1. Immunology and Inflammation
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Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation

  1. Julia M Marchingo
  2. Linda V Sinclair  Is a corresponding author
  3. Andrew JM Howden
  4. Doreen A Cantrell  Is a corresponding author
  1. Cell Signalling and Immunology Division, School of Life Sciences, University of Dundee, United Kingdom
Research Article
Cite this article as: eLife 2020;9:e53725 doi: 10.7554/eLife.53725
4 figures, 1 table, 3 data sets and 2 additional files

Figures

Figure 1 with 2 supplements
Myc controls cell growth by selectively remodelling T cell proteomes.

(A) Forward scatter area (FSC-A) of IL-7 maintained or 24 hr anti-CD3 + anti-CD28 (TCR) activated Cd4Cre+ (MycWT) and Cd4Cre+Mycfl/fl (MyccKO) T cells. (B–C, E–O) Quantitative proteomics data of ex vivo naïve WT and 24 hr TCR activated CD4+ and CD8+ T cells from MycWT and MyccKO mice. (B) Total protein content (µg/million cells). (C) Mean protein copy number per cell estimated using proteomic ruler (Wiśniewski et al., 2014) of Myc. (D) Myc expression measured by flow cytometry in 24 hr TCR activated MycWT and MyccKO CD4+ and CD8+ T cells. Proteins from 24 hr TCR activated MycWT (E) CD8+ and (F) CD4+ T cells were ranked by mass contribution and the mean cumulative protein mass was plotted against protein rank (left panel). Numbers in each quartile indicate total proteins summed with those in the quartiles below. Volcano plots show foldchange in protein copy number between TCR activated MyccKO and MycWT T cells, with proteins that contribute the top 75% of the T cell mass shown in red (right panel). (G) Heat maps of naïve and TCR activated MycWT and MyccKO CD8+ and CD4+ proteomes. Relative protein abundance is graded from low (blue) to high (yellow) per row. Input data for heatmaps is listed in Supplementary file 1. Mean protein copy number per cell for activation markers (H) IL7ra (J) CD69 and (K) CD44 and key transcription factors (I) Klf2, (L) Rel, (M) JunB, (N) Tbet, and (O) Irf4. Symbols on bar charts represent biological replicates: error bars show mean ± S.E.M. Quantitative proteomics was performed on biological triplicates. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, and TCR MycWT vs TCR MyccKO protein copy number per cell is listed in Supplementary file 1.

Figure 1—figure supplement 1
Immune activated Myc-deficient T cells fail to proliferate.

(A) CFSE labelled lymph node cells from Cd4Cre+ (MycWT) and Cd4Cre+ Mycfl/fl (MyccKO) mice were stimulated with anti-CD3 and anti-CD28 (both 0.5 µg/mL) for 48 hr and CFSE dilution was measured. Representative of technical duplicate plots. (B) Lymph node cells from GFP-MycKI mice were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (3 µg/ml) in the presence of IL-2 receptor-blocking antibody PC61 (2 µg/ml) or isotype control and GFP-Myc expression was measured over time. Symbols show biological replicates.

Figure 1—figure supplement 2
Myc-deficiency has a larger quantitative effect in CD8+ T cells.

Naïve WT and 24 hr TCR activated MycWT and MyccKO proteomic data was generated as described in Figure 1 and Materials and methods. (A) Venn diagram showing that there were a larger number of proteins that were classified as differentially regulated in CD8+ T cells compared to CD4+ T cells, where differentially regulated is defined as proteins that were more than 2-fold regulated and p<0.05 between MycWT vs MyccKO TCR activated conditions. (B) Heat maps of proteins that were Myc-regulated in CD4+ T cells only, both CD4+ and CD8+ T cells or CD8+ T cells only show similar qualitative effects of Myc-deficiency for most proteins. Relative protein abundance is graded from low (blue) to high (yellow) per row. (C) Mean copy number per cell for examples of proteins classified as Myc-regulated in CD8+ T cells only. Protein expression was much higher in MycWT CD8 T cells compared MycWT CD4+ T cells, whereas expression levels in MyccKO CD4+ and CD8+ T cells are reduced to a similar extent thus giving a larger quantitative effect of Myc-deficiency in CD8+ T cells. Examples include important transcription factors such as Nfkb1 and Nfkb2, the integrin Icam1 and cytokine receptor components such as Il2rb. Symbols show biological replicates. Mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, and TCR MycWT vs TCR MyccKO protein copy number per cell is listed in Supplementary file 1.

