to the mouse reference genome (mm10). Gene-level counts were calculated and normalized as reads per kilobase of transcript per million mapped reads (RPKM) to estimate gene expression levels. The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject \u003cb\u003ePRJNA1354648\u003c/b\u003e. Differentially expressed genes (DEGs) were identified using DESeq2, with genes showing an adjusted \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log₂ (fold change) | \u0026ge; 1 considered significantly differentially expressed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003esiRNA knockdown assay\u003c/h2\u003e \u003cp\u003eThe siRNAs targeting mouse genes were synthesized as follows:\u003c/p\u003e \u003cp\u003eTcf7-1: 5\u0026prime;- GAAGCCAGUCAUCAAGAAA-3\u0026prime;\u003c/p\u003e \u003cp\u003eTcf7-2: 5\u0026prime;- GAAAUGCAUUCGGUACUUA-3\u0026prime;\u003c/p\u003e \u003cp\u003eHcar2-1: 5\u0026prime;-GGCGAGGCAUAUCUGUGUA-3\u0026prime;\u003c/p\u003e \u003cp\u003eHcar2-2: 5\u0026prime;-CGUUCCUGACGGACAACUA-3\u0026prime;\u003c/p\u003e \u003cp\u003eScrambled siRNA: 5\u0026prime;-UUCUCCGAACGUGUCACGU-3\u0026prime;\u003c/p\u003e \u003cp\u003eBriefly, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were seeded into a six-well plate in 2 mL of RPMI 1640 medium (without FBS or antibiotics) for 24 h. Cells were then transfected with 3 \u0026micro;L of 10 \u0026micro;M siRNA per well, pre-mixed with 9 \u0026micro;L of Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Cat# 13778150) in Opti-MEM medium. After 6 h, the medium was replaced with fresh RPMI 1640 supplemented with 10% FBS. After culturing for 48 h, cells were treated with βOHB for another 48 h before being harvested for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo, Cat# CK04) according to the manufacturer\u0026rsquo;s instructions. In brief, 90 \u0026micro;L of tumor cells were plated at a concentration of 3,000 cells/well into 96-well plates. Then, 10 \u0026micro;L of CCK-8 solution was added to each well and the cells were incubated at 37\u0026deg;C for 2 h. Absorbance was measured at 450 nm using a microplate reader (VICTOR Nivo).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eβOHB measurements\u003c/h2\u003e \u003cp\u003eEach sample from the mouse tumor models was divided in two aliquots for total protein measurement and βOHB quantification, respectively. Tumor samples were washed with PBS and collected by centrifugation at 300\u0026times;g for 5 min. βOHB levels were determined using the BHB Colorimetric Assay Kit (Cayman).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9.0 software, unless otherwise indicated. All experimental data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined using a two-tailed Student\u0026rsquo;s t-test. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKD retards tumor growth by enhancing CD8\u003csup\u003e+\u003c/sup\u003e T cell functions\u003c/h2\u003e \u003cp\u003eTo explore the impact of ketone bodies on tumor growth and anti-tumor immunity, we treated C57BL/6J mice with a ketogenic diet (KD) containing 80% fat under specific pathogen\u0026ndash;free (SPF) conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Compared with mice receiving a normal diet (ND), KD-fed mice showed a substantial reduction in the growth of subcutaneously implanted B16F10 melanoma tumors, accompanied by elevated βOHB levels in serum but not in tumors, while body weight remained comparable between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u0026ndash;C). KD feeding also improved long-term survival of tumor-bearing mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD), consistent with previous studies supporting the therapeutic potential of KD in cancer (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then evaluated the effects of KD on the tumor immune microenvironment by profiling tumor-infiltrating immune cells via flow cytometry (FACS). KD feeding led to a modest increase in NK cell infiltration and had minimal effects on CD4⁺ T cells, dendritic cells, and B cells. Notably, the accumulation of CD8⁺ T cells within tumors was significantly elevated in KD-fed mice compared with the ND group as shown by FACS and immunofluorescent analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE\u0026ndash;I). Further analysis revealed that the tumor-infiltrating CD8⁺ T cells from KD-fed mice exhibited markedly reduced apoptosis, as indicated by decreased Annexin V staining, suggesting improved survival within the tumor microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, they also showed elevated expression of key cytotoxic and effector molecules, including Granzyme B, IFNγ, and TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;H), implying that KD enhances the cytotoxic potential of CD8⁺ T cells for effective tumor cell killing. The above observations demonstrate that KD suppresses tumor growth by promoting both the accumulation and functional potency of CD8⁺ T cells, underscoring an important role of CD8⁺ T cell-mediated immunity in the antitumor effects of ketogenic diets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eβOHB promotes CD8⁺ T