Trans-vaccenic acid reprograms CD8+ T cells and anti-tumour immunity

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6–8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP–PKA–CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules.However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown.Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary transvaccenic acid (TVA) directly promotes effector CD8 + T cell function and anti-tumour immunity in vivo.TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously 1 .Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter 2,3 , but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively 4,5 .Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands [6][7][8] .TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8 + T cell function.These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8 + T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids.TVA thus has translational potential for the treatment of tumours.
Diet-related nutritional availability represents a crucial element in the environmental influences that have shaped human physiology during millions of years of human evolution, which has witnessed substantial dietary changes that are associated with physiological and pathological adaptions in human 9 .However, despite extensive studies on links between diet and human health and disease, little is known about how the circulating diet-derived nutrients affect specific human physiological and pathological processes.The main reason is that mechanistic studies to decipher such links are difficult owing to the vast diversity of foods and the high complexity of diet metabolism at organismal levels.We thus approached this dilemma by assembling a blood nutrient compound library and performed screens to initially identify dietary nutrients that influence anti-tumour immunity.Here, we report that dietary TVA (also known as (11E)-octadec-11-enoic acid), promotes tumour-infiltrating and cytotoxic functions of effector CD8 + T cells, leading to enhanced anti-tumour immunity in vivo.
Our integrated temporal genomics and protein phosphorylation analyses reveal that the cell-surface receptor GPR43 is a target of TVA.GPR43 (also known as free fatty acid receptor 2 (FFAR2)) is a short-chain fatty acid (SCFA)-binding G-protein-coupled receptor (GPCR) that is activated by its agonists-including acetate, propionate and butyratewhich are SCFAs produced during dietary fibre fermentation by resident bacteria in gut microbiota 10 .GPR43 couples with both Gα i and Gα q (ref.11).GPR43 is suggested to be involved in anti-inflammation by mediating functions of its SCFA agonists including propionate 6,7 .
Although it has been reported that SCFAs require GPR41 and GPR43 to establish memory CD8 + T cells with optimized recall responses 8 , GPR41 and GPR43 have also been shown to be dispensable for SCFAs to promote CD8 + T cell anti-tumour activity 12,13 .However, whether

Nutrients that enhance T cell function
There are around 633 circulating 'nutrients' (Supplementary Table 1), including inorganic compounds, organic metabolites, lipids, dietary supplements and proteins.Our blood nutrient library for cell-based screens contains 255 compounds (Supplementary Table 2) that are commercially available.We excluded proteins that have been extensively studied and some supplements that only function at the organismal level, such as fish oils and herbs.The reported physiological range of serum levels and working levels (approximately two times the highest reported serum level) in the screens of individual nutrients are included in Supplementary Table 2.The initial screen (1a) was used to identify nutrients that enhance activation of Jurkat T cells stimulated by CD3 and CD28 antibodies (anti-CD3/CD28) (Fig. 1a, top).Screen 1b was
The results of the two screens are shown in Supplementary Tables 3  and 4. Six overlapping top candidates were further examined in the second screen using mouse primary T cells, and CD4 + or CD8 + T cells (Extended Data Fig. 1a and Supplementary Table 5).TVA was ranked top and was further confirmed to enhance IL-2 production from mouse and human primary T cells (Extended Data Fig. 1b,c) and rescue PD-L1-PD-1-dependent exhaustion of Jurkat T cells induced by co-cultured PD-L1-expressing human cancer cells (Extended Data Fig. 1d), as indicated by reversed inhibition of IL-2 production.

Dietary TVA enhances anti-tumour immunity
We found that TVA treatment enhanced the cytotoxicity of mouse melanoma B16F10 cells mediated by co-cultured mouse pmel-1-specific T cells (Extended Data Fig. 1e), whereas TVA did not alter B16F10 cell proliferation or apoptosis (Extended Data Fig. 1f), suggesting that TVA functions through T cells.In addition, the tumour growth potential of immunogenic B16F10 cells was significantly attenuated in syngeneic mice fed with TVA-enriched diet, compared to mice fed with control diet (Fig. 1b, top and Extended Data Fig. 1g).By contrast, the control group fed with either cis-vaccenic acid (CVA)-enriched diet or control diet did not show differences in tumour growth potential of B16F10 cells (Fig. 1b, bottom).The TVA and CVA diets did not alter body weights of mice (Extended Data Fig. 1h).Similar results were obtained using immunogenic MC38 colon cancer or E0771 breast cancer cells in syngeneic mice, but not in mice inoculated with poorly immunogenic LLC1 lung cancer cells (Extended Data Fig. 1i-k).Furthermore, TVA diet had minimal effects on B16F10 tumour growth in syngeneic immune-deficient nude mice, and in T cell receptor-α knockout mice (Extended Data Fig. 1l,m), or mice whose CD8 + T cells were depleted with CD8α antibodies (Fig. 1c; depletion efficiency shown in Extended Data Fig. 1n).
Together, these data indicate that TVA promotes anti-tumour immunity through regulation of CD8 + T cells.

TVA enhances CD8 + T cell function
We next analysed B16F10 tumours 11-15 days after implantation, when tumours were similar in volume (Extended Data Fig. 2a).Quantitative NMR-based measurement revealed that the mean serum TVA concentration is approximately 386 μM, and the mean TVA concentration in tumour interstitial fluid (TIF) is approximately 48 μM (Extended Data Fig. 2b).Flow cytometry analyses revealed that TVA diet, but not control CVA diet, results in an increased CD8 + T cell population in tumour-infiltrating lymphocytes (TILs).By contrast, the CD4 + T cell population in TILs was not altered (Fig. 1d).TVA diet also markedly increased tumour-infiltrating populations of neutrophils and monocytes, but not dendritic cells or macrophages (Extended Data Fig. 2c).Further, TVA diet did not alter populations of these myeloid cells in spleens or draining lymph nodes (dLNs) (Extended Data Fig. 2d,e).
The TVA diet had no effects on conventional CD4 + T cell or CD4 + FOXP3 + regulatory T (T reg ) cell (Extended Data Fig. 2f,g) populations in spleens, dLNs and tumours from B16F10 tumour-bearing mice.By contrast, TVA diet resulted in a larger percentage of CD8 + T cells as a fraction of the CD45 + leukocytes infiltrated in B16F10 tumours and dLNs but not spleens (Fig. 1e).Dietary TVA reduced exhaustion of CD8 + T cells in tumours and spleens, but not in dLNs, as indicated by decreased expression levels of the exhaustion markers PD-1 (Fig. 1f) and LAG-3 (Extended Data Fig. 2h).Further analysis of tumour-infiltrating CD8 + T cells with additional representative markers revealed that dietary TVA promotes CD8 + T cell function with increased levels of cytokines including IL-2, IFNγ (Extended Data Fig. 2i,j) and TNF (Fig. 1g), the proliferation marker Ki-67, the co-stimulatory receptor ICOS and the cytolytic molecule GZMB (Fig. 1h), as well as the stem-like CD8 + T cell survival marker TCF1 (ref.14) (Fig. 1i) but reduced expression level of the exhaustion marker TOX (Extended Data Fig. 2k).Similar results were obtained using tumour-infiltrating CD8 + T cells isolated from MC38 tumours (Extended Data Fig. 2l).By contrast, the TVA diet had no effects on CD4 + and CD8 + T cell population and function in non-tumour-bearing mice (Extended Data Fig. 2m), suggesting that the effects of dietary TVA on T cells probably depends on the induction of immune responses.Together, these results suggest that dietary TVA promotes accumulation and function of tumour-infiltrating CD8 + T cells.
By contrast, CVA diet did not alter the circulating TVA levels in mice (Extended Data Fig. 3a, left), and CVA diet in vivo or CVA treatment in vitro had minimal effects on CD8 + T cell function (Extended Data Fig. 3a-e).Consistent with these findings, anti-CD3/CD28-stimulated primary CD8 + T cells treated with TVA in vitro increased in cell number, with increased expression of Ki-67, TNF and IFNγ (Extended Data Fig. 4a,b), but decreased apoptosis, increased BCL-2 expression and decreased active caspase-3 levels (Extended Data Fig. 4c).By contrast, TVA-treated primary CD4 + T cells in vitro showed increased IL-2 production but unaltered production of effector molecules including TNF and IFNγ, proliferation and apoptosis (Extended Data Fig. 4d).Collectively, these results suggest that TVA selectively enhances function of stimulated CD8 + T cells.