Myc control of T cell metabolism is selective.

Naïve WT and 24 hr TCR activated MycWT and MyccKO CD4+ and CD8+ T cell proteomic data was generated as described in Figure 1 and Materials and methods. (A) schematic of nutrient transporters and enzymes involved in glycolysis. (B) Mean copy number per cell for glucose transporters Slc2a1 and Slc2a3. (C) Total protein content (µg/million cells) (left panel) and % contribution to total cellular protein mass (right panel) of total glycolytic enzymes. (D) Mean protein copy number per cell for lactate transporters Slc16a1 and Slc16a3. (E) Schematic of transporters and enzymes involved in Glutamine transport and metabolism. (F) Mean copy number per cell for major enzymes involved in glutamine metabolism. Symbols on bar charts represent biological replicates from biological triplicate data, error bars show mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, and TCR MycWT vs TCR MyccKO protein copy number per cell is listed in Supplementary file 1.

Figure 3 with 4 supplements
Myc induces amino acid transporter expression, a critical step for proteome remodelling.

Naïve WT and 24 hr TCR activated MycWT and MyccKO CD4+ and CD8+ T cell proteomic data was generated as described in Figure 1 and Materials and methods. (A) Mean copy number per cell of abundant amino acid transporters in T cells. (B) Fold-change in amino acid transporter protein copy number from naïve WT to 24 hr TCR activated MycWT and MyccKO, mean (min, max). Transport by system L amino acid transporters was measured by uptake of fluorescent (emission 450 nm when excited at 405 nm) Tryptophan metabolite, Kynurenine (Kyn) (Sinclair et al., 2018) in (C) 2 hr IL-7 maintained and (D-E) 4 hr IL-7 maintained or TCR activated splenic CD4+ and CD8+ WT, MyccKO and Slc7a5cKO T cells or (F) 20 hr TCR activated Myc-GFP reporter CD4+ T cells. In (C,E) system-L uptake is represented as the ratio of BCH (a system L inhibitor) untreated: treated T cells. In (E) dotted line indicates Slc7a5cKO uptake level. (G) Forward scatter and CD69 expression of IL-7 maintained or 24 hr TCR activated wild-type and Slc7a5cKO (Cd4Cre+ Slc7a5fl/fl) T cells. (H-K) Quantitative proteomics data of naïve WT and 24 hr TCR activated CD4+ and CD8+ T cells from Ly5.1 (Slc7a5WT) and Slc7a5cKO mice. Baseline naïve WT data is the same as used for the MyccKO dataset. (H) Total protein content (µg/million cells). (I) Heat map of naïve and TCR activated Slc7a5WT and Slc7a5cKO CD4+ T cell proteomes. Relative protein abundance is graded from low (blue) to high (yellow) per row. Input data for heatmaps is listed in Supplementary file 1. (J) Venn diagram showing the overlap in TCR regulated proteins that are more than 2-fold regulated and p<0.05 in MycWT vs MyccKO and Slc7a5WT vs Slc7a5cKO TCR activated CD4+ T cells. (K) Volcano plots of TCR regulated proteins comparing Slc7a5WT and Slc7a5cKO datatsets. Proteins > 2 fold different between MycWT and MyccKO TCR activated T cells are highlighted in red; proteins reduced in the MyccKO (left panel), proteins higher MyccKO (right panel). Symbols in bar charts represent biological replicates: error bars show mean ± S.E.M. Dot plot in (F) is representative of biological triplicate data. Quantitative proteomics was performed on biological triplicates. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, TCR MycWT vs TCR MyccKO, naïve WT vs TCR Slc7a5WT, naïve WT vs TCR Slc7a5cKO and TCR Slc7a5WT vs TCR Slc7a5cKO protein copy number per cell is listed in Supplementary file 1.

