Fig 2: Functional validation of the tumor cell-differentiation status and the effects on cytotoxic immunity(A) IF images of Apc-driven colonic tumor and Braf-driven proximal colon villiform metaplasia (white arrows).(B) Quantification of CD8-positive pixels from IF. Red line denotes the mean level detected in adjacent normal colon in Braf mice. Error bars represent SEM from n = 3 animals per group.(C) IF images of CD8+ T cells in tumor, villiform metaplasia (white arrows), and control colon. Dotted line demarcates border between villus and crypt compartments.(D and E) H&E (D) and ß-catenin IHC (E) of colonic tissues and tumors of tamoxifen-induced Lrig1 or Mist1 tumor mice 28 days after DSS.(F–H) UMAP of epithelial scRNA-seq data generated from mouse colonic tissues and tumors, with overlays indicating (F) cell type, (G) gene overlays, and (H) biological replicates.(I) Heatmap of genes defining human metaplastic and cell signatures in specified epithelial populations from mouse scRNA-seq.(J and K) Combined UMAP of immune cell scRNA-seq data from mouse colonic tissues and tumors, with overlays indicating (J) conditions and (K) cell type.(L and M) Quantification of (L) general immune cell types and (M) specific lymphocyte populations from Lrig1 (left) and Mist1 (right) scRNA-seq data.(N) UMAP overlays of genes related to immunosuppression or cytotoxicity in myeloid and lymphoid cells.(O) MxIF images of T cells in tumors.(P) Image quantification of T cells. Each dot represents a field of view. Error bars represent SEM from n = 3 animals per group.(Q) CytoTRACE score for TSCs from scRNA-seq.(R) Organoid formation efficiency of single cells isolated from tumors and control colons. Each dot represents data from a well with representative images shown in insets. Error bars represent SEM from n = 4 animals per tumor, 2 for control.(S) SCI, II, and III metagene signatures for TSCs from scRNA-seq.(T) Heatmap of individual antigen-presentation genes at single-cell level.(U) MHCII metagene signature expression for TSCs.(V) Quantification of DQ-OVA+/I-AI-E+ epithelial tumoroid cells from flow plots. Error bars represent SEM from n = 6 animals per condition.(W) Percentage of proliferating T cells determined by CellTrace Violet assay when co-cultured with organoids derived from colonic tumors or normal tissues (+DSS) +/— 50 mg/mL OVA peptide. Error bars represent SEM of organoids from n = 5 mice for tumors and two for normal.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S7 and Table S5.

Fig 3: 2-KG restores Got1-deficient CD8+ T cell antiviral responses in vivo.a, Experimental design. b–f, Contour plots, histograms and bar graphs show the flow cytometry staining results of Ly5.1+ donor P14 CD8+ T cells (b), PD-1 and TIGIT (c), cytokines (d), Ki-67 (e), and cleaved caspase-3 and Bim (f) in Got1-deficient and sufficient P14 CD8+ T cells. Cells were stimulated with GP33–41 peptides before flow cytometry staining (d). g,h, Bar graphs show the viral titers at day 8 (g) and day 30 (h) after infections. Data were pooled from two independent experiments (b–h). n = 10 C57BL/6 mice per group. The results are presented as mean ± s.d. **P < 0.01; ***P < 0.001; ****P < 0.0001. Comparisons were performed with one-way ANOVA. In b, P = 1.6 × 10−9, P = 3.2 × 10−11, P = 1.3 × 10−7 and P = 5.8 × 10−7; in c, both P = 1.1 × 10−14; in d, P = 4.5 × 10−14 and P = 1.1 × 10−11; in e, P = 2.5 × 10−6 and P = 0.00015972; in f, P = 1.7 × 10−14, P = 1.9 × 10−14, P = 3.7 × 10−9 and P = 4.0 × 10−9; in g, P = 0.937 and P = 0.477; in h, P = 0.002629596 and P = 0.000658958. Six-week-old female mice were used (b–h).Source data

