Graphical abstract graphic file with name fx1.jpg [77]Open in a new tab Highlights * • Plant-produced nutrient zeaxanthin enhances the cytotoxicity of CD8^+ T cells * • Orally supplemented zeaxanthin augments anti-tumor immunity * • Zeaxanthin promotes T cell receptor stimulation on CD8^+ T cell surface * • Zeaxanthin treatment improves immunotherapy efficacy __________________________________________________________________ Mechanistic insights into how dietary components regulate anti-tumor immunity remain limited. Zhang et al. identify zeaxanthin—a carotenoid in many fruits and vegetables—as an immunomodulator that enhances CD8^+ T cell function, highlighting its translational potential in supporting immunotherapy. Introduction The evolution of diet has played an essential role in shaping human physiology and pathology, including the development of the immune system and its responses to environmental stimuli.[78]^1^,[79]^2^,[80]^3 However, despite extensive epidemiological studies, the mechanistic understanding of how different diets impact the human immune system is very limited due to the intricate nature of the immune system, whole-organism metabolism, and the vast diversity of dietary components available. Emerging studies on the diet-immune axis have uncovered diet-derived nutrients that, besides performing their metabolic functions to provide energy and biosynthesis building blocks, serve immunoregulatory functions to influence diverse immune cell populations.[81]^4 We thus developed an approach to focus on the bioactivity of individual nutrients, which allows us to conduct mechanistic studies to decipher the links between diet and immunity, despite the vast diversity of foods and dietary origins. We assembled a library of “blood nutrients” that are commercially available, including dietary supplements, inorganics, organic metabolites, peptides, and lipids, and screened for circulating blood nutrients that affect CD8^+ T cell function. We uncovered that dietary nutrient trans-vaccenic acid (TVA) selectively promotes effector CD8^+ T cell function and anti-tumor immunity in vivo, through TVA-mediated antagonism of an immunomodulatory G-protein-coupled receptor, GPR43, on the cell surface of CD8^+ T cells.[82]^5 Here, using this blood nutrient compound library, we applied a co-culture-based screening approach to identify circulating nutrients that function as immunomodulators to affect the cytotoxicity of mouse Pmel-1 CD8^+ T cells against mouse B16F10 melanoma tumor cells. We uncovered zeaxanthin (ZEA), a dietary nutrient found in many fruits and vegetables, as an unexpected immunomodulator, which enhances CD8^+ effector T cell function and consequently improves anti-tumor immunity in vivo. ZEA is a non-provitamin A carotenoid synthesized only by plants and microorganisms, which can be found in grains such as corn and corn products, as well as many green leafy vegetables, and is commonly used as a feed additive and colorant for birds, swine, and fish.[83]^6^,[84]^7^,[85]^8 While more than 700 carotenoids are found in nature, only 40–50 have nutritional values and about 20 of them reach detectable levels in human circulation and tissues. ZEA and its structural isomer lutein (LUT) are among the most prevalent circulating carotenoids in the human body, but they can only be incorporated into the human circulation through consumption from diet and dietary supplements.[86]^9^,[87]^10 The chemical structures of ZEA and LUT differ only in the position of a double bond in one cyclic ring, resulting that ZEA has two β-ionone rings while LUT has a β-ionone ring and an ε-ionone ring.[88]^11 In addition, among the three stereoisomeric forms of ZEA, (3R,3′R)-zeaxanthin is the most common natural form found in many fruits and vegetables and is the predominant isomer present in the human retina. Therefore, (3R,3′R)-zeaxanthin was used in our study. Both ZEA and LUT are found to concentrate at the macular region of the human retina, with ZEA being the dominant component in the central macula and LUT distributed more broadly throughout the retina.[89]^12 Because of their blue-light-filtering and antioxidant roles, ZEA and LUT are commonly consumed as dietary supplements to protect the retina and eye tissues for vision health. Previous studies have revealed ZEA’s role in photoprotection, energy transfer, and antioxidation.