Figure 3—figure supplement 1
Myc-deficient T cells fail to induce protein translation machinery.

Naïve WT and 24 hr TCR activated MycWT and MyccKO proteomic data was generated as described in Figure 1 and Materials and methods. Diagram and mean copy number per cell of (A) translation initiation complex and (B) 43S preintiaition complex components. (C) Protein content of 40S and 60S ribosome (µg/million cells). Symbols show biological replicates. Mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, and TCR MycWT vs TCR MyccKO protein copy number per cell is listed in Supplementary file 1.

Figure 3—figure supplement 2
Amino acid transport capacity corresponds with transporter number.

Transport by system L amino acid transporters was measured by uptake of fluorescent (emission 450 nm when excited at 405 nm) Tryptophan metabolite, Kynurenine (Kyn) in 6 or 24 hr IL-7 maintained or anti-CD3/anti-CD28 (TCR) activated CD4+ and CD8+ WT and MyccKO T cells. Histograms are representative of at least three biological replicates.

Figure 3—figure supplement 3
Myc-deficient T cells fail to induce Branched-chain amino acid and Methionine metabolism.

Naïve WT and 24 hr TCR activated MycWT and MyccKO proteomic data was generated as described in Figure 1 and Materials and methods. Diagram and mean copy number per cell of proteins involved in (A) Methionine metabolism and (B) Branched-chain amino acid metabolism. Symbols show biological replicates. Mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, and TCR MycWT vs TCR MyccKO protein copy number per cell is listed in Supplementary file 1.

Figure 3—figure supplement 4
Ribosome expression is reduced in both Myc and Slc7a5 deficient T cells, but other proteins are differentially regulated between Myc and Slc7a5 deficient T cells.

(A) Protein content of ribosomes (excluding mitochondrial) from naïve WT and 24 hr TCR activated MycWT and MyccKO proteomics data (generated as described in Figure 1 and Materials and methods). (B) Protein content of ribosomes (excluding mitochondrial) and (C) Slc2a3 mean copy number per cell from naïve WT and 24 hr TCR activated Slc7a5WT and Slc7a5cKO proteomics data (generated as described in Figure 3 and Materials and methods). Symbols show biological replicates. Mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR MycWT, naïve WT vs TCR MyccKO, TCR MycWT vs TCR MyccKO, naïve WT vs TCR Slc7a5WT, naïve WT vs TCR Slc7a5cKO and TCR Slc7a5WT vs TCR Slc7a5cKO protein copy number per cell is listed in Supplementary file 1. Splenocytes from MycWT, MyccKO and Slc7a5cKO were stimulated with anti-CD3 and anti-CD28 for 24 hr and; (D) Golgi plug with or without PdBU and ionomycin was added to the cultures for the last 4 hr prior to harvest and IFNgamma expression was measured in CD8+ T cells by flow cytometry; or (E) Granzyme B was measured in CD8+ T cells by flow cytometry. Histograms are representative of at least three biological replicates.

Myc induces amino acid transport early after TCR activation, triggering a feedforward loop maintaining its own expression.