TVA signals through a GPCR-CREB axis
We next determined whether TVA functions in an extracellular or intracellular manner.Quantitative mass spectrometry detected isotope-labelled [ 13 C]TVA imported from the medium to mouse primary CD8 + T cells in a dose-dependent manner, whereas the CD36 inhibitor sulfosuccinimidyl oleate (SSO) reduced import of [ 13 C]TVA (Extended Data Fig. 4e).However, SSO treatment did not affect TVA-enhanced CD8 + T cell activation (Extended Data Fig. 4f), and withdrawal of TVA from the culture medium abolished TVA-enhanced CD8 + T cell activation, suggesting that the effects of TVA are probably extracellular and reversible (Extended Data Fig. 4g).We then performed an isotope-tracing experiment to examine whether intracellular [ 13 C] TVA is broken down by fatty acid β-oxidation and incorporated in citrate (Extended Data Fig. 4h).When primary mouse CD8 + T cells were cultured with full glucose and treated with [ 13 C]TVA, there was no labelling of [ 13 C]TVA to citrate, whereas 13 C labelling of citrate was detected in cells treated with control [ 13 C]palmitate.Similar results were obtained using cells under a glucose-deprivation condition.We also tested whether intracellular TVA is broken down to participate the TCA cycle (Extended Data Fig. 4i).TVA treatment did not alter the oxygen consumption rate (OCR) of CD8 + T cells in the presence or absence of the CPT1a inhibitor etomoxir, whereas the control palmitic acid treatment resulted in increased OCR that was abolished by etomoxir.Together, these data indicate that TVA primarily functions outside of CD8 + T cells, whereas intracellular TVA is not broken down for CD8 + T cell metabolism.
We investigated the effects of TVA on human or mouse primary CD8 + T cells using integrated, temporal mechanistic studies, including: (1) the kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach 15 to identify the initial (20 min-2 h) effect of TVA on cells by capturing global transcription dynamics; (2) phospho-antibody array to identify early (40 min-24 h) cellular signalling changes; and (3) the RNA-sequencing (RNA-seq) approach (at 24 h), for whole-transcriptome analysis.Functional enrichment of the top-ranking altered gene bodies from the genome-scale KAS-seq results (Extended Data Fig. 5a,b) revealed GPCR activities among the top-enriched ontologies in TVA-treated CD8 + T cells (Fig. 2a and Extended Data Fig. 5c).Consistent with this finding, we observed increased phosphorylation of the transcription factor CREB-a common downstream effector of GPCRs 16 -as early as 40 min after TVA treatment (Fig. 2b and Extended Data Fig. 5d; full phospho-antibody array data in Supplementary Table 6).We confirmed the increased phosphorylated CREB (p-CREB) level in mouse primary CD8 + T cells treated with TVA by flow cytometry analysis (Extended Data Fig. 5e).Phosphorylation levels of STAT1-which is important for efficient expansion of CD8 + T cells 17 -were also increased by TVA.Increased p-CREB and p-STAT1 levels in tumour-infiltrating CD8 + T cells in B16F10 tumours from syngeneic mice fed on TVA were confirmed by flow cytometry analysis (Extended Data Fig. 5f).
In addition, Gene Ontology (GO) enrichment analysis 18,19 and global gene set enrichment analysis 20 (GSEA) identified a set of upregulated signalling pathways that are responsible for the effects of TVA treatment on CD8 + T cells at 24 h, including T cell proliferation and activation, and a set of downregulated signalling pathways, including apoptosis (Extended Data Fig. 5g).Notably, TVA treatment enhanced expression of the genes enriched in effector CD8 + T cell function, E2F (Fig. 2c) and MYC pathways (Extended Data Fig. 5h), correlating with enhanced CD8 + T cell function and proliferation.TVA treatment also upregulated the expression of Creb1 (which encodes CREB) and Prkacb and Prkaca (which encodes the catalytic subunits α and β, respectively, of cAMP-dependent protein kinase A (PKA)) (Fig. 2d), with increased mRNA and protein levels (Extended Data Fig. 5i,j), and Tnf, and downregulated Lag3 and Batf, which are associated with T cell exhaustion (Extended Data Fig. 5i).Collectively, these results suggest that enhanced CD8 + T cell function induced by TVA is mediated through a GPCR-CREB pathway, with positive feedback augmenting expression of PKA and CREB at the gene level.

TVA activates the cAMP-PKA-CREB pathway
We next sought to determine which pathways downstream of GPCRs are required for TVA function (the results are summarized in Extended Data Fig. 6a).Specific inhibitors targeting downstream effectors of GPCRs, including MEK1, NFAT and RHOA (Extended Data Fig. 6b-d) did not alter TVA-dependent CD8 + T cell activation.Effectiveness and selectivities of the inhibitors are shown in Extended Data Fig. 6e.By contrast, treatment with SCH-202676 hydrobromide, a sulphydryl-reactive compound that blocks agonist and antagonist binding to GPCRs, abolished TVA-enhanced activation of CD8 + T cells (Extended Data Fig. 7a).Consistent with this finding, TVA treatment increased levels of cAMP in CD8 + T cells (Extended Data Fig. 7b), and treatment with the PKA inhibitor H-89 abolished TVA-enhanced activation of CD8 + T cells (Extended Data Fig. 7c).

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Downstream PKA effectors include CREB 21 and the lymphocytespecific tyrosine kinase LCK 22 , that are reported to positively and negatively regulate T cell activation, respectively.We found that TVA treatment promotes phosphorylation of CREB but not LCK (Extended Data Fig. 7d).In addition, treatment with the CREB inhibitor 666-15 markedly reduced TVA-enhanced activation of CD8 + T cells (Extended Data Fig. 7e).Next, we found that treatment with 666-15 alone reduced B16F10 cell proliferation and tumour growth (Extended Data Fig. 7f,g), and combined treatment with 666-15 and TVA diet resulted in a significant rescue of tumour growth potential of B16F10 syngeneic mice compared with mice treated with TVA diet alone to a similar level to that of mice treated with 666-15 alone (Extended Data Fig. 7g).These results suggest that CREB inhibition antagonizes the effects of dietary TVA on anti-tumour immunity.
Furthermore, treatment with cell-permeable 8-bromo-cAMP increased the amount of p-CREB in CD8 + T cells in a dose-dependent manner, similar to TVA (Extended Data Fig. 7h).CRISPR-Cas9-mediated knockout of CREB in OT-I cells attenuated TVA-enhanced cell proliferation, Ki-67 level, and TNF and IFNγ production, and attenuated the reduction in apoptosis in response to TVA (Extended Data Fig. 7i).Similarly, activation of mouse primary CD8 + T cells by 8-bromo-cAMP could not be further altered by TVA (Extended Data Fig. 7j).In addition, as shown in Extended Data Fig. 7k, Creb1 knockout in OT-I cells abolished the TVA-enhanced cytotoxic effects of OT-I cells on co-cultured B16-OVA cells.

The effects of TVA require CREB
We performed transcriptome-wide RNA-seq using CD8 + T cells with Creb1-knockdown control cells treated with non-targeting control (siNTC) short inhibitory RNA (siRNA) in the presence and absence of TVA.Principal component analysis revealed that cells treated with siRNA targeting Creb (siCreb1) in the presence and absence of TVA can be grouped together and are separated from the siNTC control cell group, or the group of control cells treated with TVA (Fig. 3a).GSEA analysis revealed that knockdown of CREB reverses TVA-dependent upregulation of gene sets related to effector CD8 + T cell function, and cell proliferation including E2F (Fig. 3b) and MYC pathways (Extended Data Fig. 8a).
To further identify targets downstream of TVA-CREB, we characterized genes that were upregulated or downregulated only in the siNTC plus TVA group compared with three other groups (Fig. 3c, left; full gene list is shown in Supplementary Table 7).In total, 312 upregulated genes were enriched in 11 GO categories, including cell cycle, cell proliferation, cell division, T cell aggregation, T cell activation, T cell differentiation and cytokine production (Fig. 3c, top right).By contrast, a total of 139 downregulated genes were enriched in 8 GO categories, including apoptosis and chromatin organization (Fig. 3c, bottom right).For functional validation, we selected four upregulated genes that are critical in cell proliferation and T cell function.These genes include Il18 (associated with cell proliferation, T cell activation and cytokine production), Ebi3 (associated with cell proliferation and T cell activation), Tbx21(associated with of T cell activation and cytokine production) and Ilf2 (associated with cell proliferation, T cell activation and cytokine production).We also tested Foxo4 and Bcl6, which are critical genes in apoptosis-related GO categories.Each of these six TVA-CREB target genes was knocked down by distinct siRNA probes in mouse CD8 + T cells (knockdown efficiency shown in Extended Data Fig. 8b), and in functional studies upon TVA treatment.
As shown in Fig. 3d and Extended Data Fig. 8c, knockdown of Creb1 expression by siCreb1 effectively reverses TVA-dependent changes in cell number and apoptosis, and levels of Ki-67, IL-2, TNF and IFNγ in CD8 + T cells, compared with control cells treated with siNTC.Knockdown of Il18, Ebi3, Tbx21, Ilf2, Foxo4 or Bcl6 partially reversed the TVA-dependent change in cell number (Fig. 3d, left).Knockdown of Bcl6, Foxo4 or Ebi3 partially reversed the TVA-dependent change in the apoptotic population of CD8 + T cells, whereas knockdown of Il18, Tbx21 or Ilf2 had no such effect (Fig. 3d, middle).Knockdown of Il18, Tbx21, Ilf2 or Ebi3 partially reversed TVA-dependent change of Ki-67 level, whereas knockdown of Bcl6 or Foxo4 did not have this effect (Fig. 3d, right).Knockdown of Foxo4, Tbx21, Ilf2 or Ebi3 partially reversed the TVA-dependent change in IL-2 level, whereas knockdown of Bcl6 or Il18 did not (Extended Data Fig. 8c, left).Knockdown of each of the six genes partially reversed the TVA-dependent change in TNF level (Extended  Data Fig. 8c, middle).Finally, knockdown of Bcl6, Il18, Foxo4, Tbx21 or Ebi3 partially reversed the TVA-dependent change in IFNγ level, whereas knockdown of Ilf2 had no effect (Extended Data Fig. 8c, right).Together, these results establish differentially functional contributions of diverse CREB target genes to distinct TVA-dependent changes in CD8 + T cells.