[90]^12^,[91]^13 However, the distinct immunomodulatory functions of ZEA and LUT in the context of anti-tumor immunity were previously unknown. Here, we show that ZEA interacts with the TCR complex and promotes stimulation on the cell surface of CD8^+ T cells, leading to enhanced TCR complex formation, activation of intracellular TCR signaling to augment CD8^+ effector T cell function, and consequent anti-tumor immunity. Results Carotenoid nutrient ZEA enhances cytotoxicity of CD8^+ T cells and anti-tumor immunity Using our blood nutrient library[92]^5 including dietary supplements, lipids, peptides, organic metabolites, and inorganics ([93]Figure S1A), we performed a co-culture screen to identify circulating nutrients that influence the cytotoxicity of mouse Pmel-1 CD8^+ T cells stimulated by anti-CD3/CD28 antibodies against co-cultured B16F10-luc2 mouse melanoma cells ([94]Figure 1A). The screen results ([95]Figure 1B; [96]Table S1) show that ZEA, but not its structural isomer LUT, effectively enhanced Pmel-1 mediated cell death of B16F10-luc2 cells. In addition, treatment with ZEA, but not LUT, augmented the ability of OT-I CD8^+ T cell-mediated cytotoxicity to induce cell death against co-cultured B16-OVA cells ([97]Figure 1C). Notably, ZEA exhibited no significant direct cytotoxicity toward either tumor cell line at the concentration used in the co-culture assays ([98]Figure S1B). Moreover, pre-treatment of cancer cells with ZEA did not intrinsically sensitize them to killing mediated by CD8^+ T cells ([99]Figure S1C). Figure 1. [100]Figure 1 [101]Open in a new tab Oral ZEA enhances CD8^+ T cell-mediated anti-tumor immunity (A) Schematic depicting experimental design for effector T cell cytotoxicity screen. This schematic was generated using BioRender.com. (B) Chemical structures of ZEA and LUT (upper) and volcano plot showing results from the library screen with ZEA and LUT highlighted (lower). (C) Effects of 5 μM ZEA or LUT on B16-OVA cell death when treating B16-OVA cells alone and treating OT-I primary murine T cell and B16-OVA cell co-culture (n = 3 technical replicates). (D) Schematic depicting experimental design for in vivo tumor-bearing mouse model. This schematic was generated using BioRender.com. (E) Effect of orally administered ZEA (500 mg/kg b.w.) on the growth of subcutaneous B16F10 melanoma in C57BL/6 mice (n = 11 mice). (F) Effect of orally administered ZEA (500 mg/kg b.w.) on the growth of subcutaneous MC38 colon tumors in C57BL/6 mice (n = 10 mice). (G) Schematic depicting experimental design for CD8^+ (or CD4^+) T cell depletion experiment. This schematic was generated using BioRender.com. (H) Effect of orally administered ZEA (500 mg/kg b.w.) on B16F10 tumor growth in C57BL/6 mice treated with isotype control (left) or CD8^+ T cell-depleting antibody (right) (n = 8 mice). b.w., body weight. Data are mean ± SEM (E, F, and H) or mean ± SD (C). p values are calculated using two-way ANOVA (E, F, and H) or one-way ANOVA with Dunn’s multiple comparisons test (C) (ns, not significant; ∗0.01 < p < 0.05; ∗∗0.001 < p < 0.01; ∗∗∗p < 0.001). Also see [102]Figure S1 and [103]Table S1. We next found that in vivo tumor growth of poorly immunogenic B16F10 cells ([104]Figure 1D) was significantly attenuated in syngeneic mice receiving oral gavage of ZEA ([105]Figures 1E and [106]S1D), but not in mice receiving LUT ([107]Figure S1E), compared to B16F10 syngeneic mice receiving oral gavage of control vehicle. We detected increased levels of ZEA and LUT in the tumor interstitial fluid (TIF) of tumors in the syngeneic mice ([108]Figure S1F), whereas oral administration of neither ZEA nor LUT affected body weights of syngeneic tumor-bearing mice ([109]Figure S1G). Further absolute quantification showed that oral gavage of ZEA significantly increased its levels in both mouse plasma and TIF, with overall higher concentrations observed in the TIF ([110]Figure S1H). Similar results were obtained using mouse colon cancer MC38 cells in syngeneic mice receiving oral gavage of ZEA or vehicle control ([111]Figures 1F and [112]S1I). Moreover, we found that depletion of CD8^+ T cells by anti-CD8 antibody in B16F10 syngeneic mice ([113]Figures 1G and [114]S1J) resulted in abolishment of ZEA-mediated reduction of tumor growth ([115]Figure 1H). In contrast, depletion of CD4^+ T cells by anti-CD4 antibody had minimal effects on ZEA-mediated tumor growth in B16F10 syngeneic mice ([116]Figures S1K and S1L). These data together suggest that ZEA reprograms CD8^+ T cells and consequently enhances anti-tumor immunity. Oral ZEA improves recruitment and activation of tumor-infiltrating CD8^+ T cells Flow cytometry analyses revealed that oral ZEA supplementation to B16F10 syngeneic mice significantly increased both the number and proportion of CD8^+ T cells within the CD45^+ tumor-infiltrating leukocyte (TIL) population ([117]Figure 2A). In contrast, tumor-infiltrating populations of CD4^+ T cells, B cells, dendritic cells, M1 and M2 macrophages, neutrophils, and natural killer