(A) Expression levels of Slc7a5, Slc7a1, Slc1a5 and Slc38a2 vs Myc mRNA from published single cell RNAseq dataset of OT1 T cells stimulated with SIINFEKL (N4) peptide for the indicated times (Richard et al., 2018). Dotted lines represent 95th percentile of 0 hr gene expression. Symbols represent individual cells (B) Quantitative proteomics of OT-I CD8+ T cells activated with N4 peptide for the indicated time. Mean copy number per cell of the proteins Myc, Slc7a5, Slc7a1, Slc1a5, Slc38a1 and Slc38a2. (C) Slc7a5, Slc1a5 and Slc7a1 mRNA measured by qPCR from ex vivo naïve or 4 hr TCR activated lymph node CD4+ and CD8+ T cells. mRNA levels are relative to naïve CD4+ T cells. (D) Histograms and geometric mean fluorescence intensity (gMFI) vs time of Myc protein measured with antibody by flow cytometry in IL-7 maintained or TCR activated Slc7a5WT and Slc7a5cKO lymph node CD4+ and CD8+ T cells. Representative of 4 biological replicates. (E) Myc-GFP gMFI in IL-7 maintained or GFP+ TCR activated CD4+ and CD8+ T cells ± rapamycin from lymph nodes of Myc-GFP reporter mouse. Myc-GFP reporter expression in CD4+ and CD8+ T cells maintained in IL-7 or TCR activated in (F) amino-acid free media (-aa, HBSS), vs RPMI (+aa) or (G) media deficient in a single amino acid. Data representative of at least three biological replicates. Symbols unless otherwise stated represent biological replicates. Mean ± S.E.M. Quantitative proteomics was performed on biological triplicates.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Cd4CrePMID: 27345256, PMID: 11728338
Genetic reagent (M. musculus)Cd4Cre+ Mycfl/flPMID: 19423665, PMID: 19342639, PMID: 11742404
Genetic reagent (M. musculus)Cd4Cre+ Slc7a5fl/flPMID: 23525088, PMID: 24586861
Genetic reagent (M. musculus)GFP-MycKIPMID: 18196519, PMID: 26136212, PMID: 23021216
Genetic reagent (M. musculus)OT1PMID: 8287475maintained in house as an OT1 TCR transgene heterozygote on a CD45.1 (Ly5.1) background
AntibodyAnti-CD3 (armenian hamster, monoclonal)Thermo Fisher ScientificCat # 14-0031-82, RRID:AB_467049T cell activation: 0.5 or 1 μg/ml as indicated in Materials and methods
AntibodyAnti-CD28 (syrian hamster, monoclonal)Thermo Fisher ScientificCat # 16-0281-82, RRID:AB_468921T cell activation: 0.5 or 3 μg/ml as indicated in Materials and methods
AntibodyAnti-CD4 (rat, monoclonal)BD BiosciencesCat # 553650, RRID:AB_394970; Cat# 552775, RRID:AB_394461; Cat# 553047, RRID:AB_394583cell surface staining 1:200
AntibodyAnti-CD4 (rat, monoclonal)Thermo Fisher ScientificCat# 47-0042-82, RRID:AB_1272183cell surface staining 1:200
AntibodyAnti-CD8a (rat, monoclonal)BiolegendCat# 100708, RRID:AB_312747; Cat# 100722, RRID:AB_312761
Cat# 100738, RRID:AB_11204079
cell surface staining 1:200
AntibodyAnti-CD8a (rat, monoclonal)BD BiosciencesCat# 551162, RRID:AB_394081cell surface staining 1:200
AntibodyAnti-CD69 (armenian hamster, monoclonal)ThermoFisher ScientificCat# 17-0691-82, RRID:AB_1210795cell surface staining 1:200
AntibodyAnti-CD69 (armenian hamster, monoclonal)BiolegendCat# 104514, RRID:AB_492843cell surface staining 1:200
AntibodyAnti-CD69 (armenian hamster, monoclonal)BD BiosciencesCat# 553237, RRID:AB_394726cell surface staining 1:200
AntibodyAnti-B220 (rat, monoclonal)BD BiosciencesCat# 553087, RRID:AB_394617cell surface staining 1:200
AntibodyAnti-NK1.1 (mouse, monoclonal)BiolegendCat# 108706, RRID:AB_313393cell surface staining 1:200
AntibodyAnti-CD11b (rat, monoclonal)BiolegendCat# 101206, RRID:AB_312789cell surface staining 1:200
AntibodyAnti-CD25 (rat, monoclonal)BD BiosciencesCat# 553072, RRID:AB_394604cell surface staining 1:200
AntibodyAnti-CD62L (rat, monoclonal)Thermo Fisher ScientificCat# 12-0621-83, RRID:AB_465722cell surface staining 1:200
AntibodyAnti-TCRb (armenian hamster, monoclonal)Thermo Fisher ScientificCat# 45-5961-82, RRID:AB_925763cell surface staining 1:200