TVA inactivates SCFA-binding GPR43
To identify the GPCR target of TVA, we screened the six known fatty acid-binding GPCRs, including GPR40, GPR41, GPR43, GPR84, GPR119  and GPR120 (ref.23) by siRNA-mediated knockdown (Extended Data Fig. 8d).Of these, only knockdown of GPR43 abolished TVA-enhanced TNF levels in primary mouse CD8 + T cells (Fig. 4a).Further analysis confirmed that knockdown of GPR43 in primary mouse CD8 + T cells leads to increased cAMP, p-CREB and p-STAT1 levels (Extended Data Fig. 8e) as well as increased TNF and IFNγ (Extended Data Fig. 8f) in mouse primary CD8 + T cells that cannot be further activated by TVA treatment.Similar results were obtained using OT-I T cells with siRNA-mediated GPR43 knockdown (Extended Data Fig. 8g), and OT-I T cells with knockout of Gpr43 by CRISPR-Cas9 (Fig. 4b and Extended Data Fig. 8h).These data suggest that GPR43 has a suppressive role in CD8 + T cell activation, and that TVA is likely to attenuate GPR43 function.These results are also consistent with previous reports linking activation of GPR43 with Gα i coupling and decreased cAMP levels 7,10 .Consistent with these findings, stimulation with anti-CD3/CD28 resulted in a much larger increase in GPR43 mRNA and protein expression levels in CD8 + T cells than that in CD4 + T cells (Extended Data Fig. 8i).This may partly explain why activated CD8 + T cells are sensitive to TVA treatment.
We next performed structure-activity relationship (SAR) studies on TVA.We designed 15 TVA derivatives (Extended Data Fig. 9a; synthetic procedures and NMR results of TVA derivatives and probes are presented in Supplementary Information) with changes in chain length, double bond position or number, tail group or terminal acid group, and compared them with TVA for bioactivity to enhance CD8 + T cell activation (Extended Data Fig. 9b).The results of the SAR studies revealed that the double bond (1), acid group ( 2) and a length of at least 16 carbons (3, 4, 13, 15 and 16) were crucial for maintaining TVA bioactivity.In addition, shifting the position of the double bond from C11-C12 to C9-C10 ( 6), or adding a C9-C10 double bond (7) resulted in enhanced TVA bioactivity, suggesting that a C9-C10 double bond may be optimal for TVA bioactivity.The SAR results for 9, 10, 14 and 17 also suggest that the terminal chain can be modified, and the resulting TVA derivatives retained 70-80% bioactivity.We thus designed photo-affinity probes using TVA as a parental molecule to bear a photo-reactive diazirine group and a tail alkynyl group (Extended Data Fig. 9c).All three TVA probes enhanced CD8 + T cell activity in a dose-dependent manner (Extended Data Fig. 9d).We next performed a photo-affinity labelling study 24 using mouse CD8 + T cells treated with TVA probe 3.The diazirine enables covalent photo-crosslinking to contacting protein residues and the alkynyl group enables linking the reporter (biotin) and probing TVA-binding protein target(s).Western blotting using biotin-labelled proteins pulled down by streptavidin beads demonstrated binding between the TVA probe and GPR43 (Fig. 4c).
We next found that GPR43 SCFA agonists (at 20 mM) significantly decreased mouse CD8 + T cell activation, indicated by decreased TNF expression, whereas TVA at a 1,000× lower level (20 μM) was able to reverse SCFA-dependent suppression on CD8 + T cells (Extended Data Fig. 10a).In addition, increasing concentrations of SCFA acetate up to 20 mM attenuated CD8 + T cell activation, as assessed by reduced TNF (Fig. 4d) and IFNγ (Extended Data Fig. 10b) levels; this readout was effectively reversed by 20 μM TVA.By contrast, 2 μM TVA was sufficient to reverse the suppression of CD8 + T cells by acetate at 20 mM (Fig. 4e).Consistent with these findings, suppression of CD8 + T cells with reduced TNF (Extended Data Fig. 10c, left) and IFNγ (Extended Data Fig. 10d) levels by pre-treatment with 20 mM acetate could be reversed by 20 μM TVA.By contrast, enhanced CD8 + T cell activation by pre-treatment with 20 μM TVA could not be reversed with 20 mM acetate.Similar results were obtained using other SCFA agonists, including propionate and butyrate (Extended Data Fig. 10c, middle and right).These results suggest that TVA may bind to and inactivate GPR43 by antagonizing its SCFA agonists.

TVA antagonizes effects of SCFAs on cAMP
A TVA-enriched diet did not significantly alter the diversity and patterns of gut microbial distribution in mice (Extended Data Fig. 10h,i), or serum or TIF levels of gut microbiota-derived SCFA acetate (Extended Data Fig. 10j).The concentrations of acetate, propionate and butyrate in culture medium are in the range of 100-300 μM (Extended Data Fig. 11a), suggesting that SCFAs in the culture medium activate GPR43 in CD8 + T cells and predispose them to TVA-mediated inactivation.We thus compared the opposing effects of TVA and SCFAs on cAMP signalling in CD8 + T cells.We first examined whether SCFAs inhibit CD8 + T cell activity through GPR41 and GPR43, as well as the pH sensor GPR65 (ref.25), since unlike long-chain fatty acids such as TVA, SCFAs are soluble in water and reduce the luminal pH.GPR65 is activated by low pH and signals through Gα s , increasing cAMP production and consequent CREB phosphorylation and activation 25 .Indeed, SCFAs in the culture medium reduced the pH in mouse primary CD8 + T cells (Extended Data Fig. 11b), accompanied by reduced cAMP, TNF and IFNγ expression (Extended Data Fig. 11c-e), whereas individual or combined knockdown of GPR41 or GPR43 (knockdown efficiency shown in Extended Data Fig. 11f) led to increased CD8 + T cell activity.This increase in CD8 + T cell activity could be partially decreased with additional SCFA treatment.By contrast, knockdown of GPR65 led to decreased CD8 + T cell activity, with additional SCFA treatment resulting in further reduction, whereas triple knockdown of GPR41, GPR43 and GPR65 completely abolished the SCFA-mediated effects on CD8 + T cells.Finally, SCFAs increased the intracellular Ca 2+ level in CD8 + T cells, probably through Gα q (Extended Data Fig. 11g,h).By contrast, TVA exhibited minimal effects in both assays.Together, SCFAs inhibit overall CD8 + T cell activity, consistent with our results showing that the negative regulation of cAMP mediated by GPR41 and GPR43 antagonizes the positive effects of GPR65 on cAMP levels (Extended Data Fig. 11i).
By contrast, we found that TVA diet does not alter the pH in serum and TIF samples from mice fed on control or TVA diets (Extended Data Fig. 11j), and no significant difference of pH was detected in human primary serum samples from a group of lymphoma patients with different serum levels of TVA (Extended Data Fig. 11k; detailed description in Fig. 5d).We also found that mixing TVA or control diets with water results in comparable pH values, and that TVA up to 2,000 μM does not significantly alter the pH values in culture medium (Extended Data Fig. 11l, m).Moreover, knockdown or CRISPR-Cas9-mediated knockout of Gpr65 in primary mouse CD8 + T cells in vitro leads to decreased TNF, IFNγ and p-CREB levels but had no effects on TVA-enhanced CD8 + T cell function (Extended Data Fig. 12a,b), nor does changing pH of culture medium (Extended Data Fig. 12c).Lastly, in an ACT mouse model using Cas9-expressing OT-I T cells, CRISPR-Cas9-mediated Gpr65 knockout resulted in decreased tumour growth, which, however, was further reduced by TVA diet (Extended Data Fig. 12d; note that top 4 control groups are from the same experiment described in Fig. 4f).These results demonstrate that TVA does not lower pH or signal through GPR65 to enhance CD8 + T cell function in vitro and in vivo, supporting

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our hypothesis that TVA inactivates GPR43 and antagonizes the overall negative effects of SCFAs on cAMP (Extended Data Fig. 12e).

