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Asparagine enhances LCK signalling to potentiate CD8+ T-cell activation and anti-tumour responses

Nature Cell Biology volume 23, pages 75–86 (2021)Cite this article

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Abstract

Nutrient availability is central for T-cell functions and immune responses. Here we report that CD8+ T-cell activation and anti-tumour responses are strongly potentiated by the non-essential amino acid Asn. Increased Asn levels enhance CD8+ T-cell activation and effector functions against tumour cells in vitro and in vivo. Conversely, restriction of dietary Asn, ASNase administration or inhibition of the Asn transporter SLC1A5 impairs the activity and responses of CD8+ T cells. Mechanistically, Asn does not directly alter cellular metabolic fluxes; it instead binds the SRC-family protein tyrosine kinase LCK and orchestrates LCK phosphorylation at Tyr 394 and 505, thereby leading to enhanced LCK activity and T-cell-receptor signalling. Thus, our findings reveal a critical and metabolism-independent role for Asn in the direct modulation of the adaptive immune response by controlling T-cell activation and efficacy, and further uncover that LCK is a natural Asn sensor signalling Asn sufficiency to T-cell functions.

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Fig. 1: Identification of Asn as a promotor of CD8+ T-cell activation.
Fig. 2: Asn enhances CD8+ T-cell activity and responses in vivo.
Fig. 3: CD8+ T-cell activation by Asn relies on its uptake through SLC1A5 and not its amino-acid exchange function.
Fig. 4: Asn promotes LCK activation.
Fig. 5: Asn promotes T-cell activation through LCK.
Fig. 6: Asn binds directly to LCK and modulates its activity.
Fig. 7: Asn improves CD8+ T-cell anti-tumour activity.

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Data availability

All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank X. Lin, L. Yu, X. Guo, L. Zhang, Y. Wang and X. Hu at Tsinghua University; H. Wu at Peking University; W. Du at Peking Union Medical College and C. Xu at Shanghai Institute of Biochemistry for materials and/or technical assistance. We thank H. Wang and W. Wu at Tsinghua University for their great support of this work during the COVID-19 pandemic. We thank all of the members of the Jiang laboratory for their technical assistance and/or discussions. We thank X. Liu, L. Xu, X. Wang and W. Wang for their help with the LC–MS/MS experiments. This research was supported by the National Key R&D Program of China (grant no. 2019YFA0801701), Tsinghua-Peking Center for Life Sciences and National Natural Science Foundation of China (grant nos 81930082 and 81722035) to P.J.

Author information

Author notes

  1. These authors contributed equally: Jun Wu, Gen Li.

Authors and Affiliations

  1. Tsinghua-Peking Center for Life Sciences, Beijing, China

    Jun Wu, Gen Li, Le Li & Peng Jiang

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Jun Wu, Gen Li, Le Li & Peng Jiang

  3. School of Medicine and Institute for Immunology, Tsinghua University, Beijing, China

    Dan Li & Zhongjun Dong

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  1. Jun Wu
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Contributions

J.W., G.L. and P.J. designed the experiments. J.W. and G.L. performed all of the experiments. L.L. and D.L. provided technical assistance. Z.D. provided constructive comments and supplied reagents. P.J. supervised the research and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Peng Jiang.

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Extended data

Extended Data Fig. 1 In vitro activation of CD8+ T cells by Asn.

a, 15N-Asp levels outside or within mouse naive CD8+ T cells activated in medium containing 4 mM 15N-Asp for 40 h. b-d, Surface expression of CD25 (b), CD44 (c), or CD69 (d) on CD8+ T cells stimulated in the presence of increasing amounts of Asn. e-g, Percentage of CD25 (e), CD44 (f), or CD69 (g) expression on CD8+ T cells stimulated with CD28 antibody (1 μg/ml) and increasing amounts of anti-CD3 antibody in the presence or absence of Asn. h, CFSE staining of mouse naive CD8+ T cells activated in Asn/Asp-free medium (Ctrl) or medium containing Asn for indicated time points. i and j, Proliferation of mouse CD8+ T cells treated as in (b) was determined by Ki67 staining (i), or BrdU incorporation assay (j). mFI, mean fluorescence intensity. k and l, Surface expression of CD25, CD44 and CD69 on mouse OT-I CD8+ T cells activated by OVA peptide in Asn/Asp-free medium (Ctrl) or Asn medium (k). Production of cytokines was also determined (l). m and n, Surface expression of CD69 (m) and CD44 (n) on mouse naive CD8+ T cells activated in Asn/Asp-free (Ctrl) or Asn medium containing 10% dialysed FBS. o-q, Surface expression of indicated proteins on mouse naive CD8+ T cells activated in Asn/Asp-free (Ctrl) or Asn medium containing no FBS. r and s, Representative flow cytometry plots of FSC-A versus TNF-α (r), or IFN-γ (s) staining for human CD8+ T cells treated as in Fig. 1k. Data are mean ± SEM, two-tailed Student’s t-test. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. All experiments were done at least 3 times per group or condition. Related representative FACS plots (Supplementary Figs. 9, 10) and numerical source data for a-g, i, k-p are provided.

