MAIT cells exhibit a distinct effector-memory phenotype and intrinsic cytotoxic program compared to conventional T cells.

a. Schematics showing antigen recognition by MAIT, CD8⁺ T, and CD4⁺ T cells. b. Tissue distribution of MAIT cells in healthy humans, summarized from the published database. c. FACS detection of MAIT (CD3+MR1-Tetramer⁺) and Tc (CD3+MR1-Tetramer-) cells, as well as their CD4⁺ vs CD8⁺ subsets. d. FACS analysis of effector/memory phenotype based on CD45RO and CD62L expression. Tem: effector memory T cells; Tcm, central memory T cells; Tnaive: naïve T cells; Te: effector T cells. e-h. Comparison of human MAIT and conventional CD4+ and CD8+ T cells isolated from healthy donor peripheral blood using scRNA-seq analysis. e. Schematic showing the gene profiling of T cells in in healthy donor PBMCs. f. Combined UMAP plot showing the identification of MAIT cells. Each dot represents a single cell and is colored according to its cell cluster assignment. g. Proportion of MAIT cell subset among total T cells. h-i. scRNA-seq analysis of the expression of the indicated gene signatures (h) and genes (i) in MAIT cells. j. FACS detection of surface markers, intracellular cytokines, cytotoxic molecules and transcription factors in MAIT cells. CD4+ T and CD8+ T cells were included as controls. k. Quantification of j. Representative of 1 (e-i) and 3 (c-d, j-k) experiments. Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (k). Created with BioRender.com.

Microbial metabolites selectively mobilize and expand human MAIT cells across healthy donors and cancer patients.

a. Schematic for riboflavin-derived microbial metabolite 5-OP-RU stimulation of MAIT cells in PBMCs from healthy donors or cancer patients. b. FACS plots showing MR1/5-OP-RU tetramer⁺ TCR Vα7.2⁺ MAIT cells at baseline (D0), day 7 without 5-OP-RU, and day 7 with 5-OP-RU stimulation. c. CD4 and CD8 subset distribution of MAIT cells at D0 and D7 with 5-OP-RU stimulation. d. Quantification of MAIT cell proportions in PBMCs from healthy donors (n = 6), liver cancer patients (n = 5), and ovarian cancer patients (n = 3) at D0 and D7 ± 5-OP-RU stimulation. e. Fold enrichment of MAIT cells after 5-OP-RU stimulation in healthy donors (n = 6), liver cancer patients (n = 5), and ovarian cancer patients (n = 3). f. Schematic for riboflavin-derived microbial metabolite 5-OE-RU stimulation of MAIT cells in PBMCs from healthy donors or cancer patients. g. FACS plots showing MR1/5-OP-RU tetramer⁺ TCR Vα7.2⁺ MAIT cells at baseline (D0), day 7 without 5-OE-RU, and day 7 with 5-OE-RU stimulation. h. CD4 and CD8 subset distribution of MAIT cells at D0 and D7 with 5-OE-RU stimulation. i. Quantification of MAIT cell proportions in PBMCs from healthy donors (n = 6), liver cancer patients (n = 5), and ovarian cancer patients (n = 3) at D0 and D7 ± 5-OE-RU stimulation. j. Fold enrichment of MAIT cells after 5-OE-RU stimulation in healthy donors (n = 6), liver cancer patients (n = 5), and ovarian cancer patients (n = 3). Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant, **p < 0.01, ****p < 0.0001 by one-way ANOVA (d, i). See also Supplementary Fig. 1. Created with BioRender.com.

Microbial metabolite-driven MR1 signaling enables broad MAIT cell cytotoxicity across solid tumors.

a. Schematics illustrating solid cancer types utilized in this study. b-d. Studying microbial metabolite-enhanced MAIT cell antitumor activity using human solid tumor cell lines. Tumor cells were cocultured with MAIT cells in the presence or absence of 5-OP-RU. T cells were included as a therapeutic cell control. b. FACS detection of MR1 expression on the indicated tumor cells. c. Experimental design. d. Tumor cell killing data at 24 h (n = 4). e-f. Studying microbial metabolites-enhanced MAIT cell antitumor activity using primary liver cancer patient samples. Tumor cells were cocultured with MAIT cells in the presence or absence of 5-OP-RU. e. Experimental design. f. Primary liver tumor cell killing data at 24 h (n = 4). g-h. Studying the tumor cell killing mechanisms of MAIT cells mediated by MR1 and microbial metabolite, 5-OP-RU. g. Experimental design. h. Tumor cell killing data at 24 h (E:T ratio = 5:1; n = 4). Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (f, h). See also Supplementary Fig. 2 and 3. Created with BioRender.com.

Microbial metabolites induce MAIT cytotoxic and inflammatory effector responses.

a. Schematics illustrating the mechanism of MAIT cell–mediated tumor killing in cancer patients. b-e. Studying the expression of effector molecules in MAIT cells cocultured with liver cancer cells and microbial metabolite. Tumor cells were cocultured with MAIT cells in the presence or absence of 5-OP-RU. b. Experimental design. c. FACS detection of surface CD69 and CD25 expression as well as intracellular Granzyme B production of MAIT cells under the indicated conditions. d. Quantification of c (n = 4). e. Luminex analyses of cytokine and chemokine production by MAIT cells under the indicated conditions (n = 3). Representative of 1 (e) and 3 (b-d) experiments. Data are presented as the mean ± SEM. ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (d and e). See also Supplementary Fig. 4. Created with BioRender.com.

