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Coordination of asparagine uptake and asparagine synthetase expression modulates CD8+ T cell activation
Helen Carrasco Hope, … , Mihaela Lorger, Robert J. Salmond
Helen Carrasco Hope, … , Mihaela Lorger, Robert J. Salmond
Published April 6, 2021
Citation Information: JCI Insight. 2021;6(9):e137761. https://doi.org/10.1172/jci.insight.137761.
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Research Article Immunology

Coordination of asparagine uptake and asparagine synthetase expression modulates CD8+ T cell activation

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Abstract

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses.

Authors

Helen Carrasco Hope, Rebecca J. Brownlie, Christopher M. Fife, Lynette Steele, Mihaela Lorger, Robert J. Salmond

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Figure 1

Extracellular asparagine is required to maintain viability and initiate cell growth following T cell receptor stimulation.

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Extracellular asparagine is required to maintain viability and initiate ...
OT-1 T cell receptor (TCR) transgenic T cells were stimulated with cognate SIINFEKL peptide for 24 hours in DMEM supplemented with or without asparagine and Gln, as indicated. (A) T cell viability was assessed by exclusion of Live/Dead Aqua dyes and FACS. (B) T cell growth was assessed by analysis of forward scatter and side scatter area (FSC-A/SSC-A) parameters on gated live cells by flow cytometry. (C) Nascent protein synthesis was assessed by incorporation of OPP, intracellular staining, and labeling using Click chemistry reagents and FACS analysis. Cycloheximide (CHX) was used as a negative control. (D) O-propargyl-puromycin (OPP) mean fluorescence intensity (MFI) was assessed by FACS analysis. Data are from 1 of 3 experiments, and individual data points (n = 3) represent technical replicates. *P < 0.05, ****P < 0.0001, as assessed by 1-way ANOVA, with Tukey’s multiple comparisons test.

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