cells were not altered in B16F10 syngeneic mice by oral ZEA supplementation, compared to mice receiving vehicle control ([118]Figures 2B and [119]S1O). Further analysis of tumor-infiltrating CD8^+ T cells with additional representative markers revealed that oral ZEA supplementation promoted CD8^+ T cell function with increased expression levels of the activation marker CD69; the co-stimulatory receptor ICOS; and cytokines including tumor necrosis factor (TNF)-α, interferon (IFN)γ, and interleukin (IL)-2 ([120]Figures 2C, [121]S1N, and S1P). Oral ZEA supplementation increased the proportion of Ki-67^+ cells but had no effect on the frequency of cleaved caspase-3^+ cells in tumor-infiltrating CD8^+ T cells ([122]Figure S1P). While ZEA did not significantly alter the proportions of stem-like or effector-like exhausted T cells within the tumor microenvironment, it reduced the population of terminally exhausted CD8^+ T cells ([123]Figure S1Q). In addition, ZEA supplementation significantly increased the proportion of tumor-reactive CD8^+ T cells within the TILs of MC38-OVA tumor-bearing mice ([124]Figure S1R). Oral ZEA also resulted in increased CD4^+ T helper 1 (Th1) but reduced CD4^+CD25^+Foxp3^+ Treg cell populations in B16F10 tumors, while it had minimal effects on other tumor-infiltrating CD4^+ T cell populations including Th2, Th17, Th9, and Th22 ([125]Figure 2D). In contrast, oral ZEA supplementation did not affect the cell populations of diverse immune cells in spleens or draining lymph nodes (dLNs) in B16F10 syngeneic mice ([126]Figures S1S and S1T). Lastly, oral ZEA supplementation did not significantly alter the diversity and composition of gut microbiota ([127]Figure S2). Given that Th1 cells are critical in cellular immune responses against intracellular viruses, including activation of cytotoxic T cells,[128]^14 and that Treg cells suppress CD8^+ T cells,[129]^15 these results together suggest that oral ZEA enhances recruitment and activation of CD8^+ effector T cells to tumors for improved anti-tumor immunity. Figure 2. [130]Figure 2 [131]Open in a new tab ZEA reprograms CD8^+ T cells in vivo and in vitro (A) Schematic depicting experimental setup for harvesting the spleen, tumor, and draining lymph node (dLN) to examine zeaxanthin’s effect on leukocytes. This schematic was generated using BioRender.com. (B) Effect of orally administered ZEA (500 mg/kg b.w.) on the composition of tumor-infiltrating leukocytes (n ≥ 10 mice). (C) Effects of orally administered ZEA (500 mg/kg b.w.) on tumor-infiltrating CD8^+ T cell activation, co-stimulation, and pro-inflammatory cytokine TNF-α production (n ≥ 7 mice). (D) Effects of orally administered ZEA (500 mg/kg b.w.) on tumor-infiltrating CD4^+ T cell composition (n ≥ 7 mice). (E) Schematic depicting experimental setup for in vitro primary murine CD8^+ T cells treatment. This schematic was generated using BioRender.com. (F) Effects of 5 μM ZEA or LUT treatment for 24 h on αCD3/CD28-stimulated murine CD8^+ T cell proliferation, activation, and pro-inflammatory cytokine levels (n = 3 technical replicates). (G) Chemical structures of ZEA and 6 natural structural analogs of ZEA. (H) Effect of 24-h treatment with 5 μM ZEA or ZEA derivatives on primary murine CD8^+ T cell TNF-α production (n = 3 technical replicates). Data are mean ± SD. p values are calculated using Student’s two-sided unpaired t test (B, C, and D) or one-way ANOVA with Dunn’s multiple comparisons test (F and H) (ns, not significant; ∗0.01 < p < 0.05; ∗∗0.001 < p < 0.01). Also see [132]Figure S1. Consistent with these findings, we next found that ZEA but not LUT treatment had direct effects on and effectively promoted mouse primary CD8^+ T cell function ([133]Figure 2E) with increased expression levels of proliferation marker Ki-67; activation marker CD69; and cytokines including TNF-α, IFNγ, and IL-2 ([134]Figure 2F). Given that ZEA and LUT are structurally similar but have distinct effects on CD8^+ T cells, we next explored the structure-activity relationship (SAR) by testing several natural compounds that share structural similarity with ZEA, all of which are available in dietary sources ([135]Figure 2G). Notably, only ZEA and D5 (fucoxanthin) significantly promoted CD8^+ T cell activation, despite the fact that all compounds have long chains of conjugated double bonds ([136]Figure 2H). This result suggests that the antioxidant properties of conjugated double bonds in these compounds are not sufficient to mediate enhanced activation of CD8^+ T cells. Instead, the structurally defined symmetrical conjugated polyene capped with six-membered rings is crucial for ZEA to enhance CD8^+ T cell activation. In addition, the different