cell survival and cytotoxic function\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular basis of KD-mediated enhancement of anti-tumor immunity, we investigated whether βOHB, the predominant ketone body elevated under ketogenic conditions, directly modulates effector CD8⁺ T cell activity in culture. Mouse splenic CD8⁺ T cells were activated with soluble anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) and IL-2, and cultured in complete medium supplemented with increasing concentrations of βOHB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D, treatment with βOHB reduced apoptosis of activated CD8⁺ T cells and enhanced their cytotoxic effects in a dose-dependent manner, with the strongest effects observed at 5 mM, as indicated by decreased Annexin V staining and elevated Granzyme B, IFN-γ, and TNF-α production. Notably, this effect was attenuated at 10 mM βOHB, suggesting a narrow concentration range in which βOHB optimally supports effector T cell survival and function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). In contrast, βOHB had minimal impact on na\u0026iuml;ve CD8⁺ T cells, indicating that it selectively modulates antigen-experienced T cells (Fig. S2A\u0026ndash;C). Consistent with its prosurvival effects and enhancement of effector functions, βOHB treatment also promoted proliferation of activated CD8⁺ T cells, as shown by increased EdU incorporation relative to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then examined whether acetoacetate (AcAc), another major ketone body, exerts similar effects. Treatment of activated CD8⁺ T cells with 5 mM AcAc did not affect apoptosis or effector molecule expression, indicating that βOHB, rather than AcAc, is the primary ketone body responsible for enhancing effector CD8⁺ T cell survival and function (Fig. S2D\u0026ndash;G). To further evaluate the effects of βOHB in a tumor context, tumor-infiltrating CD8⁺ T cells (CD8⁺ TILs) isolated from human colon cancer specimens were cultured with βOHB at concentrations ranging from 0 to 10 mM. Consistent with observations in activated mouse splenic CD8⁺ T cells, 5 mM βOHB elicited the strongest response, markedly reducing apoptosis and enhancing the production of key effector cytokines in CD8⁺ TILs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and Fig. S2H). Together, these results identify βOHB as a selective enhancer of effector CD8⁺ T cell survival, function, and proliferation, providing a direct link between ketogenic conditions and antitumor immunity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eβOHB potentiates CD8⁺ TILs to promote anti-tumor immunity\u003c/h2\u003e \u003cp\u003eTo investigate the role of βOHB in CD8⁺ T cell-mediated anti-tumor immunity \u003cem\u003ein vivo\u003c/em\u003e, B16F10 melanoma-bearing mice were treated with βOHB intratumorally (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). βOHB administration elevated βOHB levels within tumor tissue while serum concentrations remained largely unchanged, and markedly suppressed tumor growth without affecting body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig. S3A\u0026ndash;D). We next analyzed immune cell infiltration in tumors by flow cytometry. Compared with PBS-treated controls, βOHB-treated tumors exhibited modest increases in NK cells and dendritic cells, whereas the relative abundance of CD4⁺ T cells and B cells remained unchanged across the examined tumor samples (Fig. S3E\u0026ndash;H). Notably, intratumoral CD8⁺ T cells were substantially enriched following βOHB treatment, as confirmed by both flow cytometry and immunofluorescence staining of tumor tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Fig. S3I). This enrichment aligns with the increased CD8⁺ T cell infiltration observed in tumors from KD-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequent analysis revealed that CD8⁺ T cells isolated from βOHB-treated tumors exhibited substantially enhanced survival, whereas spleen-derived CD8⁺ T cells showed no significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These tumor-infiltrating CD8⁺ T cells also displayed elevated expression of polyfunctional effector molecules, including Granzyme B, IFN-γ, and TNF-α, indicative of enhanced cytotoxic activity and potentiated antitumor function (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;H). To determine whether the reduced tumor growth in βOHB-treated mice was mediated by CD8⁺ T cells, B16F10-bearing mice were treated with anti-CD8 antibodies to deplete CD8⁺ T cells. Remarkably, removal of CD8⁺ T cells abrogated the anti-tumor effects of βOHB, confirming that βOHB exerts its anti-tumor efficacy in a CD8⁺ T cell\u0026ndash;dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and Fig. S3J, K).