TVA augments T cell-based therapies
TVA treatment significantly reversed exhaustion of primary human bulk T cells and CD8 + T cells treated with purified recombinant PD-L1 (Extended Data Fig. 12f-i).Similar results were obtained using mouse primary T cells co-cultured with B16F10 cells overexpressing PD-L1 (Extended Data Fig. 12j-m).Moreover, dietary TVA combined with anti-PD-1 antibody, representative of immune checkpoint inhibitor therapy 26 , showed synergistic attenuation of B16F10 tumour growth (Fig. 5a).We next tested the effects of TVA on efficacy of blinatumomab, a bispecific T cell engager that targets CD19 on B cells and CD3 on T cells 27 .TVA significantly enhanced the in vitro killing efficiency of human peripheral blood mononuclear cells (PBMCs) on human B-ALL RS4;11 cells in the presence of blinatumomab in a dose-dependent manner (Fig. 5b).Furthermore, TVA increased in vitro expansion of chimeric antigen receptor (CAR) T cells derived from primary T cells from 3 patients with lymphoma aged between 42 and 47 years (Fig. 5c and Extended Data Fig. 12n, left).By contrast, cells from an older patient (age 77 years) did not respond to TVA treatment (Extended Data Fig. 12n, right), which might be owing to a lower starting cell number compared with the younger patients.Of note, in a retrospective clinical study, we found that serum TVA levels were higher in a group of patients with lymphoma that responded to CAR-T cell therapy than in non-responders (Fig. 5d; detailed timepoint information can be found in Supplementary Table 8).These findings align with the notion that dietary TVA may improve clinical responsiveness to T cell-based immunotherapies.

Discussion
Our findings reveal a mechanism during human dietary evolution by which extraorganismal TVA reprograms CD8 + T cells through extrinsic regulation to inactivate GPR43, in contrast to gut microbiota-derived, intraorganismal SCFAs as GPR43 agonists (Extended Data Fig. 12o).Our study, by comprehensive evaluation of diet-derived nutrients, advances the identification and understanding of mechanistic links between diet and human physiology and pathology.Thus, despite the vast diversity of food and diet origins, studies that focus on individual nutrients can be used to identify the molecular mechanisms underlying nutritional influences on human health and disease, as well as effectiveness of therapies.The approach used in our study could have broad implications for future elucidation of previously unknown physiological and pathological roles of circulating nutrients in human health and disease.
As a natural food component, TVA has high translational potential as a dietary element in therapeutic approaches to improve clinical outcomes of diverse anti-cancer therapies such as immune checkpoint inhibitors, T cell engagers and CAR-T and T cell receptor T cell therapy.Epidemiological studies suggest that the circulating levels of TVA in humans are associated with lower adiposity, diabetes risk and systemic inflammation, but the effects of dietary TVA on the risk of cancer and cardiovascular diseases are unclear, although in mouse models of dyslipidaemia, a TVA-enriched diet exerts hypolipidemic effects by lowering circulating triglyceride levels [28][29][30] .Nevertheless, a comprehensive understanding of the interactive and collective influences of diverse dietary nutrients on cancer risk, development and therapy responses is crucial for diet choices.For example, consuming red meat may provide TVA for improved anti-tumour immunity, but a high intake of red meat has been positively associated with risk of many cancers, including breast, colorectal, colon and rectal cancer 31 .Thus, our studies support TVA supplementation as a more targeted and efficient way than dietary changes to benefit anti-tumour immunity.
The GPR43-CREB mechanism may be cell type-specific for CD8 + T cells.For example, although TVA treatment increased IL-2 production by CD4 + T cells, TVA did not alter the production of effector molecules including TNF and IFNγ, or proliferation/apoptosis of CD4 + T cells.Thus, the overall effects of TVA on CD4 + T cells are modest compared to those on CD8 + T cells, which may be related to the low expression of GPR43 in CD4 + T cells.Future studies are warranted to determine how GPR43 switches its downstream effector pathways in different immune cells, whether TVA regulates functions of other immune cells with high levels of GPR43 expression such as monocytes, neutrophils and marginal zone B cells 7,11,32,33 , and whether TVA similarly inactivates GPR43 for activation of the cAMP-PKA-CREB axis in these cells.Finally, since TVA is bulky compared with SCFAs, it is unlikely to compete with them for their binding pocket on GPR43.It may bind to a different site and function as a negative allosteric modulator.Further studies are warranted to elucidate the underlying structural and molecular mechanisms by which TVA inactivates GPR43.

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Cell lines
All cell lines were authenticated by genomic short tandem repeat (STR) profiling at the University of Chicago Integrated Genomics Core (EIGC) upon purchase and at least annually as appropriate.Human T lymphocyte cell line Jurkat T was purchased from American Type Culture Collection (ATCC).Human Plat-E cells and mouse melanoma cancer cell line B16-OVA were provided by H.C. Human RS4;11 cells were provided by the laboratory of W.S. Mouse melanoma cancer cell line B16F10, breast cancer cell line E0771, and Lewis lung carcinoma cell line LLC1 were purchased from ATCC.Mouse colorectal adenocarcinoma cell line MC38 was purchased from Kerafast.Plat-E, B16-OVA, B16F10, E0771, and LLC1 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (Sigma, F2442) and penicillin/streptomycin. Jurkat T cells were cultured in RPMI-1640 medium with 10% FBS and penicillin/ streptomycin.All cells were cultured at 37 °C and 5% CO 2 .Cell line experiments were conducted and designed according to protocols approved by Institutional Biosafety Committee (IBC) of the University of Chicago.

Primary cells
Cas9-expressing OT-I cells were isolated from the spleen and peripheral lymph nodes (provided by the laboratory of H.C.) of Cas9;OT-I mice 34 by magnetic bead purification using EasySep Mouse naive CD8 + T Cell Isolation Kit according to the manufacturer's instructions (Stem Cell Technologies).Cas9;OT-I cells were activated in vitro for 18 h with plate-bound anti-CD3 (10 μg ml −1 ; Biolegend) and anti-CD28 (5 μg ml −1 ; Biolegend) antibodies in Click's medium at 37 °C and 5% CO 2 incubator for further experiments.

Human samples
Serum from patients who had undergone commercial CAR-T cell therapy were from the University of Chicago cell therapy biobank.Supplementary Table 8 contains relevant patient information.

Construction of PD-1 + Jurkat T cell line
Jurkat T cells were infected with pre-made lentivirus expressing human PD-1 (Gen Target, LVP1076-PBS) according to the manufacturer's instructions.After infection for 24 h, cells were selected with 2 μg ml −1 puromycin to obtain PD-1 + Jurkat T cells.PD-1 expression level was checked using western blot.

Circulating nutrient library screens
To construct circulating nutrient compound library for cell-based screening purposes, components such as antibodies that are difficult to distinguish due to a wide variety, and some supplements that only function at a whole organism level including fish oils and herbs were excluded.Physiological ranges of serum levels of individual nutrients are available in the human metabolome database (https://hmdb.ca/) and applied in the experimental design.For the initial screen (1a), Jurkat T cells were treated with nutrient library for 48 h and then activated with 2.5 μg ml −1 anti-CD3 and 0.5 μg ml −1 anti-CD28 antibodies for another 12 h, followed by measurement of IL-2 level in medium supernatant using an ELISA kit (Biolegend).For screen 1b, 1 × 10 5 PD-1 + Jurkat T cells were co-cultured with 2 × 10 4 H596 (PD-L1 + ) cells in a well of 96-well plate for 60 h, and then activated with 2.5 μg ml −1 anti-CD3 and 0.5 μg ml −1 anti-CD28 antibodies for another 12 h, followed by measurement of IL-2 level in medium supernatant using an ELISA kit.Please also refer to Supplementary Tables 1-4 for more details.

