Extended Data Figure 5. Restoring glutamine influx enhances mitochondrial function in ascites-exposed CD4+ T cells.
a-b, Immunoblot (a) and densitometric quantification (b) for ASCT2 and SNAT1 protein levels in human CD4+ T cells exposed to OvCa ascites at the indicated concentrations for 16 h. β-ACTIN was used as loading control. Data are shown as the relative expression compared with untreated (0%) controls. n = 4 for 10% ascites; n = 4 for 50% ascites; n = 2 for 100% ascites. Data were generated from two independent experiments. c-d, Human CD4+ T cells were activated via CD3/CD28 stimulation for 16 h in the absence or presence of 25% OvCa ascites supernatants, and DMKG (5 mM) was added to the cell culture during the last 4 h of incubation. OCR profile (c) and quantification of maximal OCR (d). Data are presented as relative expression compared with untreated controls incubated in the absence of ascites (n = 17 total from two independent experiments). e-f, Human CD4+ T cells activated via CD3/CD28 stimulation and IL-2 (50 U/ml) for 36 h were transduced with GFP-expressing retroviruses harboring no insert (control) or the gene encoding human SNAT1. GFP+ cells were sorted 3 days post-transduction and expanded for an additional 48 h in the presence of CD3/CD28 stimulation and IL-2 (50 U/ml). After 20 h of resting, cells were restimulated with CD3/CD28 antibodies in the absence or presence of OvCa ascites supernatants for 16 h. e, Sorting plots showing GFP expression by CD4+ T cells that were left untreated or transduced with either control or SNAT1-expressing viruses. Representative plots from two experiments. f, Relative SLC38A1 expression levels in sorted cells after transduction (n = 6 total from two independent experiments). n values represent biologically independent samples (b, d, f). Data are shown as mean ± s.e.m (b, c, f). One-way ANOVA with Bonferroni’s multiple comparisons test (b); Two-tailed paired Student’s t-test (d); Two-tailed Student’s t-test (f). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.