\u003c/p\u003e \u003cp\u003eWe then assessed the reproducibility of these findings in another syngeneic tumor model. In mice bearing CT26 colon carcinoma, βOHB administration similarly suppressed tumor growth, enhanced CD8⁺ T cell survival within the tumor microenvironment, and promoted effector cytokine production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;E). These results suggest that the immunopotentiating effects of βOHB on CD8⁺ TILs are conserved across distinct tumor contexts. Besides, when B16F10 and CT26 cancer cells were cultured with βOHB for 48 hours \u003cem\u003ein vitro\u003c/em\u003e, only minimal changes were observed in cell viability, apoptosis, or proliferation rates, indicating that βOHB exerts its anti-tumor effects primarily through potentiation of CD8⁺ T cell-driven immune responses rather than through direct cytotoxicity against tumor cells (Fig. S4A\u0026ndash;D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eβOHB enhances CD8⁺ T cell anti-tumor function though Tcf7-dependent transcriptional regulation\u003c/h2\u003e \u003cp\u003eTo dissect the molecular mechanisms by which βOHB modulates tumor-infiltrating CD8⁺ T cell function, we performed RNA sequencing of CD8⁺ T cells isolated from tumors and cultured in the presence or absence of βOHB. Transcriptomic profiling revealed broad reprogramming of pathways associated with metabolism, effector function, and survival (Fig. S5A). Gene set enrichment analysis (GSEA) showed a strong upregulation of T cell activation\u0026ndash;associated genes (\u003cem\u003eGzma\u003c/em\u003e, \u003cem\u003eGzmb\u003c/em\u003e, \u003cem\u003ePrf1\u003c/em\u003e, \u003cem\u003eIl12rb2\u003c/em\u003e, \u003cem\u003eIfng\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e) and enrichment of TCR signaling and cytokine pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Transcription factors (TFs) that orchestrate CD8⁺ T cell differentiation and effector programming, including Tcf7, Tbx21, and Eomes, were also markedly upregulated following βOHB stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Consistently, qPCR validation confirmed that the expression of representative genes (\u003cem\u003eTcf7\u003c/em\u003e, \u003cem\u003eLck\u003c/em\u003e, \u003cem\u003eTbx21\u003c/em\u003e, \u003cem\u003eEomes\u003c/em\u003e, \u003cem\u003eNfκb2\u003c/em\u003e, and \u003cem\u003eNfκbiz\u003c/em\u003e) was substantially elevated in βOHB-treated CD8⁺ T cells compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D and Fig. S5B\u0026ndash;D). In agreement with these transcriptional changes, Western blot analysis demonstrated that βOHB treatment also increased Tcf7, Lck, and NF-κB protein levels in both splenic effector and tumor-infiltrating CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong these transcriptional regulators, Tcf7 stood out due to its well-established role in maintaining CD8⁺ T cell stemness, self-renewal, and effector function in response to viral or tumor antigens (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To investigate whether Tcf7 mediates the tumor-suppressive effects of βOHB, CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated from C57BL/6 mice spleens and activated with anti-CD3, anti-CD28 and IL-2. Small interfering RNA (siRNA) targeting \u003cem\u003eTcf7\u003c/em\u003e was used to knock down \u003cem\u003eTcf7\u003c/em\u003e expression in activated CD8⁺ T cells (Fig. S5E and F). Compared with control cells transfected with scrambled siRNA, siTcf7 CD8⁺ T cells exhibited markedly reduced expression of Lck and NF-κB, key mediators of antitumor immunity, consistent with the established role of Tcf7 in regulating T cell effector function, and βOHB treatment failed to enhance their expression in si\u003cem\u003eTcf7\u003c/em\u003e CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Moreover, Tcf7 knockdown significantly attenuated the effects of βOHB on CD8⁺ T cell apoptosis and effector cytokine production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;F and Fig. S6A\u0026ndash;B). Taken together, these data suggest that βOHB enhances CD8⁺ T cell anti-tumor activity through Tcf7-dependent transcriptional reprogramming and activation of the Lck\u0026ndash;NFκB signaling axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHcar2 is required for βOHB-mediated Tcf7 upregulation\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanisms by which βOHB upregulates \u003cem\u003eTcf7\u003c/em\u003e, we comprehensively evaluated potential regulatory pathways in activated mouse splenic CD8⁺ T cells. We first assessed whether HDAC inhibition by βOHB contributes to \u003cem\u003eTcf7\u003c/em\u003e induction. Although βOHB treatment modestly increased global H3K27ac levels, the extent of acetylation was much lower than that induced by the potent HDAC inhibitor Trichostatin A (TSA). Notably, TSA could not upregulate \u003cem\u003eTcf7\u003c/em\u003e expression as observed with βOHB treatment instead it led to a reduction in \u003cem\u003eTcf7\u003c/em\u003e protein levels (Fig. S6C and D). These results suggest that βOHB-induced Tcf7 expression is independent of its HDAC-inhibitory activity. NOTCH signaling has been reported to induce \u003cem\u003eTcf7\u003c/em\u003e and its downstream targets such as \u003cem\u003eBcl11b\u003c/em\u003e, \u003cem\u003eGata3\u003c/em\u003e, \u003cem\u003eLck\u003c/em\u003e, and \u003cem\u003eLat\u003c/em\u003e (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, the levels of Notch1 responsible for downstream transcriptional activation, remained unchanged following βOHB treatment, suggesting that NOTCH signaling is not involved in βOHB-mediated upregulation of \u003cem\u003eTcf7\u003c/em\u003e (Fig. S6E).