six-membered ring structures at both ends of the double bonds account for the differential bioactivity of ZEA compared to D1, D2, D4, and D5 for enhanced CD8^+ T cell activation, while D3 with an open-chain structure and D6 with a short-chain monomer lacked bioactivity ([137]Figure 2H). These data together suggest that the distinct structure and conformation of ZEA are vital for its bioactivity to enhance CD8^+ T cell activation and function. ZEA engages the T cell receptor complex and augments CD8^+ T cell surface TCR stimulation To determine the mechanisms underlying ZEA’s bioactivity in enhancing CD8^+ T cell function, we performed kethoxal-assisted single-stranded DNA sequencing (KAS-seq) (40 min–2 h)[138]^16 to investigate the initial influences of ZEA treatment on mouse primary CD8^+ T cells by capturing global transcription dynamics and enhancer activity. Functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the top-ranking altered genes from the genome-scale KAS-seq results ([139]Figure 3A; [140]Table S2) revealed the T cell receptor (TCR) signaling pathway among the top-enriched ontologies in ZEA-treated CD8^+ T cells. Figure 3. [141]Figure 3 [142]Open in a new tab ZEA augments CD8^+ T cell surface TCR stimulation (A) KEGG pathway enrichment for upregulated genes generated from KAS-seq analysis of αCD3/CD28-stimulated murine CD8^+ T cells treated with 5 μM ZEA compared to cells treated with DMSO control (n = 3 biological replicates). (B) Schematic depicting experimental setup for flow cytometry and confocal microscopy detection of αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8^+ T cells with or without 5 μM ZEA or LUT treatment with different stimulation strategies. This schematic was generated using BioRender.com. (C) Flow cytometry analysis showing the effects of 5 μM ZEA or LUT on αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8^+ T cells at 5, 15, and 30 min after treatment and stimulation with αCD3ε and αCD28 antibodies (n = 3 technical replicates). (D) Representative confocal microscopy images showing the effects of 5 μM ZEA on αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8^+ T cells at 5, 15, and 30 min after treatment and stimulation with αCD3ε and αCD28 antibodies (n = 3 technical replicates). Scale bars represent 5 μm. (E) Quantification of fluorescence intensities from confocal microscopy images shown in (D). TCRβ, CD3, and CD28 antibody signals were measured at 5, 15, and 30 min after treatment and stimulation with CD3 and CD28 antibodies (n = 3 technical replicates). (F) The chemical structure of the ZEA-based photo-affinity labeling (PAL) probe and schematic workflow for validating its binding to the TCR complex. (G) Pull-down of the TCR complex using the biotinylated ZEA probe. Mouse primary CD8^+ T cells were incubated with DMSO and 50 μM ZEA probe or pre-treated with 250 μM ZEA for 1 h followed by 50 μM ZEA probe. After UV-induced crosslinking, samples were subjected to click chemistry with a biotin tag, followed by streptavidin pull-down and immunoblotting. Data are mean ± SD. p values are calculated using two-way ANOVA (ns, not significant; ∗∗∗p < 0.001). Also see [143]Figure S3 and [144]Table S2. TCR signaling in CD8^+ T cells is triggered when the cell surface TCR binds to a major histocompatibility complex (MHC) class I-bound peptide antigen. This interaction triggers the assembly of the TCR signaling complex and activates downstream intracellular signaling cascades to turn on CD8^+ effector T cell function including cytokine production and cytotoxicity.[145]^17 Given that polar carotenoids including ZEA and LUT can insert into plasma membranes and alter the membrane fluidity,[146]^18^,[147]^19 we examined the effects of ZEA or LUT treatment on TCR complex formation on the cell surface of mouse primary CD8^+ T cells upon stimulation with anti-CD3/CD28 antibodies or on the cells with overnight pre-stimulation ([148]Figure 3B). Flow cytometry analysis revealed that ZEA treatment for 5, 15, and 30 min effectively enhanced TCR complex formation on mouse primary CD8^+ T cells assessed by increased levels of TCRβ, CD3ε, and CD28 on CD8^+ T cell surface upon stimulation ([149]Figure 3C; left two panels). In contrast, LUT treatment had minimal effects on TCR complex formation on mouse primary CD8^+ T cell surface upon stimulation ([150]Figure 3C; right two panels). Moreover, we performed confocal microscopy to visualize cell surface TCR complex formation on mouse primary CD8^+ T cells upon stimulation with anti-CD3/CD28 antibodies. ZEA treatment for 5, 15, or 30 min effectively promoted TCR complex formation with increased detection of cell surface TCRβ, CD3ε, and CD28 on mouse primary