Source data

Extended Data Fig. 2 Effect of ASNase, Glu and Asn on CD8+ T cell responses.

a-d, Mouse CD8+ T cells cultured in RPMI 1640 medium, or medium containing 10 µg/ml ASNase were left untreated (naive) or stimulated with anti-CD3/CD28 antibodies for 4 h or 12 h. Relative levels of Asn, Asp, Gln and Glu within and outside cells were measured by LC-MS/MS. e and f, Mouse naive CD8+ T cells stimulated with anti-CD3/CD28 antibodies in control Glu-free medium (Ctrl) or medium containing Glu in the presence of dialysed FBS. Percentage of CD44+CD8+ T cells (e), CD62LlowCD44high CD8+ T cells and CD62LhighCD44high CD8+ T cells (f) relative to total CD8+ T cells were measured. g-j, Jurkat cells were stimulated with anti-CD3/CD28 antibodies in the absence or presence of L-Alb, PP2, or increasing amounts of Asn for 24 h (for detecting CD69 expression) or 40 h (for detecting CD25 expression). Surface expression of CD69 (c) or CD25 (d) was measured. Production of TNF-α (e), and IFN-γ (f) by the activated Jurkat cells was determined by FACS analysis. All data are mean ± SEM, n = 3 independent wells. Two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Representative FACS plots for e-j (Supplementary Fig. 11) and numerical source data for a-j are provided.

Source data

Extended Data Fig. 3 Effect of extracellular and de novo Asn on T activation.

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.

Source data

Extended Data Fig. 4 Increasing Asn promotes T cell activity in vivo.

a-j, C57BL/6 mice intraperitoneally injected with Asn or PBS (Ctrl) every two days for a period lasting one month. Spleens were excised, weighted and pictured (a). Relative numbers of cells in spleens and lymph nodes (b), weight of mice (c), Percentage of CD4+ T cells and CD8+ T cells relative to total cells in spleens and lymph nodes (d, e), effector (e) and central memory-like (f) CD8+ T cells relative to total CD8+ T cells in spleens and lymph nodes, CD25+CD4+ T cells (h), and effector (i) and central memory-like (j) CD4+ T cells relative to total CD4+ T cells in spleens and lymph nodes were assessed. k-r, C57BL/6 mice intraperitoneally injected with ASNase or PBS (Ctrl) every two days for a period lasting one month. Spleens were excised, weighted and pictured (k). Relative numbers of cells in spleens and lymph nodes (l), percentage of CD4+ T cells and CD8+ T cells relative to total cells in spleens and lymph nodes (m), effector memory-like (n) and central memory-like (o) CD8+ T cells relative to total CD8+ T cells in spleens and lymph nodes, percentage of CD25+CD4+ T cells (p), effector memory-like (q) and central memory-like (r) CD4+ T cells relative to total CD4+ T cells in spleens and lymph nodes were assessed. s, Surface expression of CD25 and CD44 on mouse naive CD8+ T cells activated with serum from normal diet mice intraperitoneally injected with PBS (Ctrl) or ASNase (n = 6 mice per group). All data are mean ± SEM, in a-r, Ctrl, n = 7 mice; Asn, n = 6 mice. Two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Related representative FACS plots (Supplementary Figs. 13, 14) and numerical source data for a-s are provided.