\u003c/p\u003e \u003cp\u003eWe next explored whether βOHB regulates \u003cem\u003eTcf7\u003c/em\u003e expression through receptor-mediated signaling pathways. βOHB functions as a signaling metabolite by engaging its cell-surface receptor Hcar2 (Gpr109a), which has been implicated in immune regulation and transcriptional control (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). To determine whether Hcar2 mediates βOHB-induced \u003cem\u003eTcf7\u003c/em\u003e expression, CD8⁺ T cells were transiently transfected with siRNA targeting Hcar2 or with scrambled control siRNA and subsequently treated with βOHB or PBS for 48 hours. qPCR analysis showed that βOHB markedly upregulated \u003cem\u003eTcf7\u003c/em\u003e expression in control cells, whereas this induction was nearly abolished upon Hcar2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and Fig. S6F). These results identify Hcar2 as a key receptor mediating βOHB-induced activation of Tcf7 transcription and CD8⁺ T cell-dependent antitumor immunity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eβ-hydroxybutyrate (βOHB), the major ketone body elevated during fasting or ketogenic states, has been shown to suppress tumor growth, in part by modulating the tumor immune microenvironment(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e); however, its impact on CD8⁺ T cell-mediated antitumor immunity remains poorly understood. In this study, we revealed that βOHB inhibits tumor progression by enhancing CD8⁺ T cell accumulation, survival, and effector function within the tumor microenvironment. Mechanistically, βOHB upregulates the transcription factor Tcf7 and its downstream Lck\u0026ndash;NFκB signaling cascade via the βOHB receptor Hcar2, independently of its canonical HDAC-inhibitory activity or Notch signaling. The identified βOHB\u0026ndash;Hcar2\u0026ndash;Tcf7 axis establishes a direct molecular link between metabolic signaling and transcriptional regulation in effector T cells, highlighting βOHB as a key immunometabolic cue that sustains CD8⁺ T cell persistence and effector potency within the tumor microenvironment.\u003c/p\u003e \u003cp\u003eKetogenic diets can inhibit tumor growth and metastasis by restricting energy availability to cancer cells, inhibiting angiogenesis, alleviating oxidative stress in normal cells, and modulating oncogenic signaling pathways (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). KDs have also been shown to enhance T cell-mediated antitumor immunity by inhibiting immunosuppressive signals in TME, such as KLF5-dependent CXCL12 expression decreased intratumoral accumulation of immunosuppressive cells, increased infiltration of NK and cytotoxic T cells (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). CD8⁺ T cells, the principal cytotoxic effectors of antitumor immunity, are essential for tumor clearance and immune surveillance. Previous work reported the ketogenic diet enhances immune function in healthy individuals by inducing ketone body-driven metabolic reprogramming toward mitochondrial oxidation, thereby strengthening CD4⁺, CD8⁺, and regulatory T-cell activity and memory formation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). However, whether βOHB directly regulates cytotoxic CD8⁺ T cell activity in tumors remained unclear. Our study indicates that KD and βOHB treatment in mouse tumor models change the tumor immune microenvironment by markedly enhancing CD8⁺ T cell infiltration and effector functions, resulting in increased antigen-specific cytotoxicity and improved tumor suppression. These observations align with recent studies showing that ketogenic diets and ketone bodies can augment the efficacy of PD-1 blockade in mouse tumor models (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), supporting the hypothesis that ketogenic interventions may potentiate CD8⁺ T cell-mediated antitumor immunity and help overcome resistance to immune checkpoint blockade.\u003c/p\u003e \u003cp\u003eRecent single-cell RNA sequencing analyses in melanoma patients have shown that CD8⁺ TILs consist of progenitor-like (Tex-stem) and terminally differentiated (Tex-term) subsets (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Tex-stem cells display self-renewal, durability, and the capacity to differentiate into terminal effector cells, with these properties critically dependent on TCF1, a transcription factor encoded by \u003cem\u003eTcf7\u003c/em\u003e that maintains the stemness and long-term persistence of antigen-responsive CD8⁺ T cells (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In our study, βOHB treatment significantly upregulated Tcf7 expression in mouse CD8⁺ TILs, along with its downstream targets Lck and NFκB. Silencing \u003cem\u003eTcf7\u003c/em\u003e abrogated βOHB-mediated enhancement of CD8⁺ TIL functions, suggesting that βOHB may promote the stem-like properties and functional capabilities of CD8⁺ T cells within the tumor microenvironment through Tcf7 signaling. The presence of TCF1/Tcf7⁺ stem-like CD8⁺ TILs within tumors is associated with enhanced therapeutic efficacy in immune checkpoint blockade (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Our findings that βOHB increases Tcf7 expression and enhances CD8⁺ T cell\u0026ndash;mediated antitumor immunity suggest that dietary or pharmacological βOHB supplementation could serve as an adjuvant to checkpoint inhibitors to promote durable immune responses in cancer patients. Moreover, βOHB supplementation during \u003cem\u003eex vivo\u003c/em\u003e T cell expansion may enhance the generation and persistence of therapeutic T cells, such as CAR-T cells, thereby augmenting their antitumor potency and long-term survival in patients.\u003c/p\u003e \u003cp\u003eOur study further reveals that βOHB upregulates Tcf7 expression in CD8⁺ T cells through receptor-mediated signaling, rather than through its traditional role in HDAC inhibition. Knockdown of Hcar2, a known cell surface receptor for βOHB, abolished this effect, establishing Hcar2 as an essential mediator of βOHB-induced Tcf7 expression. βOHB binding to Hcar2 activates a G-protein coupled receptor signaling cascade, but the specific intracellular signaling pathways downstream of Hcar2 that regulate Tcf7 expression remain to be further elucidated. In colorectal cancers, previous studies indicate that βOHB activates the anti-tumorigenic transcription regulator Hopx via Hcar2 signaling, independently of HDAC inhibition, suggesting that Hcar2 signaling plays an important role in the anti-tumor effects of βOHB (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). These results also underscore the potential of targeting the βOHB\u0026ndash;HCAR2 axis, such as by combining PD-1 blockade with selective HCAR2 agonists, to enhance the efficacy of immune checkpoint therapy and advance immunometabolic interventions. Beyond cancer therapy, these mechanisms may also be relevant to chronic infections and immune-mediated disorders characterized by T cell exhaustion and metabolic stress, providing a framework for restoring immune function through metabolite-driven transcriptional regulation.\u003c/p\u003e \u003cp\u003eIn summary, our work demonstrates that βOHB reinforces antitumor immunity by enhancing CD8⁺ T cell persistence and effector function, which is mediated through activation of the Hcar2\u0026ndash;Tcf7 axis and the downstream Lck\u0026ndash;NFκB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings provide a mechanistic basis for metabolite-driven immunomodulation of CD8⁺ T cell, offering a rationale for integrating ketogenic or ketone-based therapies with current immunotherapeutic strategies to enhance cancer treatment outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eData and materials availability\u003c/h2\u003e \u003cp\u003eAll data is present in the manuscript and supplemental figures. RNA-seq data reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number \u003cb\u003ePRJNA1354648\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declared that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e All the animal experiments were conducted in accordance with the ARRIVE guidelines and approved by the Laboratory Animal Ethics Committee of the Ninth People\u0026rsquo;s Hospital, Shanghai Jiao Tong University School of Medicine (SH9H-2022-A8-1). Human samples were obtained after written informed consent from all participants. All experiments involving human specimens were conducted in accordance with the ethical principles of the Declaration of Helsinki and were approved by the Institutional Review Board (IRB) of Renji Hospital, Shanghai Jiao Tong University School of Medicine (RA-2022-411).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2022YFA1302800), the National Natural Science Foundation of China (32022036), The Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212300 and JWAIZD-2).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.B. performed all ex vivo experiments and analyzed the data; Y.B., H.X., and Y.P. performed the animal experiments; J.T. performed the collection of operative samples; Y.B, Yuj. B, and M.W. prepared the figures and helped with manuscript preparation; J.X., and Y.W. provided valuable advice on the project and the manuscript; J.H. and J.X. designed and supervised the research; J.H., Y.B., and J.X. wrote the manuscript with help from all other authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to the staff of the core facilities at Shanghai Institute of Precision Medicine for their instrument support and technical assistance, as well as to the patients who generously donated tissue samples for this study. We sincerely thank F.X. and X.C. for insightful discussions related to the animal studies, and Q.D., Y.C., and S.L. for constructive suggestions on the flow cytometry experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data is present in the manuscript and supplemental figures. RNA-seq data reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number **PRJNA1354648** .