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The cytotoxic T cell proteome and its shaping by the kinase mTOR

Nature Immunology volume 17, pages 104–112 (2016)Cite this article

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Abstract

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

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Figure 1: The cytotoxic T cell proteome.
Figure 2: Abundance of key CTL proteins.
Figure 3: Comparison of the CTL transcriptome and CTL proteome.
Figure 4: The mTORC1-regulated CTL proteome.
Figure 5: mTORC1 regulation of cellular pathways.
Figure 6: Comparison of the mTORC1-controlled transcriptome and proteome in CTLs.
Figure 7: Selective control of CTL metabolism by mTORC1.
Figure 8: mTORC1 represses PtdIns(3,4,5)P3 production and controls the requirement for mTORC2 in the activation of Akt.

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Acknowledgements

We thank past and present colleagues of the Cantrell Group for advice and discussions; C. Feijoo-Carnero for help with microarray analysis; T. Ly for help with peptide fractionation using hSAX; and D. Lamont and the team of the MS facility at the College of Life Sciences of the University of Dundee and the Finnish DNA microarray Centre at the Centre for Biotechnology (Turku, Finland). Supported by the Wellcome Trust (093713/Z/10/A to J.L.H., 073980/Z/03/Z and 105024/Z/14/Z to A.I.L., 065975/Z/01/A and 097418/Z/11/Z to D.A.C.).

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Authors and Affiliations

  1. Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee, UK

    Jens L Hukelmann, Linda V Sinclair, Katarzyna M Grzes & Doreen A Cantrell

  2. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK

    Jens L Hukelmann, Alejandro Brenes Murillo & Angus I Lamond

  3. Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, UK

    Karen E Anderson, Phillip T Hawkins & Len R Stephens

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  1. Jens L Hukelmann
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Contributions

J.L.H., design and performance of proteomic and transcriptomic experiments and most other experiments; K.E.A., measurement of PtdIns(3,4,5)P3; L.V.S., glucose uptake assay; K.M.G., lactate output assay; A.B.M., Encyclopedia of Proteome Dynamics; P.T.H. and L.R.S., experimental design for measurement of PtdIns(3,4,5)P3; A.I.L., experimental design; D.A.C., experimental design and manuscript authorship (with input from J.L.H.).

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Correspondence to Doreen A Cantrell.

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Integrated supplementary information

Supplementary Figure 1 Experimental design for analysis of the CTL proteome.

Splenocytes from P14 TCR transgenic mice were activated for 48 h with gp33 in the presence of IL-2 and IL-12. Cells were expanded in IL-2/IL-12 for 4 days. CTL were lysed in urea buffer and digested with either lysyl endopeptidase (LysC) or combined LysC and trypsin and subjected to SAX chromatography prior to Orbitrap mass spectrometer analysis. Andromeda and MaxQuant were used for data normalisation, peak picking, database searching, peptide and protein identification. Further analysis was done using R, Perseus, Excel.

Supplementary Figure 2 Glutaminolysis in CTLs.

Representation of the glutaminolytic pathway: enzymes catalyzing glutaminolytic reactions in purple, metabolic intermediates in black. Glutamate dehydrogenase (GLUD1) as rate limiting step highlighted.

Supplementary Figure 3 Modes of Akt activation in CTLs.

Different mechanisms of Akt activation. Low PtdIns(3,4,5)P3 (PIP3) levels in the plasma membrane cause Akt to be locked in an inactive state. IL-2 receptor and nutrient signaling in CTL maintain basal PI(3)K and mTOR signaling and thus enable the PIF-pocket dependent activation of membrane-bound Akt via its upstream kinase PDK1. Acute mTOR inhibition leads to dephosphorylation of Akt p-S473 and thus disrupts the colocalisation of Akt and PDK1 via p-S473-PIF-pocket interaction. Chronic mTOR inhibition leads to the accumulation of PtdIns(3,4,5)P3 and allows colocalisation of Akt and PDK1 via their respective PH domains and subsequent activation of Akt via T308 phosphorylation regardless of dephosphorylation of Akt p-S473.

Supplementary Figure 4 Effects of treatment with rapamycin or KU-0063794 on the CTL proteome.

Scatter plot depicting correlation of mean fold changes in protein expression of DMSO (vehicle) treated CTL vs CTL treated with rapamycin (x-axis) or KU-0063794 (y-axis) obtained by stable isotope labelling of amino acid in cell culture (SILAC) based proteomics. 4934 rapamycin:DMSO vs KU-0063794:DMSO pairs are plotted. Statistically different proteins (p ≤ 0.01, p-value determined by unpaired, two-tailed, unequal variance t-test on SILAC ratios) are depicted as filled black circles. All proteins with p ≤ 0.01 show a less than 2-fold difference between the inhibitor induced effects (area within the two dashed diagonal lines). Furthermore, when compared to control CTL, all of these proteins show a less than 2-fold up or down-regulation with either rapamycin (area between the two dashed vertical lines) or KU-0063794 (area between the two horizontal dashed lines).

Supplementary Figure 5 Encyclopedia of Proteome Dynamics web-based data-sharing tool.

Screen shots of several sections of the EPD (http://www.peptracker.com/epd). (a) Homepage with search input accepting Uniprot accession, gene term, protein description, protein name or gene ontology terms. (b) Data derived from multiple databases such as Uniprot or STRING (e.g. in this case for perforin) are displayed with links to these and further external resources. (c) Histogram showing mean estimated copy numbers of the selected protein (as calculated by the proteomic ruler) highlighted in the context of the copy numbers distribution of the entire dataset. (d) Volcano plot for the 48h rapamycin treated CTL. The mean fold change for the selected protein is highlighted in the volcano plot and shown in the context of all protein groups of the dataset.

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Hukelmann, J., Anderson, K., Sinclair, L. et al. The cytotoxic T cell proteome and its shaping by the kinase mTOR. Nat Immunol 17, 104–112 (2016). https://doi.org/10.1038/ni.3314

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