Mouse tumour models
For C57BL/6 mice and the TCRα-knockout mice tumour model, mice were anaesthetized with isoflurane, shaved at the injection site, and then injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10, MC38 or LLC1 cells, or in the mammary gland with 2 × 10 5 E0771 cells.C57BL/6 nude mice were injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10 cells.The tumour-bearing mice were assigned to TVA-enriched diet (1% TVA, special order from Research Diets), CVA-enriched diet (1% CVA, special order from Research Diets) or control diet (Research Diets) as of the day of tumour inoculation, with mice body weight monitored.Tumours were measured using a calliper every 2-3 days.Tumour volumes were calculated using the following formula: length × width × [(length × width) × 0.5] × π/6.Mice were euthanized at humane endpoints or day 11-15 for tissue collection.
For the 666-15 treatment mouse model, 6-8-week-old C57BL/6 mice were anaesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10 cells for tumour development.The tumour-bearing mice were assigned to TVA-enriched diet or control diet as of the day of tumour inoculation.When the tumour volume reached approximately 100 mm 3 , the mice were treated with either vehicle or 666-15 at 20 mg kg −1 .The 666-15 was dissolved in 1% N-methylpyrrolidone, 5% Tween-80 in H 2 O.The mice were treated once a day for 5 consecutive days per week for 2 weeks.Tumours were measured using a calliper every 2-3 days.Tumour volumes were calculated using the following formula: length × width × [(length × width) × 0.5] × π/6.Mice were euthanized at humane endpoints.
For the anti-PD-1 treatment mouse model, 6-8-week-old C57BL/6 mice were anaesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10 cells for tumour development.The tumour-bearing mice were assigned to TVA-enriched diet or control diet as of the day of tumour inoculation.On day 3, 6, 9, 12 and 15, 250 μg anti-PD-1 (BioXCell) or IgG control (BioXCell) was injected intraperitoneally.Tumours were measured the tumours using a calliper every 2-3 days.Tumour volumes were calculated using the following formula: length × width × [(length × width)0.5]× π/6.Mice were euthanized at humane endpoints.
For the Gpr43 and Creb1-knockout Cas9;OT-I cells adoptive transfer mouse model, 4 × 10 5 B16-OVA cells were injected subcutaneously in the abdominal flank of 6-8-week-old C57BL/6 nude mice on day 0, and tumour were allowed to grow for 6 days.Target genes were knocked out in Cas9;OT-I cells, 5 × 10 6 cells were transferred to tumour-bearing C57BL/6 nude mice on day 6 by tail vein injection.The tumour-bearing mice were assigned to TVA-enriched diet or control diet on day 6, tumours were measured using a calliper every 2-3 days.
For the Gpr43 −/− mouse tumour model, 6-8-week-old Gpr43 −/− or littermate control mice were anaesthetized with isoflurane, shaved at the injection site, and then injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10 cells for tumour development.The tumour-bearing mice were assigned to TVA-enriched diet or control diet as of the day of tumour inoculation, tumours were measured using a calliper every 2-3 days.Tumour volumes were calculated using the following formula: length × width × [(length × width) × 0.5] × π/6.Mice were euthanized at humane endpoints.
For the Gpr43 −/fl Cd8a cre mouse tumour model, Gpr43 −/− , Gpr43 fl/fl and Cd8a cre mice were used for breeding to get Gpr43 Cd8 conditionalknockout mice (Gpr43 −/fl Cd8a cre ).Six-to eight-week-old Gpr43 −/fl Cd8 cre or littermate control mice were anaesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 1 × 10 5 B16F10 cells for tumour development.The tumour-bearing mice were assigned to TVA-enriched diet or control diet as of the day of tumour inoculation, tumours were measured using a calliper every 2-3 days.Tumour volumes were calculated using the following formula: length × width × [(length × width) × 0.5] × π/6.Mice were euthanized at humane endpoints.

Diet formula
Detailed information on the full compositions of the control, TVA-and CVA-enriched diets is summarized in Supplementary Table 9.

Secreted cytokines level
Human or mouse T cell secreted IL-2, TNF and IFNγ were detected by ELISA MAX Standard Set (Biolegend) per the manufacturer's instructions.

Pmel-1 killing assay
Pmel-1 cells were isolated from a Pmel mouse and seeded at density of 1 × 10 6 cells/well in 6-well plate (pre-coated with anti-CD3 and anti-CD28) for 18 h.One million activated Pmel-1 cells were then co-cultured with 2 × 10 5 B16F10 cells for 24 h with or without 20 μM TVA.All cells in suspension were collected, stained with anti-mouse CD45 and PI, and analysed by flow cytometry.

Cell proliferation assay
Cell proliferation assays were conducted by seeding 5 × 10 4 cells in a 6-well plate.Cell numbers were recorded daily within 5 days using TC20 Automated Cell Counter (Bio-Rad).

Extraction and quantification of TVA levels by NMR
TIF and serum from tumour-bearing animals were isolated as described 35 .In brief, tumours were dissected away from the euthanized animal, rinsed in a saline containing Petri dish, and then placed on top of a 20-μm mesh nylon filter (Spectrum Labs, 148134) that was affixed to the conical tube (Falcon, 1495949 A).After the conical tube lid was placed on top without screwing and taped using laboratory tape in place, tumour-containing conical tube was subjected to centrifugation at 4 °C for 10 min at 106g using a refrigerated clinical centrifuge.In total, 10-50 μl of fluid was obtained in the bottom of the conical tube.To prepare the mouse serum, once the tumours were dissected, blood was isolated from the mouse heart by cardiac puncture using a 1-ml 25 G TB syringe, allowed to clot by leaving it undisturbed at room temperature, and then centrifuged at 1,500g for 10 min in a refrigerated centrifuge.The resulting supernatant was designated serum.Human serum samples were obtained from the University of Chicago cell therapy biobank.The TIL and serum samples are lyophilized and subjected to quantification of TVA levels by 1 H-NMR spectroscopy using a Bruker Ascend TM 600 spectrometer equipped with CryoProbeProdigy.In a typical procedure, 350 μl deuterated methanol (methanol-d4) with 0.03% tetramethylsilane (Oakwood Chemical) was added to a lyophilized sample to dissolve TVA.After being vortexed, the sample was then centrifuged at 10,000g for 5 min to collect the supernatant. 1H-NMR spectrum of the supernatant (250 μl) in a 335-pp Precision 3 mm NMR tube (Wilmad-Lab Glass) was acquired with delay time (d1) set to 2 s for 3,072 scans.TVA concentration was calculated based on the integral of peak at 1.97 ppm and the tetramethylsilane internal standard.

CD45 + tumour-infiltrating leukocyte isolation
Tumour tissue were dissected from euthanized tumour-bearing mice, minced into small pieces ( ≤ 2 mm) using a scalpel in a dish, and then transferred to a 14-ml round-bottom tube containing 5 ml tumour digestion medium (500 μl collagenase/hyaluronidase solution, 750 μl 1 mg ml −1 DNase I solution, and 3.75 ml RPMI-1640 medium).After incubation at 37 °C for 25 min on a shaking platform, the digested tumour tissues were transferred into a 70-μm mesh nylon strainer on a 50 ml conical tube, pushed through the strainer using the rubber end of a syringe plunger, and rinsed with the recommended medium.After centrifugation at 300g for 10 min at room temperature with the brake on low, the resulting cell pellets were added 10 ml of ammonium chloride solution for incubation at room temperature for 5 min, followed by centrifugation at 300g for 10 min at room temperature with the brake on low.The resulting cell pellets were re-suspended at 1-10 × 10 6 cells per ml in PBS and then subjected to CD45 + tumour-infiltrating leukocytes isolation by magnetic bead purification using EasySep Mouse TIL (CD45) Positive Selection Kit according to the manufacturer's instructions (Stemcell Technologies).

Mouse TIL isolation
Freshly excised mouse tumour tissues were minced into small pieces (volume smaller than 1 mm 3 ) by scissors in PBS, digested by Collagenase IV (1 mg ml −1 ) and DNase I (200 U ml −1 ) at 37 °C for 20 min in PBS with gentle rocking.The digested tumour tissues were added 5 times volume 0.01 M EDTA, filtered into a new tube through 200 mesh screen (100 μm strainer), and then centrifuged at 300g for 5 min at room temperature.The resulting cell pellets were re-suspended with 3 ml PBS, laid gently on 3 ml lymphocytes isolation liquid (pre-warmed to room temperature), and subjected to density gradient centrifugation (300g, 30 min, room temperature, accelerate 6, decelerate 2).The middle layer of cells was moved carefully to a new tube, added PBS to 15 ml, and centrifuged (300g, 10 min, room temperature).The cell pellets (lyse red cells if necessary) were designated TILs and used for the following experiments.

Mouse spleen lymphocyte isolation
Mouse spleens were disrupted with syringe plunger in 1 ml PBS in a 40-μm strainer filtered to a 15-ml tube, washed with PBS and centrifuged at 300g for 5 min.The resulting cell pellets were re-suspended the with 2 ml red cell lysis buffer (Invitrogen), incubated at room temperature for 10 min, and centrifuged at 300g for 5 min after adding 13 ml PBS.The resulting cell pellets were designated splenocytes and used for following experiments.