AntibodyAnti-CD44 (rat, monoclonal)BD BiosciencesCat# 559250, RRID:AB_398661cell surface staining 1:200
AntibodyAnti-Thy1.2 (rat, monoclonal)BD BiosciencesCat# 553006, RRID:AB_394545cell surface staining 1:200
AntibodyAnti-CD45.1 (mouse, monoclonal)BiolegendCat # 110714, RRID:AB_313503cell surface staining 1:200
AntibodyAnti-CD45.2 (mouse, monoclonal)BiolegendCat # 109816, RRID:AB_492868cell surface staining 1:200
AntibodyAnti-mouse CD16/CD32 Fc Block, (rat, monoclonal)BD BiosciencesCat # 553141, RRID:AB_394656Fc block 1:100
Antibodyc-Myc (D84C12) XP (rabbit, monoclonal)Cell Signaling TechnologiesCat# 5605, RRID:AB_1903938intracellular staining 1:200
AntibodyAnti-rabbit A647 (goat)Cell Signaling TechnologiesCat # 4414, RRID:AB_10693544intracellular staining 1:1000
AntibodyAnti-IFNg (rat, monoclonal)BiolegendCat # 505810, RRID:AB_315404intracellular cytokine staining 1:100
AntibodyAnti-Granzyme BThermo Fisher ScientificCat# 17-8898-82, RRID:AB_2688068intracellular cytokine staining 1:200
Chemical compound, drugDAPIThermo Fisher ScientificD13061 µg/mL
Chemical compound, drugKynurenineSigmaCat# K8625200 µM
Chemical compound, drugBCHSigmaCat# A790210 mM
Chemical compound, drugRapamycinMerck/CalbiochemCat# 55321120 nM
Commercial assay or kitRneasy minikitQiagenCat # 74104
Commercial assay or kitiScript cDNA Synthesis kitBioradCat#1708891
Commercial assay or kitiTaq Universal SYBRGreen SupermixBioradCat# 1725121
Commercial assay or kitEZQ protein quantitation kitThermo Fisher ScientificR33200
Commercial assay or kitSera-Mag SpeedBead Carboxylate-modified magnetic particles (hydrophilic)GE Lifesciencescat# 45152105050250
Commercial assay or kitSera-Mag SpeedBead Carboxylate-modified magnetic particles (hydrophobic)GE Lifesciencescat# 65152105050250
Commercial assay or kitCBQCA protein quantitation kitThermo Fisher ScientificC6667
Commercial assay or kitHiPPR Detergent Removal Spin Column KitThermo Fisher ScientificCat# 88305
Commercial assay or kitEasySep CD8 T cell isolation kitSTEMCELL Technologies, UKCat # 19853
Commercial assay or kitGolgi PlugBD BiosciencesCat# 555029
Commercial assay or kiteBioscience Intracellular Fixation and Permeabilization Buffer SetThermo Fisher ScientificCat# 88-8824-00
Commercial assay or kitCFSEThermo Fisher Scientific/InvitrogenCat# C345545 µM
Peptide, recombinant proteinIL7PeprotechCat# 217–175 ng/mL
Peptide, recombinant proteinIL2Novartis, UKProleukin20 ng/ml
Peptide, recombinant proteinIL12PeprotechCat#210–122 ng/ml
Sequence based reagentSlc7a5 forward primerAAG GCT GCG ACC CGT GTG
Sequence based reagentSlc7a5 reverse primerATC ACC TTG TCC CAT GTC CTT CC
Sequence based reagentSlc7a1 forward primerGGA GCT TTG GC CTT CAT CAC T
Sequence based reagentSlc7a1 reverse primerCAG CAC CCC AGG AGC ATT CA
Sequence based reagentSlc1a5 forward primerGCC ATC ACC TCC ATC AAC GAC T
Sequence based reagentSlc1a5 reverse primerAGA GCG GAA GGC AGC AGA CAC
Sequence based reagentTBP forward primerGTG AAT CTT GGC TGT AAA CTT GAC CT
Sequence based reagentTBP reverse primerCGC AGT TGT CCG TGG CTC T
Software, algorithmFlowJo softwareTreestarversions 9 and 10
Software, algorithmMaxquanthttps://www.maxquant.org, PMID: 19029910version 1.6.2.6
Software, algorithmPerseushttps://www.maxquant.org/perseus, PMID: 27348712version 1.6.6.0
OtherRPMI 1640Thermo Fisher Scientific/GIBCOCat# 21875–034
OtherRPMI - glutamineThermo Fisher Scientific/GIBCOCat# 42401–018
OtherRPMI - methionineDC Biosciences Ltdcustom made RPMI without methionine and arginine - supplemented back the arginine (0.2 g/L) to RPMI levels
OtherRPMI - leucineSigmaCat# R1780 SAFCsupplemented back arginine (0.2 g/L) and lysine (0.04 g/L)to RPMI levels
OtherHBSSThermo Fisher Scientific/GIBCOCat# 14025–050used this as amino acid-free media
OtherFBSThermo Fisher Scientific/GIBCOCat # 10270106
OtherFBS, dialyzedThermo Fisher Scientific/GIBCOCat# 26400044
OtherArginineSigmaCat# A5006
OtherLysineSigmaCat# L5501