Pharmacologic MR1 activation enables MAIT cell–mediated tumor control in vivo.

a-e. Studying the in vivo antitumor efficacy of MAIT cells in an orthotopic liver cancer xenograft NSG mouse model. 5-OP-RU was administrated to experimental mice weekly. a. Experimental design. BLI, live animal bioluminescence imaging; i.p., intraperitoneal injection. b. BLI images measuring tumor loads in experimental mice over time. c. Quantification of b (n = 5). d. Kaplan–Meier survival curves (n = 5). e. Studying the in vivo functionality of MAIT cells in the presence or absence of 5-OP-RU. FACS analyses of T cell activation marker CD69 and CD25 at Day 14 (n = 5). f-h. Studying the in vivo antitumor efficacy of MAIT cells in a subcutaneous liver cancer xenograft NSG mouse model. 5-OP-RU was administrated to experimental mice semi-weekly. f. Experimental design. s.c., subcutaneous injection. g. Measurement of tumor weight on day 34 (n = 5). h. Measurements of tumor size over time (n = 5). Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test (e), by one-way ANOVA (c, g), or by log rank (Mantel-Cox) test adjusted for multiple comparisons (d). See also Supplementary Fig. 5.

Microbial metabolite signaling drives cytotoxic state conversion in MAIT cells.

a. Schematic showing the experimental design to study the gene profiling of MAIT cells using scRNA-seq. MAIT cells were cultured alone (MAIT), cocultured with tumor cells (MAIT+Tumor) or cocultured with tumor cells in the presence of 5-OP-RU (MAIT+Tumor+5-OP-RU). Cells were subsequently isolated by FACS and subjected to scRNA-seq analysis. b. Combined UMAP plot showing the formation of four major MAIT cell clusters: (1) Cytotoxic MAIT cells, (2) Proliferating MAIT cells, (3) Effector/memory MAIT cells, and (4) Resting MAIT cells. Each dot represents a single cell and is colored according to its cell cluster assignment. c. Individual UMAP plots showing cell cluster composition of the indicated samples, and and pie charts showing cell cluster proportions of the indicated samples. Each dot represents a single cell and is colored according to its cell cluster assignment. d. Violin plots showing the expression distribution of cytotoxic, proliferating and exhausted gene signatures in the indicated samples. e. Violin plots showing the expression levels of the metabolic pathways (glycolysis, cholesterol synthesis, and fatty acid metabolism) in the indicated samples. f. Violin plots showing the expression levels of representative pro-inflammatory genes in the indicated samples. g. Venn diagram illustrating the numbers of shared and unique differentially expressed genes (DEGs) of MAIT+Tumor and MAIT+Tumor+5-OP-RU samples relative to non–tumor-challenged MAIT cells. The pathway analyses were conducted using the shared and unique DEGs from each comparison. The indicated pathway in each category are shown in the bar plots. The experiment was performed once; cells collected from three repeated experiments were combined for analyses. In the violin plots (d, e, and f), box and whisker plots exhibit the minimum, lower quartile, median, upper quartile and maximum expression levels of each sample. See also Supplementary Fig. 6. Created with BioRender.com.

Microbial metabolite enables MAIT-mediated elimination of immunosuppressive myeloid cells.

a-b. Studying microbial metabolite-mediated TME targeting by MAIT cells using primary liver cancer patient samples. Primary samples were cocultured with MAIT cells in the presence or absence of 5-OP-RU. T cells were included as a therapeutic cell control. Donor-matched patient T and B cells were included as target cell controls. a. Experimental design. b. TAM and MDSC killing data by MAIT cells at 24 h (n = 4). c-d. In vitro generation and polarization of human monocyte-derived M2 macrophages from healthy donor PBMCs. c. Experimental design. M-CSF, macrophage colony-stimulating factor; MDM, monocyte-derived macrophage; Mφ, macrophage. d. FACS analyses of MR1 on M2-polarized macrophages. Donor-matched T and B cells were included as controls. e-g. Studying microbial metabolite-mediated macrophage targeting by MAIT cells using an in vitro mixed macrophage/MAIT cell (Mφ/MAIT) reaction assay. M2-polarized macrophages were cocultured with MAIT cells in the presence or absence of 5-OP-RU. T cells were included as a therapeutic cell control. Anti-MR1 antibody was added into the coculture to block MAIT TCR recognition. e. Experimental design. f. M2-polarized macrophage killing data by MAIT cells at 24 h (n = 4). g. FACS detection of T cell activation marker CD69 on MAIT cells at 24 h (n = 4). h-j. Studying microbial metabolite-mediated TAM targeting by MAIT cells using an ex vivo 3D TME mimicry culture. M2-polarized macrophages and liver cancer cells were cocultured with MAIT cells in the presence or absence of 5-OP-RU. T cells were included as a therapeutic cell control. h. Experimental design. i. Tumor cell killing data by MAIT cells at 24 h (n = 4). j. M2-polarized macrophage killing data by MAIT cells at 24 h (n = 4). k-m. Studying microbial metabolite-mediated TME targeting by MAIT cells using humanized NSG-SGM3 myeloid cell–bearing xenograft model. k. Experimental design. l. FACS plots showing GFP⁺ liver tumor cells, human CD14+ myeloid cells, and MAIT cells in mouse peritoneal cavity collected from the indicated groups at day 11. m. Quantification of l (n = 5). Representative of 2 (k-n) and 3 (a-j) experiments. Data are presented as the mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (b, d, f, g, i, j, m). Note the comparison between MAIT and MAIT + 5-OP-RU in m is analyzed by Student’s t test. See also Supplementary Fig. 7 and 8. Created with BioRender.com.