Source data

Extended Data Fig. 5 Asn enhances OVA-induced CD8+ T cell activation in vivo and a role for SLC1A5 in Asn uptake in CD8+ T cells.

a-i, C57BL/6 mice orally administrated with normal (Ctrl) or Asn-supplemented water (Asn) were infected with LmOVA for 7 days (a). spleens were excised, weighted and pictured (b, c). Percentage of CD8+ T cells and CD4+ T cells (d-g), and OVA-TET+ CD8+ T cells in spleens and lymph nodes (h, i) were determined. n = 5 mice per group. j-l, Related to Fig. 2m,n. Asn-free diet CD45.1 mice intraperitoneally injected with PBS (Ctrl) or Asn were adoptively transferred with naive CD45.2+ OT-I CD8+ T cells and injected with B16-OVA cells as indicated (j). 16 days later, percentage and number (#) of CD44+CD8+ T cells (k), and percentage of central and effector memory-liked CD8+ T cells relative to total CD8+ T cells in lymph nodes (l) were analysed (n = 5 mice per group). m, mRNA levels of potential transporters for Asn uptake and exchange in Jurkat cells. n and o, SLC1A5 and SLC38A2 expression in mouse naive CD8+ T cells activated in Asn/Asp-free (Ctrl) or Asn medium was analysed by qRT-PCR (n) and western blotting (o). p, Mouse CD8+ T cells activated by anti-CD3(5 μg/ml) and anti-CD28(1 μg/ml) in Asn/Asp-free (-) or Asn medium supplied with 10% dialysed FBS and 2 mM Gln were treated with or without 17 μM V9302 for 12 h. Surface expression of CD25 and CD69 relative to total CD8+ T cells were quantified. All data are mean ± SEM. Data in o are representative of three independent experiments, two-tailed Student’s t-test. *P < 0.05; ***P < 0.001; ***P < 0.001; ***P < 0.0001; ns, not significant. Related representative FACS plots (Supplementary Fig. 15), uncropped blots for o, and numerical source data for c-i, k-n, p, are provided.

Source data

Extended Data Fig. 6 Effect of mTORC1 and Gln on Asn-mediated CD8+ T cell activation.

a, Phosphorylation of the mTORC1 effector S6 (phospho-S6 Ser 235/236) in mouse CD8+ T cells left unstimulated (naive) or activated in the presence of increasing amount of Asn. b, Western blot analysis of mouse naive CD8+ T cells left untreated (naive), or activated in Ctrl (Asn/Asp-free) medium or Asn medium supplemented with increasing amounts of rapamycin for 12 h. c, Surface expression of CD69, CD25, and CD44 on mouse naive CD8+ T cells stimulated in Asn/Asp-free medium (Ctrl) or Asn medium with 10% dialysed FBS and increasing amounts of rapamycin (n = 3 independent wells). d, Mouse naive CD8+ T cells stimulated in Asn/Asp-free (-) or Asn-supplied (Asn) medium without (0) or with increasing amounts of Gln for 24 h were treated with 40 μg/ml Puromycin for another 10 min. Medium was added with 10% dialysed FBS. Puromycin incorporation was measured by FACS analysis using FITC-conjugated anti-Puromycin antibody (n = 3 independent wells). Representative flow cytometry plots are shown. e, Mouse naive CD8+ T cells were stimulated in the indicated medium with 10% dialysed FBS for 24 h. MG132 (10 μM) was added 2 h before harvesting for western blot analysis. f, Surface expression of CD69, CD25, and CD44 on mouse naive CD8+ T cells stimulated in Asn/Asp-free (-) or Asn medium containing no (0) or increasing amounts of Gln for 24 h (n = 3 independent wells). All medium was added with 10% dialysed FBS. All data are mean ± SEM, two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001; ns, not significant. Data in a, b and e are representative of three independent experiments. Related representative FACS plots (Supplementary Figs. 16, 17), uncropped blots for a, b, e, and numerical source data for c, d, f, are provided.

Source data

Extended Data Fig. 7 Impact of Asn on CD8+ T cell metabolism and TCR signalling.