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Raw mass spec data files and MaxQuant analysis files for naïve WT, and TCR activated MycWT, MyccKO, Slc7a5WT and Slc7a5cKO T cells are available on the ProteomeXchange data repository and can be accessed with identifier PXD016105 (https://www.ebi.ac.uk/pride/archive/projects/PXD016105). Raw mass spec data files and MaxQuant analysis files for OT-1 TCR time-course data are available on the ProteomeXchange data repository and can be accessed with identifier PXD016443 (https://www.ebi.ac.uk/pride/archive/projects/PXD016443).

The following data sets were generated
  1. 1
    PRIDE
    1. JM Marchingo
    2. LV Sinclair
    3. AJM Howden
    4. DA Cantrell
    (2019)
    ID PXD016105. Proteome of naive and TCR activated wild-type, Myc-deficient and Slc7a5-deficient T cells.
  2. 2
    PRIDE
    1. JM Marchingo
    2. LV Sinclair
    3. AJM Howden
    4. DA Cantrell
    (2019)
    ID PXD016443. OT1 T cell activation time course.
The following previously published data sets were used
  1. 1
    ArrayExpress
    1. AC Richard
    2. ATL Lun
    3. WWY Lau
    4. B Gottgens
    5. JC Marioni
    6. GM Griffiths
    (2018)
    ID E-MTAB-6051. Single-cell RNA sequencing of OT-I CD8+ T cells after stimulation with different affinity ligands.

Additional files

Supplementary file 1

Copy number, protein content, fold-change calculations, statistical tests and heatmap input from proteomics data.

https://cdn.elifesciences.org/articles/53725/elife-53725-supp1-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/53725/elife-53725-transrepform-v2.docx

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