Article
Mouse dLNs lymphocyte isolation dLNs were disrupted with syringe plunger in 1 ml PBS in a 40 μm strainer filtered to a 15-ml tube, washed with PBS, and centrifuged at 300g for 5 min.The resulting cell pellets were designated LN lymphocytes and used for following experiments.

Primary CD8 + or CD4 + T cell isolation and activation
Mouse primary CD8 + or CD4 + T cells were isolated from the spleen and peripheral lymph nodes of C57BL/6 mice by magnetic bead purification using EasySep Mouse CD8 + or CD4 + T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies).Human primary CD8 + T cells were isolated from PBMC (Zen-Bio) by human CD8 + T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies).Isolated primary CD8 + or CD4 + T cells were activated in vitro for 18 h with plate-bound anti-CD3 (10 μg ml −1 ; Biolegend) and anti-CD28 (5 μg ml −1 ; Biolegend) antibodies in Click's medium at 37 °C and 5% CO 2 incubator.Activated CD8 + or CD4 + T cells were ready for further experiments.A naive CD8 + T cells control was maintained in Click's medium containing 10 ng ml −1 IL-7 (BioLegend).

Flow cytometry
Mouse primary cells isolated from tumour, spleen and dLNs were stained with fluorescent antibodies and analysed by flow cytometry.
For experiments with live/dead criteria, cells were first stained with Fixable Viability Dyes (FVD) (Thermo Fisher Scientific) according to the manufacturer's instructions.Subsequent surface marker staining was performed in Flow Cytometry Staining Buffer (Thermo Fisher Scientific).Intracellular staining for flow panels containing nuclear proteins was performed using the eBioscience FoxP3/ Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions.For intracellular staining of cytoplasmic proteins, the Fixation/Permeabilization Solution Kit (BD Biosciences) was used according to the manufacturer's instructions.
For phospho-antibody staining, cells were incubated with FVD (cell viability dye) for 15 min at room temperature in a tube, re-suspended with 200 μl pre-warmed 1× BD Phosflow Lyse/Fix buffer directly into the tube, and incubated at 37 °C for 10-15 min, followed by centrifugation at 300g for 5 min.The resulting cell pellets were washed once with FACS buffer, permeabilized with 200 μl of BD Phosflow Perm Buffer III for 45 min on ice, and centrifuged at 300g for 5 min.The cell pellets were washed again with FACS buffer, centrifuged at 300g for 5 min, and incubated with antibodies in FACS buffer for 45 min-1hour at room temperature.

Microbiome 16S sequencing
Gut faeces of control and TVA group (7 samples per group) B16F10 tumour-bearing mice were collected at day 16, and then subjected to microbiome 16S sequencing by Zymo Research.In brief, The ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research) was used to extract DNA using an automated platform.Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S NGS Library Prep Kit (Zymo Research).The bacterial 16S primers amplified the V3-V4 region of the 16S rRNA gene.The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation.The final PCR products were quantified with quantitative PCR fluorescence readings and pooled together based on equal molarity.The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator (Zymo Research), then quantified with TapeStation (Agilent Technologies) and Qubit (Thermo Fisher Scientific).The ZymoBIOMICS Microbial Community Standard (Zymo Research) was used as a positive control for each DNA extraction, if performed.The final library was sequenced on Illumina MiSeq with a v3 reagent kit (600 cycles).The sequencing was performed with 10% PhiX spike-in.For Bioinformatics Analysis, unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline 36 .Potential sequencing errors and chimeric sequences were also removed with the Dada2 pipeline.Chimeric sequences were also removed with the DADA2 pipeline.Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16S database that is internally designed and curated, as reference.Composition visualization, alpha-diversity, and betadiversity analyses were performed with Qiime v. 1.9.1 (ref.37).
[ 13 C 1 ]TVA metabolic flux analysis by gas chromatography-mass spectrometry One million activated mouse primary CD8 + T cells were cultured for 24 h in RPMI-1640 medium containing 0, 20, 50 μM [ 13 C 1 ]TVA (Sigma), rinsed with 0.9% saline solution, and lysed with lysis buffer (400 μl of cold methanol, 300 μl of 0.88% (w/v) KCl).Lysed cells were scraped off the plate into a glass vial, 800 μl of cold dichloromethane was added and vortexed for 15 min at 4 °C, followed by centrifugation at maximum speed for 10 min at 4 °C.Samples were kept at room temperature after centrifugation to form two distinct phases.In total, 650 μl of the bottom dichloromethane fraction was transferred into a new glass tube and dried with nitrogen gas to obtain a lipid fraction pellet.FAME Derivatization was performed as previously described 38 .In brief, the dried lipid pellet was dissolved in 100 μl of toluene, added 200 μl of 2% sulfuric acid in methanol for incubation at 50 °C overnight and then added 500 μl of 5% NaCl to extract twice with 500 μl hexane.FAME cleanup was applied if analysing animal tissues (FAME Cleanup: a Florisil column was pre-conditioned with 3 ml of hexane, added methyl ester, and eluted twice with 1 ml isohexane-diethyl ether (95:5 v/v), with drying down in between elutions).Otherwise, samples were dried down under nitrogen and re-suspended in 50 μl of hexane to load onto gas chromatography-mass spectrometry (GC-MS).Derivatized samples were injected into a GC-MS using DB-FastFAME column (Agilent Technologies, G3903-63011) installed in an Agilent GC system.TVA-FAME has retention time of 9.6 min and m/z of 264 and 292, 13C1-TVA-FAME has retention time of 9.6 min and m/z 265.Metabolite levels and mass isotopomer distributions of derivatized fragments were analysed with an in-house MATLAB script, which integrated the metabolite fragment ions and corrected for natural isotope abundances.

KAS-seq and data analysis
Mouse and human CD8 + T cells were isolated, activated, and treated with or without 20 μM TVA for 20 min, 40 min, and 2 h.500 mM N3-kethoxal was then supplemented into the medium followed by brief, vigorous shaking to fully homogenize the solution.The 6-well plates were then moved into the cell incubator (37 °C, 5% CO 2 ) for 10 min to promote labelling of genomic ssDNA.Cell suspensions were then transferred to 15 ml conical tubes and centrifuged at 300g for 5 min at 4 °C.The supernatant medium was discarded, and genomic DNA was then extracted.The ssDNA with N3-kethoxal label was biotinylated, enriched, and fragmented following the established KAS-seq protocol 15,45 .Dual index libraries were constructed for high throughput sequencing using an Accel-NGS Methyl-seq DNA library kit and then sequenced at the Genomics Facility (University of Chicago) via Illumina NovaSeq 6000 (SP flowcell, 100 bp cassette) in two separate runs.Raw sequencing data under each condition was then catenated from the two runs, and adapter trimming; read deduplication and mapping; and differential KAS-seq analysis (comparing TVA-treated to untreated cells) was performed following the KAS-pipe analysis pipeline 15,45 .Volcano plots were subsequently generated in RStudio.For GO enrichment analysis, a list of gene bodies exhibiting differential ssDNA levels following TVA treatment was generated for each timepoint.M. musculus and H. sapiens CD8 + T cell KAS-seq data were assessed separately.The list of differentially expressed gene bodies was then submitted for GO enrichment and visualization, which was performed via the Gene Ontology project 18,19 and REVIGO 46 and Cytoscape 47 , respectively.

Phospho-antibody array
To analyse signaling pathways, mouse primary CD8 + T cells were isolated, activated, and treated with TVA for indicated time, followed by Phospho Antibody Array (R&D Systems) according to the manufacturer's instructions.The quantification for pixel density in each spot of the array was carried out by subtracting background signals from the spot intensity using software ImageJ (ImageJ, RRID: SCR_003070).

Quantitative real-time PCR
Total RNA was extracted with TRIzol Reagent (Invitrogen) and then used for synthesizing the first strand cDNA with PrimeScript 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's instructions.Quantitative RT-PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad).

RNA-mediated interference with Accell siRNA
Mouse primary CD8 + T cells were isolated and cultured in replete medium (RPMI-1640 medium or Click's medium containing 15% FBS, 55 μM 2-mercaptoethanol, 2 mM glutamine, penicillin/streptomycin, and either PHA, CD3 or IL-2) for 24 h, followed by incubation with Accell delivery mix (Accell siRNA Delivery Media (Horizon Discovery) with Article 1 μM siRNA, 20 IU ml −1 IL-2 and 1% FBS) for 72 h.Cells were collected for subsequent function analysis as well as depletion efficiency validation using RT-PCR.

