Extended Data Fig. 6: N-acetylglucosamine and IL-7 synergize to raise N-glycan branching in human T cells.
a, b) PBMCs from nine healthy female donors (28-45 years old) were cultured with or without rhIL-7 (50 ng/ml) and/or GlcNAc (10 mM or 40 mM) for 9 days, then analyzed for L-PHA binding by flow cytometry, gating on CD4+ TEM (CD45RA−CD45RO+CCR7−) cells (a) or CD8+ TEM (CD45RA−CD45RO+CCR7−) cells (b). c) Mouse plasma from female mice of the indicated ages was analyzed for HexNAc levels by LC-MS/MS. d, e) Flow cytometric analysis of human PBMCs stimulated by anti-CD3 in the presence or absence of kifunensine as indicated for 24 hours to analyze for activation marker CD69 (d) or 72 hours to assess proliferation by CFSE dilution (e), gating on CD4+ T cells. f) Female human PBMCs were treated in vitro with kifunensine for 24 hours, followed by analysis of L-PHA binding on CD4+ TN cells by flow cytometry. Data shown is representative of three independent experiments with different donors. g) Female human PBMCs were treated in vitro with kifunensine for up to four days, followed by analysis of L-PHA binding on CD4+ TN cells by flow cytometry. P-values by Kruskal–Wallis with Dunn’s multiple comparisons test (a, b), two-tailed Mann–Whitney (c), and one-tailed Mann–Whitney (d, e). Error bars indicate mean ± s.e.m.