a and b, Mouse naive CD8+ T cells were left untreated (naive) or activated in Asn/Asp-free medium (Ctrl), or medium supplied with Asn or Asp for indicated times. All medium was added with 10% dialysed FBS. The abundance of cellular metabolites at different time points were analysed by LC-MS/MS. The heat map shows the relative abundance of metabolites over time in CD8+ T cells stimulated in Ctrl, Asn- or Asp-medium. Log2 fold changes (FC) are relative to relative to cells stimulated in Asn/Asp-free (Ctrl) medium at corresponding time points. Data are representative of three independent experiments. Structural isomers were condensed and counted only once as the employed technology cannot distinguish metabolites with identical molecular weight. c, TCR signalling that initiates T cell activation. d, Related to Fig. 4e. Scheme of OT-I adoptive transfer and OVA-peptide treatment experiment. e, Western blot analysis of mouse CD8+ T cells activated in medium containing increasing amounts of Asn in the absence or presence of PP2 (10 µM). Data are representative of three independent experiments. f, Related to Fig. 5e–k. CD45.1 C57BL/6 mice maintained on an Asn-free diet were intraperitoneally injected with Asn or PBS (Ctrl), or PP2 (30 µg/mouse) or GDC-0032 (100 µg/mouse) every two days. 8 days later, mice were adoptively transferred with naive CD45.2+ OT-I CD8+ T cells and injected with OVA peptide as indicated. g, Related to Fig. 5l–o, Scheme of Asn-free diet CD45.1 C57BL/6 mice i.p. with Asn and/or PP2 and then adoptively transferred with naive CD45.2+ OT-I CD8+ T cells and infected with LmOVA as indicated. Uncropped blots for e and numerical source data for a, b, are provided.

Source data

Extended Data Fig. 8 LCK is a natural sensor for Asn.

a, Recombinant LCK proteins were treated with ATP and Asn or Asp. LCK kinase activity was determined by ELISA. b and c, BIAcore measurement of the interaction between Asn or Asp and purified mutant LCK (Y394F) (b) or LCK (Y505F) (c). d-u, Screening the binding specificity of recombinant LCK proteins to different amino acids by BIAcore assay. Graphs of equilibrium response units (RU) and compound concentrations are shown. Gln, glutamine; Thr, threonine; Isoleu, isoleucine; Trp, tryptophan; Met, methionine; Leu, leucine; Ala, alanine; Val, valine; Ser, serine; Pro, proline; Gly, glycine; His, histidine; Cys, cysteine; Tyr, tyrosine; Phe, phenylalanine; Lys, lysine; Glu, glutamate; Arg, arginine. v, BIAcore assay of the interaction between purified ZAP70 proteins and Asn or Asp. w, CETSAs exhibit the binding affinity of LCK to Asn, but not Gln, in Jurkat T cells. Relative LCK band intensities were quantified and plotted against corresponding incubation temperatures. x, Direct binding of LCK to Asn, but not Gln in Jurkat cells measured by DARTS assays. y, In vitro kinase assay for LCK and its mutants in the presence of increasing amounts of Asn. Kinase activity is expressed as relative values to phosphorylation of LCK(Y394). z, In vitro kinase assay using purified LCK and its mutants left untreated (Ctrl), or incubated with ATP and increasing amounts of Asn for 15 min. In b-v, Graphs of equilibrium response units (RU) and compound concentrations are shown. In b, c and v, Purified proteins were analysed by SDS-PAGE followed by Coomassie Blue staining. Data are mean ± SEM, in a, n = 3 biological replicates. Two-way ANOVA followed by Sidak’s multiple comparisons. Data are representative of three independent experiments throughout. Uncropped blots for x, z, and numerical source data for a-w, y, are provided.

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Extended Data Fig. 9 Asn binds to the LCK kinase domain and enhances CD8+ T cell anti-toumor activity.

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.

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Extended Data Fig. 10 Gating strategies used in FACS analysis.

a, Gating strategy to identify APC+ in mouse spleen isolated CD8+ T cells which were activation such as Fig. 1a and Supplemental Fig. 1b. b, Gating strategy to analyse CFSE-stained or BrdU-stained activation CD8+ T cells analysis presented on Extended Data Fig. 1h,1j and 3n. c, Gating strategy to analyse annexin V stained activation CD8+ T cells in apoptosis analysis presented on Extended Data Fig. 3k,m. d, Gating strategy to analyse the population of CD4+ T cells or CD8+ T cells in mouse lymph nodes or spleens presented on such as Extended Data Fig. 4m. e, Gating strategy to analyse the population of OT-I CD8+ T cells in mouse lymph nodes or spleens presented on such as Figs. 2m, 2n, 5e–o, and Extended Data Fig. 5k,l.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1. Information about the antibodies used in this work. Supplementary Table 2. List of the primer pairs used in this study. Supplementary Table 3. Information about the human participants.

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Wu, J., Li, G., Li, L. et al. Asparagine enhances LCK signalling to potentiate CD8+ T-cell activation and anti-tumour responses. Nat Cell Biol 23, 75–86 (2021). https://doi.org/10.1038/s41556-020-00615-4

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