RNA sequencing
To check the effect of TVA treatment on gene expression of mouse CD8 + T cells, primary mouse CD8 + T cells were isolated, activated, and then treated with or without 20 μM TVA (TVA group versus control group) for 24 h, followed by RNA extraction using the PureLink RNA Mini Kit as the manufacturer's instructions.RNA samples in triplicate were used for Illumina Next Generation Sequencing.To check effect of Creb1 knockdown on TVA-dependent CD8 + T cell gene expression changes, mouse primary CD8 + T cells were isolated, activated, and then transfected with siNTC or siCreb1 using Accell siRNA method, followed by treatment with or without 20 μM TVA for 24 h.RNA samples from four groups (siNTC, siNTC+TVA, siCreb1, siCreb1 + TVA) were extracted and used for Illumina Next Generation Sequencing.Data processing and analysis were performed as previous described 48 .

cAMP level
Mouse primary CD8 + T cells were isolated, activated, and then treated with 20 μM TVA for 0, 20 min, 40 min, and 2 h.cAMP-Screen Cyclic AMP Immunoassay System (Fisher Scientific Company) was used to check cAMP level according to the manufacturer's instructions.
[ 13 C]Fatty acid tracing in vitro Activated mouse primary CD8 + T cells were cultured for 6 or 24 h in Click's medium with or without glucose, containing either 20 μM 13 C 1 -TVA or 20 μM 13 C 1 -palmitate acid.The cells were then rinsed with 0.9% saline solution and lysed using lysis buffer (500 μl of 80% cold methanol).Samples were either freeze/thawed in N 2 /Ice or sonicated.Following this, 400 μl of dichloromethane was added and vortexed for 60 s.The mixture was then set on ice for 10 min to initiate partition, followed by centrifugation at 2,000g for 15 min at 4 °C.The polar (top) layer was carefully removed into a separate labelled tube and frozen, while the organic layer was dried down using N 2 stream.After drying, 40 μl of methoxyamine (10 mg ml −1 ) in pyridine was added to each tube of dried, extracted metabolites.The solids were then re-suspended with sonication/vortexing and centrifuged briefly to remove insoluble debris.The pyridine solution was then transferred to autoinjector vials containing an insert and cooked at 70 °C for 10-15 min.Subsequently, 80 μl of TBDMS was added, briefly vortexed, and then cooked for 1 h at 70 °C.Finally, the samples were loaded for injection onto the GC-MS.

Seahorse fatty acid oxidation assay
Mouse primary CD8 + T cells were isolated, activated, and then treated with 20 μM TVA or palmitic acid.The impact of TVA or palmitic acid treatment on FAO via assessing changes in OCR was checked by Seahorse XF Long Chain Fatty Acid Oxidation Stress Test Kit (Agilent Seahorse XF Sub OX) according to the manufacturer's instructions.
Ca 2+ level of CD8 + T cells Mouse primary CD8 + T cells were isolated, activated, and then treated with TVA or SCFAs for 24 h.Bound and unbound Ca2+ levels were measured by Fluo-4 kit (Thermo Fisher Scientific) and Fura-2 kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

CRISPR editing of mouse OT-I cells
CRISPR editing of mouse primary CD8 + T cells was performed as previously described 34 .In brief, LMPd-sgNTC-mPGK-Ametrine, LMPd-sgGPR43#1-mPGK-Ametrine, LMPd-sgGPR43#2-mPGK-Ametrine, and LMPd-sgGPR43#3-mPGK-Ametrine were generated and co-transfected with pCL-Eco (Addgene, 12371) into Plat-E cells using TransIT-LT1 transfection reagent to produce retrovirus.Virus harvest medium was changed 18 h after transfection and then collected 48 h later.Primary Cas9-expressing OT-I (Cas9;OT-I) cells were derived from the spleen and peripheral lymph nodes of Cas9;OT-I mice by magnetic bead purification using EasySep Mouse T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies).In total, 2-5 million Cas9;OT-I cells were activated, placed into one well of 24-well plate, and supplemented with retrovirus supernatant containing 10 μg ml −1 polybrene, followed by spin infection (800g, brake 3) for 3 h.After infection, cells were moved to 37 °C cell culture incubator for another 2 h and then changed with new complete Click's medium (containing 20 IU ml −1 human IL-2, 2.5 ng ml −1 mouse IL-7, 25 ng ml −1 mouse IL-15) for 72 h.Cells were harvested, sorted with flow cytometry to enrich virus-transduced cells (Ametrine-positive), and subjected to subsequent function analysis as well as knockout efficiency validation using reverse transcription with PCR.
Pull-down assay to identify the crosslinked protein-TVA complexes Mouse primary CD8 + T cells were isolated and activated.10 million cells were collected, pelleted, and re-suspended in 0.3 ml ice-cold PBS containing EDTA-free protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, A32961), followed by sonication on ice.Control probe N-1 or TVA Probe 3 was added and incubated at 37 °C for 2 h under dark conditions.After probe labelling, the sample was irradiated under 365 nm UV light for 6 min on ice, diluted with 1% SDS and sonicated on ice.The proteome concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) on a microplate reader (Bio-Rad) and normalized to 1.5 mg ml −1 .700 μl protein sample was conjugated with a biotin tag by click chemistry (100 μM biotin-azide, 100 μM TBTA, 1 mM CuSO 4 and 1 mM TCEP) in the dark at room temperature for 1 h.The sample was added 4 volumes of acetone, chilled to −20 °C, vortexed, and then incubated for 3 h at −20 °C to completely precipitate the proteins and remove unreacted biotin-azide.The sample was centrifuged at 17,000g for 15 min at 4 °C to pellet the precipitated proteins.The resulting protein pellets were resolved in 1% SDS by sonication, PBS was added to dilute the concentration of SDS to 0.2% and then added 60 μl pre-washed high-capacity streptavidin C1 beads, followed by incubation overnight at 4 °C with rotation.The beads were washed 3 times with 0.1% SDS in PBS and once with PBS, 2× SDS sample buffer was added and boiled for western blot analysis of TVA-binding proteins.

Co-culture assay with Blinatumomab
The RS4;11 target cells were stained using the CellTrace Far Red Cell Proliferation Kit (Invitrogen, C34564), and then co-cultured for 24 h with PBMC in flat bottom 24-well plate at a 1:5 ratio (2 × 10 5 target cells to 10 6 PBMC) at indicated concentrations of Blinatumomab (0, 10, 100, 1,000 pg ml −1 ) derived from the leftover of infusions (provided by the laboratory of W.S.).The cell mixture was re-suspended in a flow cytometry staining buffer, stained using a Fixable Viability Dye780 (R&D), and then analysed by flow cytometry.

CAR-T cell expansion assay
In total, 63,000 anti-human CD19-CD28z-GFP CAR-T cells (provided by the laboratory of J.K.) were cultured in T cell expansion medium (StemCell) with IL-7 (5 ng ml −1 ) and IL-15 (5 ng ml −1 ) in the presence or absence of 20 μM TVA.Medium was changed to fresh medium (with IL-7 and IL-15 in the presence or absence of 20 μM TVA) in Day 2, 5, 7 and  9. Cell number was counted on the days 7, 9 and 10.

March 2021
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
Animal experiments were conducted and designed according to protocols approved by the Institutional Animal Care and Use Committee of The University of Chicago.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry Plots
Confirm that: The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Tumor tissue were dissected from euthanized tumor-bearing mice, minced small pieces (≤ 2 mm) using a scalpel in a dish, and then transferred to a 14 mL round-bottom tube containing 5 mL tumor digestion medium (500 μL Collagenase/ Hyaluronidase Solution, 750 μL 1 mg/mL DNase I Solution, and 3.75 mL RPMI 1640 Medium).After incubation at 37°C for 25 min on a shaking platform, the digested tumor tissues were transferred into a 70 μm mesh nylon strainer on a 50 mL conical tube, pushed through the strainer using the rubber end of a syringe plunger, and rinsed with the recommended medium.After centrifugation at 300×g for 10 min at room temperature with the brake on low, the resulting cell pellets were added 10 mL of ammonium chloride solution for incubation at room temperature for 5 minutes, followed by centrifugation at 300×g for 10 minutes at room temperature with the brake on low.The resulting cell pellets were re-suspended at 1-10 ×106 cells/ mL in PBS and then subjected to CD45+ tumor-infiltrating leukocytes isolation by magnetic bead purification using EasySep™ Mouse TIL (CD45) Positive Selection Kit according to the manufacturer's instructions (Stemcell Technologies).

Cell population abundance
The purities of the sorted cells were more than 98%.

