Extended Data Fig. 1. Asn restriction suppresses CD8+ T cell activation but enhances inflammation and proliferation.
a-b) CD8+ T cells were activated in a medium with or without Asn and were collected in the early phase (24 hr) and late phase (72 hr). The protein content (a) (n = 3 experimental replicates, P < 0.0001 and P = 0.9368 at 24 and 72 h respectively) and cell viability (b) (n = 5 experimental replicates, (P = 0.003 and P = 0.7328 at 24 and 72 h respectively) was determined by 7AAD staining by flow cytometry. Data are representative of 3 independent experiments. c) Cell cycle, TNF-α, inflammatory response, and effector T cell function associated gene sets were analyzed by GSEA. d-f) Indicated cytokine was quantified by intracellular staining by flow cytometry as indicated condition (n = 4 experimental replicates, Pmel T cells: P = 0.007 and P = 0.002 for IFN-γ and TNF-α respectively; GD2 CAR-T: P = 0.0013 and P = 0.01 for IFN-γ and TNF-α respectively; Pmel T cells: P = 0.01 and P = 0.19 for no IL-2 and IL-2 respectively). g) Schematic diagram of the experiment. h) cell number (n = 4 experimental replicates, P = 0.01, P = 0.01 and P = 0.9 for group 1 vs. 2, 1 vs. 3 and 1 vs. 4 respectively). i) The indicated cytokines were quantified by flow cytometry (n = 3 experimental replicates, IFN-γ: P = 0.0009, P = 0.0006, and P = 0.3992; TNF-α: P = 0.0032, P = 0.004, and P = 0.668 for group 1 vs. 2, 1 vs. 3 and 1 vs. 4 respectively). j) The cytolysis was determined by eSight (n = 3 experimental replicates, P < 0.0001, P < 0.0001 and P = 0.675 in 1 vs. 2, 1 vs. 3 and 1 vs. 4 respectively). Data from d-j are representative of 3 independent experiments. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, *** P < 0.001, Statistical differences were determined by unpaired Two-tail Student’s t-test (a, b, d-f and h-i) and Two-way ANOVA (j). k) Conceptual model showing that Asn restriction suppressed T cell activation but conferred robust proliferation and effector function to CD8+ Teff cells at the late time point.