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePuchalska P, Crawford PA (2017) Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics. Cell Metab 25:262\u0026ndash;284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2016.12.022\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2016.12.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuura TR, Puchalska P, Crawford PA, Kelly DP (2023) Ketones and the Heart: Metabolic Principles and Therapeutic Implications. Circ Res 132:882\u0026ndash;898. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/CIRCRESAHA.123.321872\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.123.321872\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeber DD, Aminzadeh-Gohari S, Tulipan J, Catalano L, Feichtinger RG, Kofler B (2020) Ketogenic diet in the treatment of cancer - Where do we stand? Mol Metab 33:102\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molmet.2019.06.026\u003c/span\u003e\u003cspan address=\"10.1016/j.molmet.2019.06.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKesarwani P, Kant S, Zhao Y, Miller CR, Chinnaiyan P (2022) The Influence of the Ketogenic Diet on the Immune Tolerant Microenvironment in Glioblastoma. Cancers (Basel). 14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers14225550\u003c/span\u003e\u003cspan address=\"10.3390/cancers14225550\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHusain Z, Huang Y, Seth P, Sukhatme VP (2013) Tumor-derived lactate modifies antitumor immune response: effect on myeloid-derived suppressor cells and NK cells. J Immunol 191:1486\u0026ndash;1495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.1202702\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1202702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei R, Zhou Y, Li C et al (2022) Ketogenesis Attenuates KLF5-Dependent Production of CXCL12 to Overcome the Immunosuppressive Tumor Microenvironment in Colorectal Cancer. Cancer Res 82:1575\u0026ndash;1588. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-21-2778\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-21-2778\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai X, Bu X, Gao Y et al (2021) Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade. Mol Cell. 81: 2317-31 e6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2021.03.037\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2021.03.037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrere G, Tidjani Alou M, Liu P et al (2021) Ketogenic diet and ketone bodies enhance the anticancer effects of PD-1 blockade. JCI Insight. 6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/jci.insight.145207\u003c/span\u003e\u003cspan address=\"10.1172/jci.insight.145207\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDmitrieva-Posocco O, Wong AC, Lundgren P et al (2022) beta-Hydroxybutyrate suppresses colorectal cancer. Nature 605:160\u0026ndash;165. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-022-04649-6\u003c/span\u003e\u003cspan address=\"10.1038/s41586-022-04649-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang C, Tan H, Wang J et al (2024) beta-hydroxybutyrate restrains colitis-associated tumorigenesis by inhibiting HIF-1alpha-mediated angiogenesis. Cancer Lett 593:216940. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.canlet.2024.216940\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2024.216940\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimazu T, Hirschey MD, Newman J et al (2013) Suppression of oxidative stress by beta-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339:211\u0026ndash;214. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1227166\u003c/span\u003e\u003cspan address=\"10.1126/science.1227166\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhilip M, Schietinger A (2022) CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol 22:209\u0026ndash;223. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41577-021-00574-3\u003c/span\u003e\u003cspan address=\"10.1038/s41577-021-00574-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWherry EJ (2011) T cell exhaustion. Nat Immunol 12:492\u0026ndash;499. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ni.2035\u003c/span\u003e\u003cspan address=\"10.1038/ni.2035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcLane LM, Abdel-Hakeem MS, Wherry EJ (2019) CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol 37:457\u0026ndash;495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-immunol-041015-055318\u003c/span\u003e\u003cspan address=\"10.1146/annurev-immunol-041015-055318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeltra JC, Manne S, Abdel-Hakeem MS et al (2020) Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms. Immunity. 52: 825\u0026thinsp;\u0026ndash;\u0026thinsp;41 e8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2020.04.014\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2020.04.