Gating strategy
For all experiments FSC-A vs. SSC-A gates of the starting cell population were used to identify viable cells.Singlet cells were identified using FSC-A vs. FSC-H gating.Positive populations were determined by the specific antibodies, which were distinct from negative populations.Isotype control was used to distinguish between background and marker-positive events.Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

Fig. 2 |
Fig. 2 | TVA has a regulatory function through a GPCR-CREB axis.a, Human or mouse primary CD8 + T cells were isolated, activated and treated with or without TVA for different durations, followed by integrated, temporal mechanistic studies.GO term enrichment graphs generated from KAS-seq differential analysis of H. sapiens (left) and M. musculus (right) CD8 + T cell gene bodies (treated with 20 μM TVA versus untreated).Specifically, gene bodies exhibiting differential single-stranded DNA (ssDNA) levels for all timepoints (cut-off for individual timepoints of P < 0.4 (H.sapiens) or P < 0.5 (M.musculus)) are shown.Colour indicates fold-enrichment and size of GO term circles denotes the number of differentially expressed genes (DEGs) from KAS-seq data for that term.n = 3. Det. of chem.stim., determination of chemical stimulation.b, Scatter plot of a phospho-antibody array representing relative pixel density after TVA treatment for 40 min versus corresponding −log 10 (P value).Phosphoproteins with relative pixel density greater than 110% or less than 90% with P < 0.05 are highlighted (n = 3).c, GSEA of upregulated effector CD8 + T cells (top) and E2F targets (bottom) induced by TVA treatment in CD8 + T cells (n = 3).NES, normalized enrichment score.d, Heat map showing relative expression of PKA-CREB pathway genes in CD8 + T cells comparing TVA treatment group to the control ones (n = 3).Student's two-sided unpaired t-test (b).Nominal P values were adjusted by the Benjamini-Hochberg method (a,c).

Fig. 3 |
Fig.3| TVA's effect on CD8 + T cells is primarily mediated through CREB and its target gene sets.a, Principal component analysis of genes from RNA-seq analysis of siRNA-mediated transient knockdown of Creb1 (siCreb1) in CD8 + T cells with or without TVA treatment, compared to non-targeting control siRNA (siNTC) (n = 3).b, GSEA of upregulated effector CD8 + T cells (left) and E2F targets (right) with siNTC and TVA treatment compared with siCreb1 and TVA treatment group.n = 3. Nominal P values were adjusted by the Benjamini-Hochberg method.c, Heat map of differentially expressed genes from RNA-seq analysis of siCreb1 CD8 + T cells comparing with siNTC with or without TVA treatment.The up-or downregulated genes in the siNTC plus TVA group compared to the other three groups were gated with orange boxes (left) and enriched for GO analysis (right) (n = 3).d, Creb1 target genes validation.log 2 fold changes of cell number (left), apoptosis (middle) and Ki-67 expression (right) after TVA treatment in CD8 + T cells with individually transient knockdown of Creb1 (n = 5), Il18 (left, n = 3; middle and right, n = 6), Tbx21, Ilf2, Bcl6, Foxo4 and Ebi3 (n = 3).Data are mean ± s.d.Student's two-sided unpaired t-test.Ctrl, control.

Fig. 5 |
Fig. 5 | TVA augments the effectiveness of multiple T cell-based anti-cancer therapies.a, Effect of anti-PD-1 antibody on B16F10 tumour growth in C57BL/6 mice fed with TVA-enriched diet or control diet (n = 8).b, Box plots representing combined effect of blinatumomab and TVA at indicated concentrations on specific lysis of RS4;11 target cells in the presence of PBMCs by flow cytometry (n = 5).The central line is the mean, whiskers extend to minimum and maximum values and the box edges show mean ± s.d.c, Effects of treatment with 20 μM TVA on in vitro expansion of anti-CD19-CD28z CAR-T from patients with lymphoma.d, Top, schematic depicting experimental design for serum collection from patients with CAR-T cell therapy.This schematic was generated using BioRender.com.Bottom, violin plots showing serum TVA levels of 10 patients with lymphoma who have undergone commercial CAR-T cell therapy.Blood was collected from each patient at four different timepoints (detailed information can be found in Supplementary Table8).Red violin plots represent the patients who have complete response to CAR-T cell therapy, and blue violin plots represent patients who have progressive disease under CAR-T cell therapy.Data are mean ± s.e.m (a) or mean ± s.d.(b,d).Two-way ANOVA (a) or Student's two-sided unpaired t-test (b,d).
Gpr43 knockdown efficiency checked by RT-PCR (n = 4).e, Gpr43 knockdown efficiency in CD8 + T cells was checked by RT-PCR (left) (n = 6).Effects of Gpr43 transient knockdown on cAMP (middle left), p-CREB (middle right), and p-STAT1 (right) levels in mouse CD8 + T cells treated with or without TVA were shown (n = 3).f, Effects of Gpr43 transient knockdown on TNF-α (left) and IFN-γ (right) levels in mouse CD8 + T cells treated with or without TVA (n = 3).g, Gpr43 knockdown efficiency in OT-I cells was checked by RT-PCR (left).Effect of Gpr43 transient knockdown on TNF-α level (right) in OT-I cells treated with or without TVA was shown (n = 3).h, Knockout efficiency of Gpr43 in Cas9;OT-I cells was checked by RT-PCR (upper left) (n = 4).Effects of individually knockout of Gpr43 with three sgRNAs on p-CREB level (upper right) and apoptosis (lower) in mouse Cas9;OT-I cells treated with or without TVA were shown (n = 3).i, Relative Gpr43 mRNA level (n = 6) and protein level (n = 3) in CD8 + T cells and CD4 + T cells with or without anti-CD3/CD28 stimulation.Data are mean ± SD (b-i).Nominal P values were adjusted by the Benjamini-Hochberg method (a), Student's two-sided unpaired t test (b-i).Extended Data Fig. 9 | TVA derivatives and probes exhibit distinct bioactivity to enhance CD8 + T cell function.a, Chemical structures of TVA and 15 TVA derivatives.b, Effects of treatment with 20 μM TVA or TVA derivatives on TNF-α (upper) and IFN-γ (lower) levels in CD8 + T cells (n = 3).c, Chemical structures of the three synthetic designed photo-affinity probes of TVA.d, Effects of treatment with 20, 40, or 100 μM TVA probes on TNF-α level in mouse CD8 + T cells (n = 3).Extended Data Fig. 11 | SCFAs affect mouse CD8 + T cell activity through GPR41, GPR43, and GPR65.a, SCFAs level in Click's medium (n = 5).b, Medium pH level with different doses of SCFAs.c, Effects of Gpr41, Gpr43, and Gpr65 transient knockdown on cAMP level in mouse CD8 + T cells treated with or without SCFAs (n = 4).d, Effect of Gpr41, Gpr43, and Gpr65 transient knockdown on TNF-α level in mouse CD8 + T cells treated with or without SCFAs (n = 4).e, Effect of Gpr41, Gpr43, and Gpr65 transient knockdown on IFN-γ level in mouse CD8 + T cells treated with or without SCFAs (n = 4).f, Gpr41 (left), Gpr43 (middle), and Gpr65 (right) knockdown efficiency checked by RT-PCR (n = 5).g, Flow cytometry and quantification of Ca 2+ -bound Fluo-4 level (n = 3).h, Flow cytometry and quantification of free unbound Fura-2 level (n = 3).i, Working model of effect of SCFAs on cAMP level in CD8 + T cells (n = 5).The figure was generated using BioRender.j, pH level of B16F10 tumor-bearing mouse serum (left) and TIF (right) (n = 4).k, Serum pH level of 10 lymphoma patients that have undergone commercial CAR-T cell therapy.Each patient has blood collections at 4 different timepoints (detailed information can be found in Supplementary Table

8
). l, pH levels of TVA and CON diet.m, Medium pH level with different doses of TVA.Data are mean ± SD (a, c-k).Student's two-sided unpaired t test (a, c-k).Extended Data Fig. 12 | TVA-enhanced CD8 + T cell function independent of pH sensor GPR65.a, Gpr65 knockdown efficiency in CD8 + T cells was checked by RT-PCR (left) (n = 5).Effect of Gpr65 transient knockdown on TNF-α (middle left), IFN-γ (middle right), and p-CREB (right) levels (n = 3) in mouse CD8 + T cells treated with or without TVA were shown.b, Knockout efficiency of Gpr65 in Cas9;OT-I cells was checked by RT-PCR (left) (n = 4).Effect of Gpr43 knockout with two different sgRNAs on TNF-α (middle left), IFN-γ (middle right), and p-CREB (right) levels (n = 3) in mouse Cas9;OT-I cells treated with or without TVA were shown.c, Effects of Gpr65 transient knockdown on p-CREB levels in mouse CD8 + T cells treated with or without TVA under different pH (n = 3).d, Cas9;OT-I cells