Fig 4: Antiviral CD8+ T cell responses require GOT1 during chronic infection.a, Illustration of the experimental design. b, Fluorescence-activated cell sorting (FACS) gating strategies used in c–l. c–l, Contour plots, histograms and bar graphs show the flow cytometry staining results of Ly5.1+ donor P14 CD8+ T cells (c,d), PD-1 and TIGIT (e,f), cytokines (g,h), Ki-67 (i,j) and cleaved caspase-3 and Bim (k,l) in Got1-deficient and sufficient P14 CD8+ T cells. Cells were stimulated with GP33–41 peptides before the flow cytometry staining (g–h). Data were pooled from two independent experiments (c–l) with ten C57BL/6 mice in each group receiving Got1-deficient and sufficient donor P14 CD8+ T cells. The results are presented as mean ± s.d. **P < 0.01; ***P < 0.001; ****P < 0.0001. Comparisons were performed with the two-tailed Mann–Whitney test (percentage of Ly5.1+ donor cells in d and i, cleaved caspase-3 MFI in k and Bim MFI in l; data points were not normally distributed) or a two-tailed Student’s t-test (other comparisons). In c, P = 1.14 × 10−10 (left) and P = 9.48 × 10−12 (right); in d, P = 1.08 × 10−5 (left) and P = 9.08 × 10−7 (right); in e, P = 0.0048; in f, P = 1.0 × 10−15; in g, P = 2.47 × 10−5; in h, P = 6.99 × 10−6; in i, P = 2.17 × 10−5; in j, P = 0.0003; in k, P = 1.08 × 10−5 (left) and P = 8.84 × 10−11 (right); in l, P = 8.70 × 10−9 (left) and P = 1.08 × 10−5 (right). Six-week-old female mice were used (b–l).Source data

Fig 5: Tex cells require GOT1 to produce 2-KG by an ‘ammonia-neutral pathway’.a, Electron flow through mitochondrial complexes. b–d, WT donor P14 CD8+ T cells were isolated from C57BL/6 host mice that were infected with LCMV clone 13 or LCMV Armstrong 30 d earlier. Tex or Tmem donor P14 CD8+ cells were FACS-purified and permeabilized with PMP. Mitochondrial complex inhibitors (rotenone and antimycin A inhibit complexes I and III, respectively) and substrates (succinate, a substrate for complex II; Asc and TMPD, substrates for complex IV) were supplemented sequentially into the T cell culture as indicated. Line graphs show the OCR values of PMP-permeabilized Tex cell versus Tmem cells (b), Tmem cell versus Tmem cells treated with antimycin A and azide (c), or Tmem cell versus Tmem cells treated with NH4OH (d) in Seahorse electron flow assays. e, Illustration of the incorporation of the tracer 13C from 13C-malate into 13C-aspartate. f, Bar graphs show amounts of 13C-aspartate in Tmem cells and Tex cells. ‘m + 4’ indicates aspartate with four 13C atoms. g, Illustration of the incorporation of the tracer 13C from 13C-glutamine into 13C-glutamate or 13C-2-KG. h,i, Bar graphs show amounts of 13C-glutamate (h) or 13C-2-KG (i) in Tmem cells and Tex cells. ‘m + 5’ indicates glutamate (h) or 2-KG (i) with five 13C atoms. Data are combined from two experiments with six mice (b–d), 15 host mice containing Got1-sufficient donor P14 CD8+ T cells, and 30 host mice containing Got1-deficient donor P14 CD8+ T cells (f,h,i). Data are mean ± s.d. *P < 0.05; ****P < 0.0001. Comparisons were performed with two-way ANOVA (f,h,i). In f, P = 2.0 × 10−15; in i, P = 0.066993749, P < 1.0 × 10−15, P = 1.0 × 10−14, P = 6.7 × 10−7, P = 0.025248392 and P = 2.2 × 10−8. Six-week-old female mice were used (b–d,f,h,i).Source data