CD8^+ T cells ([151]Figures 3D and 3E). Similar results were obtained from flow cytometry ([152]Figure S3A) and confocal microscopy ([153]Figure S3B) analyses using mouse primary CD8^+ T cells with overnight pre-stimulation by anti-CD3/CD28 antibodies prior to treatment with ZEA or LUT. These findings prompted us to investigate whether ZEA binds to the TCR complex. To this end, we synthesized a ZEA photo-affinity labeling (PAL) probe. The ZEA PAL probe was incubated with intact mouse primary CD8^+ T cells. Upon UV crosslinking, followed by click chemistry, we performed either in-gel fluorescence (via TAMRA labeling) or streptavidin pull-down (via biotin tagging) to assess probe-protein interactions ([154]Figure 3F). TAMRA fluorescence revealed strong probe binding in wild-type CD8^+ T cells, but significantly reduced signal in TCRα knockout cells, suggesting TCR-dependent binding ([155]Figure S3C). Pull-down assays confirmed that the probe enriched TCRα, TCRβ, and CD3ζ subunits, and these interactions were specifically outcompeted by excess unmodified ZEA ([156]Figure 3G). Functionally, ZEA-induced activation was completely abrogated in TCRα-deficient CD8^+ T cells ([157]Figure S3D). These results together suggest that ZEA engages the TCR complex and facilitates or stabilizes its formation at the cell membrane, thereby enhancing TCR-mediated signaling and CD8^+ T cell activation. ZEA-mediated CD8^+ T cell activation involves intracellular Ca^2+ signaling and nuclear factor κB pathway We next sought to explore the downstream intracellular TCR signaling cascades that are crucial for ZEA-enhanced CD8^+ T cell activation and function using mouse primary CD8^+ T cells ([158]Figure 4A). We designed integrated, temporal mechanistic studies, including (1) TCR-specific phospho-antibody array (5 min) for changes of proximal TCR signaling cascades following ZEA treatment, (2) conventional phospho-antibody array (40 min–6 h) for downstream cellular signaling changes, and (3) RNA sequencing (RNA-seq) (24 h) for whole-transcriptome analysis. TCR complex assembly upon antigen recognition activates tyrosine kinase Lck, leading to consequent phosphorylation and activation of downstream signaling molecules including ZAP-70 and phospholipase Cγ (PLCγ). This initiates activation of transcription factors such as nuclear factor κB (NF-κB) and NFAT, as well as a signaling cascade involving Ca^2+ release from endoplasmic reticulum for CD8^+ T cell functions including cytokine production and cytotoxicity.[159]^20^,[160]^21^,[161]^22^,[162]^23 Indeed, TCR phospho-antibody array analysis on mouse primary CD8^+ T cells treated with ZEA revealed increased phosphorylation levels of ZAP-70, Lck, PLC, LAT, and NF-κB and reduced phosphorylation levels of NFAT as activation and nuclear localization of NFAT require dephosphorylation by Ca^2+-dependent phosphatase calcineurin[163]^24 ([164]Figure 4B; [165]Table S3). In contrast, LUT treatment had minimal effects on phosphorylation levels of these key proximal and downstream signaling molecules of the TCR complex in CD8^+ T cells ([166]Figure S4A). The results of the conventional cell phospho-antibody array ([167]Figures S4B and S4C; [168]Table S4) also revealed increased phosphorylation and activation of proteins in the JAK-STAT and ERK pathways, which are known to be activated by TNF in CD8^+ T cells[169]^25^,[170]^26 and in mouse primary CD8^+ T cells treated with ZEA, but not in cells treated with LUT. Figure 4. [171]Figure 4 [172]Open in a new tab ZEA enhances TCR signaling cascades and effectiveness of T cell-based immunotherapies (A) Schematic depicting temporal, mechanistic studies performed with murine CD8^+ T cells treated with and without 5 μM ZEA or LUT. (B) Volcano plot showing the effects of 5 min ZEA treatment on TCR-related protein phosphorylation (left). A list of changed protein phosphorylation in TCR signaling pathways (right). (C) Chord diagram showing KEGG pathway enrichment of differentially expressed genes between ZEA treatment and DMSO in RNA-seq (n = 3 biological replicates). (D) GSEA analysis of the gene set of IFNα-stimulated CD8^+ T cell upregulated genes after 24 h ZEA or LUT treatment (n = 3 biological replicates). (E) Effect of LCK inhibitor PP2 (200 nM) treatment on ZEA-dependent murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). (F) Effect of PLCγ inhibitor 3-nitrocoumarin (500 nM) treatment on ZEA-dependent murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). (G) Effect of 5 μM ZEA or LUT treatment on calcium indicator Fluo-4 levels in murine CD8^+ T cells. Cells were stimulated with soluble anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL) in the presence of 5 μM