Extended Data Fig. 3: Effect of extracellular and de novo Asn on T activation.
From: Asparagine enhances LCK signalling to potentiate CD8+ T-cell activation and anti-tumour responses
a, mRNA expression in naive CD8+ T cells. b and c, Mouse naive CD8+ T cells were left untreated (naive) or activated for different time points. ASNS expression (b) and Asn levels (c) were measured. d, Surface expression of CD25 and CD44 on pre-activated mouse naive CD8+ T cells cultured in Asn/Asp-free (Ctrl) or Asn medium. e-h, Mouse naive CD8+ T cells were activated as indicated. Intracellular (e) and extracellular (f) Asn levels, surface expression of CD25 and CD44 (g), and cytokine production (h) were measured. i and j, Survival (i) and apoptosis (j) of mouse naive CD8+ T cells activated in Asn/Asp-free (Ctrl) or Asn medium containing exogenous IL-2. k, Annexin V staining of naive CD8+ T cells activated in Asn/Asp-free (Ctrl) or Asn medium without exogenous IL-2. l-n, Naive CD8+ T cells pre-activated in 1640 IL-2-supplemented medium were cultured in IL-2-free medium without (Ctrl) or with Asn. Living cells (l), apoptotic (m) and proliferating (n) T cells were assayed at day 3 post IL-2 withdrawal. o and p, Mouse naive CD8+ T cells activated in ASNase-supplied or -free conditioned medium from H1299 cells or Asn/Asp-free medium (Ctrl) were analysed for activation and Asn levels. q, Asn levels in medium cultured from Gln-fed or starved H1299 and EG-7 cells. r, Mouse naive CD8+ T cells were stimulated in medium cultured from Gln-fed or starved EG-7 cells in the presence of 2 mM Gln. CM, conditioned medium. Data are mean ± SEM, n = 3 independent samples per group/condition. Two-tailed Student’s t-test or a two-way ANOVA followed by Sidak’s multiple comparisons (i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Related representative FACS plots (Supplementary Fig. 12), uncropped blots for b, and numerical source data for a-m, o-r, are provided.