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang JT, Wherry EJ, Goldrath AW (2014) Molecular regulation of effector and memory T cell differentiation. Nat Immunol 15:1104\u0026ndash;1115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ni.3031\u003c/span\u003e\u003cspan address=\"10.1038/ni.3031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIntlekofer AM, Takemoto N, Wherry EJ et al (2005) Effector and memory CD8\u0026thinsp;+\u0026thinsp;T cell fate coupled by T-bet and eomesodermin. Nat Immunol 6:1236\u0026ndash;1244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ni1268\u003c/span\u003e\u003cspan address=\"10.1038/ni1268\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Y, Fu L, Li Y et al (2021) Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8(+) T cell immunity. Cell Metab. 33: 988\u0026ndash;1000 e7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2021.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2021.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKallies A, Xin A, Belz GT, Nutt SL (2009) Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity 31:283\u0026ndash;295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2009.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2009.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui W, Liu Y, Weinstein JS, Craft J, Kaech SM (2011) An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8\u0026thinsp;+\u0026thinsp;T cells. Immunity 35:792\u0026ndash;805. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2011.09.017\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2011.09.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan Q, Hu S, Chen X, Danahy DB, Badovinac VP, Zang C, Xue HH (2021) Ectopic Tcf1 expression instills a stem-like program in exhausted CD8(+) T cells to enhance viral and tumor immunity. Cell Mol Immunol 18:1262\u0026ndash;1277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41423-020-0436-5\u003c/span\u003e\u003cspan address=\"10.1038/s41423-020-0436-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X, Yu S, Zhao DM, Harty JT, Badovinac VP, Xue HH (2010) Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity 33:229\u0026ndash;240. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2010.08.002\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2010.08.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLotscher J, Marti ILAA, Kirchhammer N et al (2022) Magnesium sensing via LFA-1 regulates CD8(+) T cell effector function. Cell. 185: 585\u0026ndash;602 e29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2021.12.039\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2021.12.039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGnanaprakasam JNR, Kushwaha B, Liu L et al (2023) Asparagine restriction enhances CD8(+) T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response. Nat Metab 5:1423\u0026ndash;1439. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s42255-023-00856-1\u003c/span\u003e\u003cspan address=\"10.1038/s42255-023-00856-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGounari F, Khazaie K (2022) TCF-1: a maverick in T cell development and function. Nat Immunol 23:671\u0026ndash;678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41590-022-01194-2\u003c/span\u003e\u003cspan address=\"10.1038/s41590-022-01194-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGermar K, Dose M, Konstantinou T et al (2011) T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc Natl Acad Sci U S A 108:20060\u0026ndash;20065. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1110230108\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1110230108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Shan Q, Xue HH (2022) TCF1 in T cell immunity: a broadened frontier. Nat Rev Immunol 22:147\u0026ndash;157. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41577-021-00563-6\u003c/span\u003e\u003cspan address=\"10.1038/s41577-021-00563-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeber BN, Chi AW, Chavez A, Yashiro-Ohtani Y, Yang Q, Shestova O, Bhandoola A (2011) A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476:63\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature10279\u003c/span\u003e\u003cspan address=\"10.1038/nature10279\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaggart AK, Kero J, Gan X et al (2005) (D)-beta-Hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. J Biol Chem 280:26649\u0026ndash;26652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.C500213200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.C500213200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Pei T, Hu X et al (2024) Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell. Cell Rep Med 5:101718. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.xcrm.2024.101718\u003c/span\u003e\u003cspan address=\"10.1016/j.xcrm.2024.101718\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng S, Wang H, Liu J, Aa J, Zhou F, Wang G (2019) Multi-dimensional roles of ketone bodies in cancer biology: Opportunities for cancer therapy. Pharmacol Res 150:104500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.phrs.2019.104500\u003c/span\u003e\u003cspan address=\"10.1016/j.phrs.2019.104500\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan S, Zhou X, Xie F, Zhou F, Zhang L (2025) Ketogenic diet and cancer: multidimensional exploration and research. Sci China Life Sci 68:1010\u0026ndash;1024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11427-023-2637-2\u003c/span\u003e\u003cspan address=\"10.1007/s11427-023-2637-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirschberger S, Strauss G, Effinger D et al (2021) Very-low-carbohydrate diet enhances human T-cell immunity through immunometabolic reprogramming. EMBO Mol Med 13:e14323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15252/emmm.202114323\u003c/span\u003e\u003cspan address=\"10.15252/emmm.202114323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng W, Ma Y, Su Z et al (2021) Single-cell RNA-sequencing analyses identify heterogeneity of CD8(+) T cell subpopulations and novel therapy targets in melanoma. Mol Ther Oncolytics 20:105\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.omto.2020.12.003\u003c/span\u003e\u003cspan address=\"10.1016/j.omto.2020.12.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSade-Feldman M, Yizhak K, Bjorgaard SL et al (2018) Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell. 175: 998\u0026ndash;1013 e20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2018.10.038\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2018.10.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrishna S, Lowery FJ, Copeland AR et al (2020) Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 370:1328\u0026ndash;1334. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.abb9847\u003c/span\u003e\u003cspan address=\"10.1126/science.abb9847\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"β-hydroxybutyrate, CD8 + T cell, anti-tumor therapy, Tcf7, Lck","lastPublishedDoi":"10.21203/rs.3.rs-8428666/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8428666/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKetogenic diets (KDs) have been reported to influence tumor progression through metabolic and immunological modulation of the tumor microenvironment. β-hydroxybutyrate (βOHB), the predominant ketone body elevated by KD, functions not only as an energy substrate but also as a potent signaling metabolite. Despite its role in modulating the tumor microenvironment, the direct impact of βOHB on the function of CD8⁺ T cell, a key mediator of anti-tumor immunity, remains incompletely understood. Here, we demonstrate that βOHB suppresses tumor growth in multiple mouse tumor models by enhancing the accumulation, survival, and effector function of tumor-infiltrating CD8⁺ T cells. In contrast, acetoacetate does not exert comparable immunomodulatory effects. Mechanistically, βOHB upregulates the Tcf7\u0026ndash;Lck signaling pathway by engaging with the cell surface receptor Hcar2, rather than through its role as an HDAC inhibitor. Knockdown of either Tcf7 or Hcar2 in CD8\u003csup\u003e+\u003c/sup\u003e T cells abolishes the promoting effect of βOHB on CD8\u003csup\u003e+\u003c/sup\u003e T function. Our findings elucidate a metabolite-immune axis that directly regulates the functional state of tumor-infiltrating CD8⁺ T cells and provide experimental evidence linking ketone metabolism to anti-tumor immune regulation.\u003c/p\u003e","manuscriptTitle":"β-hydroxybutyrate potentiates anti-tumor immunity by modulating cytotoxic CD8+ T cell responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 19:25:15","doi":"10.21203/rs.3.rs-8428666/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-23T01:29:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T20:25:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271432585760111708421149806173158400316","date":"2026-01-12T16:21:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254051172396692023230455597980672181963","date":"2026-01-09T16:50:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261608187262527710179611011389953967735","date":"2026-01-09T09:47:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303582167662782441837677961090264816269","date":"2026-01-09T08:21:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32339320739998510497439319924496963147","date":"2026-01-09T04:29:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T16:56:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300738804482563257314103258695586829606","date":"2026-01-08T09:26:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151521739151054691531876712904163424331","date":"2026-01-07T08:52:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198248105740955269083682897444254142903","date":"2026-01-07T08:29:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T02:08:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T11:11:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T11:11:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-12-22T22:54:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ae59360-7347-4b85-a93b-8cb0b3e72fe8","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T14:55:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 19:25:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8428666","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8428666","identity":"rs-8428666","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}