ZEA, 5 μM LUT, or DMSO control for 30 min, followed by flow cytometry analysis (n = 3 technical replicates). (H) Effect of calcineurin inhibitor cyclosporin A (7 nM) treatment on ZEA-dependent murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). (I) Effect of NFAT inhibitor (1 μM) treatment on ZEA-dependent murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). (J) Effect of NF-κB inhibitor INK4 (2 μM) treatment on ZEA-dependent murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). (K) Effect of TVA (10 μM) and ZEA (5 μM) combined treatment on murine CD8^+ T cell effector function assessed by TNF-α levels (n = 3 technical replicates). CI, combination index. (L) Effect of αPD-1 antibody on B16F10 tumor growth in C57BL/6 mice orally supplemented with (CI = 0.886) or without ZEA (n ≥ 9 mice). CI, combination index (M) Effect of aPD-1 antibody on MC38 tumor growth in C57BL/6 mice orally supplemented with (CI = 0.885) or without ZEA (n ≥ 7 mice). CI, combination index. (N) Effect of 5 μM ZEA or LUT on human CD8^+ T cell effector function assessed by IFNγ, IL-2, and TNF-α levels in human CD8^+ T cells isolated from peripheral blood mononuclear cells of healthy donors (n = 10 biological replicates). (O) Schematic depicting experimental design for human TCR-T transduction and ex vivo co-culturing with tumor cells to assess TCR-T cell-mediated cytotoxicity. This schematic was generated using BioRender.com. (P) Flow cytometry analysis showing the effects of 5 μM ZEA or LUT on human 19305DP-TCR-T cell-mediated cytotoxicity against A375 melanoma cells and A375 melanoma cells alone. Human CD8^+ cells were derived from a healthy donor (n = 3 technical replicates). (Q) Flow cytometry analysis showing the effects of 5 μM ZEA or LUT on A375 melanoma cells alone, human untransduced (mock) CD8^+ cells against A375 melanoma cells, T cell human 19305DP-TCR-T cell-mediated cytotoxicity against A375 melanoma cells, and human AL-TCR-T cell-mediated cytotoxicity against A375 cells. Human CD8^+ cells were derived from a second healthy donor (n = 3 technical replicates). Data are mean ± SEM (L and M) or mean ± SD (E–J, K, and M–Q). p values are calculated using two-way ANOVA (E, F, H–M, and Q), one-way ANOVA with Dunn’s multiple comparisons test (G and P), or Student’s two-sided paired t test (N) (ns, not significant; ∗0.01 < p < 0.05; ∗∗0.001 < p < 0.01; ∗∗∗p < 0.001). Also see [173]Figure S4 and [174]Tables S3, [175]S4, [176]S5, [177]S6, and [178]S7. Consistent with these findings, principal-component analysis of RNA-seq results revealed that control mouse primary CD8^+ T cells and cells treated with LUT can be grouped together and are separated from mouse primary CD8^+ T cells treated with ZEA ([179]Figure S4D), suggesting that ZEA selectively alters CD8^+ T cells while LUT has minimal effects. Moreover, functional KEGG pathway analysis[180]^27 and global gene set enrichment analysis (GSEA)[181]^28 of RNA-seq results revealed that ZEA treatment enhanced the expression of the genes enriched in the TCR signaling pathway ([182]Figure 4C; [183]Tables S5, [184]S6, and [185]S7). Notably, ZEA treatment upregulated the expression of genes enriched in interferon-stimulated effector CD8^+ T cells more effectively than LUT did ([186]Figure 4D), correlating with enhanced CD8^+ T cell function following ZEA treatment. We next sought to determine which downstream pathway(s) of the TCR complex is required for ZEA function. ZEA but not LUT treatment significantly increased phosphorylation levels of TCR downstream signaling molecules including CD3ζ, Lck, ZAP-70, LAT, and S6 kinase that is a downstream effector of the PI3K-AKT pathway ([187]Figures S4E–S4I). Consistent with these findings, Lck or PLCγ inhibitors abolished ZEA-dependent enhancement of CD8^+ T cell function ([188]Figures 4E and 4F). PLCγ is a downstream signaling effector of TCR for rapid release of Ca^2+ from the endoplasmic reticulum lumen to cytosol.[189]^29 Indeed, we found that ZEA but not LUT treatment effectively increased intracellular Ca^2+ level in CD8^+ T cells, assessed by increased Ca^2+-bound indicator Fluo-4 ([190]Figures 4G and [191]S4J). Consistent with this finding, treatment with specific inhibitors targeting Ca^2+-dependent phosphatase calcineurin and its downstream substrate transcription factor NFAT obliterated ZEA-enhanced CD8^+ T cell function, assessed by TNF-α production ([192]Figures 4H and 4I). Moreover, we found that treatment with NF-κB inhibitor ([193]Figure 4J), but not inhibitors targeting ERK or JAK that are activated by TNF ([194]Figures S4K–S4N), also abolished ZEA-enhanced CD8^+ T cell function assessed by TNF-α production. Taken together, our results reveal that