Figure 2. OvCa ascites limits glucose uptake and causes IRE1α/XBP-mediated mitochondrial dysfunction in human CD4+ T cells.
a-f, T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of OvCa ascites supernatants at the indicated concentrations. Histograms (a) and quantification (b) of XBP1s staining (n = 16); Iso, isotype control. c, SLC2A1 expression was determined via qRT-PCR (n = 48). Immunoblot and quantification (d) of GLUT1 in ascites-exposed CD4+ T cells. Density of GLUT1 was normalized to β-ACTIN, and data are shown as the relative expression compared with the untreated control (n = 4 for 10% and 50% ascites; n = 2 for 100% ascites, all from two independent experiments). e, Glucose uptake was assessed using 2-NBDG and XBP1 was determined in the same sample. Symbols depict ascites from 3 independent patients tested at increasing concentrations on CD4+ T cells from multiple donors (n = 37). Baseline ECAR (f) and OCR profile (g) of CD4+ T cells exposed to ascites (n = 16). CD4+ T cells were treated with 4μ8C (h, i) or GlcNAc (j) for 1 h and then stimulated via CD3/CD28 for 16 h in the presence of 10% ascites. h, XBP1s determined by FACS (n = 7). i, OCR profile in 4μ8C-treated T cells exposed to ascites (n = 9). j, OCR for GlcNAc-treated T cells exposed to ascites (n = 5). Data are shown as mean ± s.e.m (b, c, d, f, g, i, j). n-values represent biologically independent samples (b-k). One-way ANOVA with Tukey’s post-test (b); Two-tailed Student’s t-test (c, f); One-way ANOVA with Bonferroni’s post-test (d). Spearman’s rank correlation test, 95% CI −0.9076 to −0.6760 (e); Two-tailed paired Student’s t-test (h); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MFI, Mean fluorescence intensity.