Extended Data Fig. 9: Asn binds to the LCK kinase domain and enhances CD8+ T cell anti-toumor activity.
From: Asparagine enhances LCK signalling to potentiate CD8+ T-cell activation and anti-tumour responses
a and b, BIAcore measurement of the interaction between Asn and purified region AA220-390 (a) or AA320-509 (b). Graphs of equilibrium response units (RU) and compound concentrations are shown. c, Mapping the Asn binding site on LCK. The region responsible for Asn binding and results of Asn binding analysis are shown. d, OVA-specific OT-I CD8+ T cells pre-activated in Asn/Asp-free (Ctrl) or Asn medium were cultured with B16-OVA cells for 18 h. B16-OVA cell apoptosis was analysed (n = 3 independent wells). e-p, C57BL/6 mice maintained on a normal (Ctrl) or Asn diet (e-j), or on a normal diet yet subcutaneously injected with PBS (Ctrl) or ASNase (0.2 mg/mouse) were subcutaneously injected with B16-F10 cells near the inguinal lymph node for 12 days. Percentage of CD44+CD8+ T cells (e, k), effector (f, l) and central memory-like (g, m), naive (h, n) CD8+ T cells, and production of cytokines (i, j, o, p) in lymph nodes were measured (n = 7 or 8 mice per group). q, Related to Fig. 7b and c. Schematic view of the B16-OVA and activated OT-I CD8+ T cell adoptive transfer experiment. r, Naive OT-I CD8+ T cells primed in vitro in Asn or Asn/Asp-free medium (Ctrl) were injected into C57BL/6 mice subcutaneously injected (sc) with B16-OVA cells as indicated. Tumour burden were analysed over time. Data are representative of three independent experiments (n = 5 mice per group). s, Schematic of the experimental approach in Fig. 7h–j. All data are mean ± SEM. In d-p, two-tailed Student’s t-test, and in r, two-way ANOVA followed by Sidak’s multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Related representative FACS plots (Supplementary Fig. 18) and numerical source data for a, b, d-p, r, are provided.