TCR signaling cascades involving the Lck-PLC-Ca^2+-NFAT and NF-κB pathways are crucial for ZEA-enhanced CD8^+ T cell function. ZEA augments T cell-based therapies Our studies show that animal-derived TVA[195]^5 and plant-derived ZEA enhance CD8^+ effector T cells and anti-tumor immunity through different molecular and signaling mechanisms. Consistent with these findings, combined treatments with different dosages of TVA and ZEA resulted in synergistic effects to enhance primary mouse CD8^+ T cell function in vitro ([196]Figures 4K, [197]S4N, and S4O). We found that oral ZEA supplementation combined with anti-PD-1 antibody, a representative form of immune checkpoint inhibitor therapy,[198]^30 synergistically suppressed tumor growth in both B16F10 and MC38 tumor models ([199]Figures 4L and 4M). In addition, ZEA treatment was significantly more potent than LUT treatment in enhancing human CD8^+ T cell activation and function assessed by the production of cytokines, including IFNγ, IL-2, and TNF-α ([200]Figure 4N). Furthermore, ZEA treatment improved in vitro cytotoxicity of human TCR-engineered T (TCR-T) cells derived from primary CD8^+ T cells of healthy donors ([201]Figure 4O). For example, ZEA treatment was significantly more potent than LUT in enhancing cytotoxicity of A∗02-restricted NY-ESO-1 tumor antigen-specific 19305DP[202]^31 TCR-T cells derived from a healthy donor against co-cultured A∗02^+NY-ESO-1^+ A375 human melanoma cells ([203]Figure 4P; upper), while both ZEA and LUT have minimal direct cytotoxic effects on A375 cells ([204]Figure 4P; lower). ZEA but not LUT also effectively promoted cytotoxicity of both 19305DP and A∗02-restricted NY-ESO-1-specific AL-TCR-T[205]^31 cells derived from another healthy donor against co-cultured A375 cells ([206]Figure 4Q). Moreover, consistent with our findings using mouse primary CD8^+ T cells, treatment with PLCγ inhibitor abolished ZEA-enhanced cytotoxicity of AL-TCR-T cells on A375 cells ([207]Figure S4P). Additionally, we included a third donor and co-cultured TCR-T cells with A∗02^+NY-ESO-1^+ A375, A∗02^+NY-ESO-1^+ U266 (multiple myeloma), and A∗02^+NY-ESO^-U87 (glioblastoma) cell lines under ZEA treatment to demonstrate its anti-tumor effects on human T cells across different tumor types. ZEA enhanced the cytotoxic activity of TCR-T cells against all three tumor cell lines ([208]Figure S4Q), while exhibiting minimal direct cytotoxicity toward the tumor cells themselves at the concentration used in the co-culture treatment ([209]Figure S4R). These findings together align with the concept that ZEA supplementation potentially improves clinical responsiveness to T cell-based immunotherapies. Discussion Hereby our findings reveal a previously unknown immunomodulatory function of ZEA, a carotenoid antioxidant known for its role in eye health. Dietary ZEA can be found in many fruits and green leafy vegetables such as kale, spinach, broccoli, peas, lettuce, durum wheat, and corn, as well as egg yolks, because common feeds for chickens contain grains such as corn and vegetables enriched with ZEA.[210]^32 It is widely recognized that plant-based or vegan diets can potentially benefit the human immune system.[211]^33 However, the underlying mechanistic link between plant-derived nutrients and human immunity remains largely unknown. Our strategy focusing on individual diet-derived nutrients despite the vast diversity of food and diet origins makes mechanistic studies feasible to explore nutritional influences on human health and the immune system. Using this approach, we successfully identified dietary TVA derived from animal-based diets[212]^5 and hereby ZEA as a plant-produced nutrient, both of which have direct yet mechanistically distinct effects on CD8^+ effector T cells and anti-tumor immunity. Therefore, our approach has broad implications to uncover unprecedented roles of nutrients in human health and pathologic responses. Our findings support the high translational potential of ZEA, as a natural food component, to serve as a dietary supplement that enhances clinical outcomes of immunotherapies such as ICIs and TCR-T cell therapy. By investigating the physiological influences of diverse diet-derived nutrients on the immune system, our studies contribute to a comprehensive understanding of how dietary nutrients shape human biology, which is crucially informative for dietary choices that may be beneficial for maintaining human health, reducing disease risk, and improving response to therapies. Our studies on TVA from animal-based diets[213]^5 and ZEA from plant-based foods demonstrate that the scientific and mechanistic understanding of how nutrients from different diets influence human health are still limited. The finding that TVA and ZEA have mechanistically distinct effects on CD8^+ effector T cells and anti-tumor immunity suggests that a balanced diet may provide complementary benefits to human health. TCR signaling plays a vital role in activation and differentiation of CD8^+ T cells, while stronger signaling strength of TCR generally promotes cytotoxic activity.