Extended Data Fig. 1: Reduced glucose uptake and defective N-linked protein glycosylation promotes IRE1α–XBP1 activation in human CD4⁺ T cells exposed to ascites.

a, Human CD4⁺ T cells were activated by CD3 and CD28 stimulation for 16 h in the absence or presence of supernatants from ovarian cancer ascites at the indicated concentrations. XBP1 expression was determined by qPCR (10%, n = 58; 50%, n = 25; 100%, n = 30). Data were normalized to endogenous expression of ACTB in each sample. Results are presented as per cent increase in expression compared with untreated controls. b, Histograms for FACS-based XBP1s staining in CD4⁺ T cells treated as indicated. Tm, tunicamycin; 4μ8C, inhibitor of the IRE1α RNase domain. Data were validated by three independent experiments. c, d, CD4⁺ T cells were treated with vitamin E (VitE, 50 μM) or vehicle (ethanol, 0.1%) for 1 h and then stimulated with anti-CD3 and anti-CD28 beads for 16 h in the presence of ascites. Intracellular reactive oxygen species staining by DCFDA (c) and XBP1 expression (d) (n = 10). Data are expressed as per cent response change compared with untreated controls. e, Glucose concentration in regular culture medium (cRPMI) and in seven independent samples of ovarian cancer ascites. Each dot represents the mean of two measurements. f, XBP1 expression in the samples described in a are displayed, based on the final glucose concentration in the culture after addition of ascites. Three independent ascites samples were used: A10 (triangles), A15 (circles) and A17 (squares) at three different concentrations (10, 50 and 100%). g, GLUT1 surface expression on the indicated cell types present in ovarian cancer ascites from six independent patients was determined by GLUT1.RBD staining (n = 6). Quantitative analysis (left) and representative histograms (right). h, Glucose uptake and XBP1s protein expression in activated CD4⁺ T cells exposed to three different ascites samples at 10 and 100% for 16 h (n = 14). Results are presented as relative to untreated controls. i, CD4⁺ T cells were activated with anti-CD3 and anti-CD28 beads in the presence or absence of ascites, for 16 h. Cells were lysed and the enriched glycoprotein fractions were analysed for N-linked glycosylation events by LC–MS/MS. Total ion chromatograms for N-glycosylation at amino acid 315 in DPP2 are shown. Numbers in blue indicate abundance of each glycan through quantification of the corresponding peak area. Data are representative of two independent experiments with similar results. j, CD4⁺ T cells were treated with 10 mM GlcNAc for 1 h and stimulated by CD3 and CD28 for 16 h in the presence of 10% ascites. Quantitative analyses for XBP1s protein by FACS (left, n = 6) and XBP1 gene expression by qPCR (right, n = 15) are presented as per cent response change compared with untreated controls. k, Relative basal and maximal OCR for CD4⁺ T cells exposed to 10% ascites analysed in Fig. 2g (n = 16 total from five independent experiments). Data are expressed as per cent response change compared with untreated controls. l, m, CD4⁺ T cells were activated by CD3 and CD28 stimulation for 16 h in the absence or presence of tunicamycin (Tm) at the indicated concentrations (n = 3). l, XBP1 expression was determined by qPCR. m, OCR profile of Tm-treated CD4⁺ T cells are shown relative to the untreated control. n, o, Relative quantification of basal (left) and maximal (right) OCR (n), and ECAR measurements (o) in all independent samples analysed in Fig. 2i (n = 9 total from three independent experiments). p, q, Relative quantification of basal (left) and maximal (right) OCR (p), and ECAR measurements (q) for the specimens described in Fig. 2j. (n = 5 total from two independent experiments). Data are presented as relative expression compared with matching controls that were not exposed to ascites. Data are shown as mean ± s.e.m. (a, c, d, f, g, k–m). n values represent biologically independent samples (a, c–h, j–q). One-way ANOVA with Bonferroni’s multiple comparisons test (a, l); two-tailed Student’s t-test (c, k); one-way ANOVA with Tukey’s multiple comparisons test (d, g); two-tailed paired Student’s t-test (j, n–q); nonparametric Spearman’s rank correlation test, Spearman coefficient (r) with P value (two-tailed); 95% confidence interval −0.8811 to −0.1626 (h). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant; MFI, mean fluorescence intensity; gMFI, geometric mean fluorescence intensity.

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