Coordination of asparagine uptake and asparagine synthetase expression modulates CD8+ T cell activation
Helen Carrasco Hope, Rebecca J. Brownlie, Christopher M. Fife, Lynette Steele, Mihaela Lorger, Robert J. Salmond
Helen Carrasco Hope, Rebecca J. Brownlie, Christopher M. Fife, Lynette Steele, Mihaela Lorger, Robert J. Salmond
View: Text | PDF
Research Article Immunology

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

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

×

Figure 4

T cells lose requirement for extracellular asparagine upon prolonged activation.

Options: View larger image (or click on image) Download as PowerPoint
T cells lose requirement for extracellular asparagine upon prolonged act...
OT-1 T cells were stimulated with cognate SIINFEKL peptide for 72 hours (A–G) or were differentiated for 6 days (H and I) in DMEM supplemented with or without asparagine and/or Gln or complete IMDM (J and K), as indicated. T cell viability (A) and proportions of blasting cell populations (B) were assessed by exclusion of Live/Dead Aqua dyes and analysis of forward scatter and side scatter area (FSC-A/SSC-A) parameters by flow cytometry, respectively. For analysis of TCR-induced proliferation, cells were labeled with Cell Trace Violet (CTV) prior to stimulation. Histograms are representative of data from 2 repeated experiments (C). Division indices were calculated using FlowJo software (D). Proportions of live-gated T cells expressing CD25 (E), CD71 (F), or granzyme B (G and H) were determined by flow cytometry. (I and J) Effector cytotoxic T lymphocytes (CTLs) were differentiated for 6 days in DMEM with or without asparagine/Gln, then restimulated in complete IMDM for 4 hours. Proportion of IFN-γ+ and TNF+ cells were determined by intracellular staining and FACS analysis. (K) Effector CTLS were differentiated in complete IMDM for 5 days, before a 24-hour washout in DMEM with or without asparagine, then were restimulated in DMEM with or without asparagine for 24 hours. IFN-γ levels in supernatants were determined by ELISA. Data are representative of 2 (C and D) or 3 repeated experiments. In all graphs, individual data points represent technical replicates (n = 3). **P < 0.01, ****P < 0.0001, as determined by 1-way ANOVA with Tukey’s multiple comparisons test (A, B, D–F, and H) or 2-way ANOVA with Sidak’s multiple comparisons test (I–K).