[214]^34 Our findings suggest that ZEA enhances cytotoxicity of CD8^+ T cells through multiple intracellular TCR signaling cascades, which may act in concert to achieve fulfillment of effective cytotoxicity. Our results demonstrate a crucial coordination between the Lck-PLC-Ca^2+-NFAT axis and the NF-κB pathway to mediate the pro-cytotoxic effects of ZEA on CD8^+ T cells. Targeting either of them results in abolishment of ZEA-enhanced CD8^+ T cell function, suggesting that the Lck-PLC-Ca^2+-NFAT axis and the NF-κB pathway are indispensable to each other. In contrast, the ERK and JAK-STAT pathways are likely downstream effectors of TNF that is produced by CD8^+ T cells when TCR signaling is activated upon antigen recognition; thus, treatment with specific inhibitors of ERK or JAK did not abolish ZEA-enhanced CD8^+ T cell function assessed by TNF-α production. These findings also support a crucial role of TCR signaling cascades involving Ca^2+ signaling and the NF-κB pathway in promoting the cytotoxic activity of CD8^+ T cells. Limitations of the study ZEA and LUT as polar carotenoids can insert into plasma membranes and alter the membrane fluidity.[215]^18^,[216]^19 Future studies are warranted to elucidate the potential effects of ZEA on plasma membrane properties that may contribute to the alteration of TCR clustering on the cell surface of CD8^+ T cells, leading to enhanced formation of TCR complex. We noted that, although tumor-infiltrating CD8^+ T cells were the only immune cell population showing an increased proportion among the cell types analyzed following oral ZEA supplementation, we cannot completely rule out the possibility that ZEA may also modulate the function of other immune cells, which could in turn indirectly influence CD8^+ T cell responses. Another limitation of our study is that, although CD4^+ T cell depletion did not affect the anti-tumor efficacy of ZEA in vivo, the differential effects of ZEA on CD8^+ versus CD4^+ T cells remain to be fully elucidated. This discrepancy may arise from differences in their TCR co-receptors—CD8 and CD4—which interact with distinct MHC molecules and influence TCR signaling strength. It will also be crucial to determine the structural basis that distinguishes the differential bioactivities of ZEA and LUT to promote cytotoxicity of CD8^+ T cells, despite their structural similarity. Our studies on several natural homologs of ZEA revealed that the antioxidant properties of conjugated double bonds in their long chains are not sufficient for enhancing cytotoxicity of CD8^+ T cells. The specific structure of ZEA with symmetrical conjugated polyene and two specially arranged six-membered rings at both ends is vital for its bioactivity to promote CD8 T cell activation. Note that D5 (fucoxanthin) has more potent effects than ZEA to enhance TNF-α production by CD8^+ T cells. Fucoxanthin is also a plant-produced carotenoid nutrient found in marine products such as seaweed, which has anti-cancer effects.[217]^35^,[218]^36 Future studies are warranted to further evaluate fucoxanthin as a natural, dietary nutrient for its translational potential to enhance CD8^+ T cell function and related immunity in vivo. Future detailed SAR studies on ZEA and fucoxanthin will provide insights into the design and development of derivatives with improved potency for CD8^+ T cell activation and for optimizing efficacy of T cell-based therapies as a dietary supplement. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jing Chen (jingchen@uchicago.edu). Materials availability All reagents generated in this study are available from the [219]lead contact with a completed materials transfer agreement. Data and code availability * • 16S amplicon sequencing data have been deposited in the NCBI Sequence Reach Archive (SRA) : [220]PRJNA1189274 and are publicly available as of the date of publication. The KAS-seq data have been deposited in the GEO : [221]GSE282686 and are publicly available as of the date of publication. The RNA-seq data have been deposited in the GEO : [222]GSE282703 and are publicly available as of the date of publication. * • This paper does not report original code. * • Any additional information required to reanalyze the data reported in this paper is available from the [223]lead contact upon request. Acknowledgments