1. Immunology and Inflammation
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Antigen receptor control of methionine metabolism in T cells

  1. Linda V Sinclair  Is a corresponding author
  2. Andrew JM Howden
  3. Alejandro Brenes
  4. Laura Spinelli
  5. Jens L Hukelmann
  6. Andrew N Macintyre
  7. Xiaojing Liu
  8. Sarah Thomson
  9. Peter M Taylor
  10. Jeffrey C Rathmell
  11. Jason W Locasale
  12. Angus I Lamond
  13. Doreen A Cantrell  Is a corresponding author
  1. University of Dundee, United Kingdom
  2. Duke University, United States
  3. Vanderbilt University Medical Center, United States
Research Article
Cite this article as: eLife 2019;8:e44210 doi: 10.7554/eLife.44210
7 figures, 1 table, 3 data sets and 2 additional files

Figures

T cell activation and differentiation requires a sustained supply of extracellular methionine.

(a) Flow cytometry plots show the forward (FSC) and side (SSC) scatter profiles of CD4+ T cells stimulated through the TCR (CD3/CD28) for 18 hr ± methionine. (b) Cell counts of CD4+ T cells over 48 hr after TCR-stimulation (CD3/CD28) in the presence or absence of methionine. (c) Flow cytometry plots CD69 expression of CD4+ T cells stimulated through the TCR (CD3/CD28) for 18 hr ± methionine. (d-f) CD4+ T cells activated with CD3/CD28 antibodies + IL2/IL12 for 3 days in indicated methionine concentrations. (d) CD44 surface staining and intracellular IFNγ cytokine staining. The % CD4+ T cells producing IFNγ is indicated on the plot. The percentage (e) and MFI (f) of IFNγ producing CD4+ T cells from three biological replicates. (g–l) CD4+ T cells were stimulated through the TCR (CD3/CD28) for 20 hr before culturing in reducing concentrations of methionine for a further 2 hr. The histograms (left panel) show de novo protein synthesis as measured by incorporation of the puromycin analog (OPP) (g) or RNA synthesis as determined by EU incorporation (h). The right panels show the MFIs over the expanded dose response. Cyclohexamide (CHX) treatment or Actinomycin D (ActD) treatment are included as negative controls for protein and RNA synthesis. (i) The histograms (left panel) show the frequency of CD4+ T cells undergoing DNA synthesis as measured by EdU incorporation. The right panel shows the frequency of EdU positive cells over the expanded dose response. (j–l) EC50 values were calculated from dose response curves (using logged concentration values). Goodness of fit is represented by the R2 values. (m–o) Th1 effector cells were expanded for 5 days before culturing in reducing concentrations of methionine for a further 5 hr. The histograms show (m) de novo protein synthesis (n) RNA synthesis and (o) DNA synthesis. (Plots are representative of 3 biological replicates. Gating strategies are provided in Supplementary file 1. Error bars are mean ± s.d of: three biological replicates; Points on the graphs indicate biological replicates.).

https://doi.org/10.7554/eLife.44210.003
Methionine metabolism in T cells.

(a) 3H radioactivity measured in TCA precipitated protein from isolated CD4+ T cells stimulated through the TCR (CD3/CD28) in the presence of 3H-methionine for the indicated times. (b) Metabolomic analysis of metabolites in the de novo pathway and the salvage pathway of the methionine cycle. The graphs show metabolite intensity derived from integrated peak areas of MS intensity from naïve CD4+ T cells and TCR-stimulated CD4+ T cells (CD3/CD28, for 16 hr). Enzymes are indicated adjacent to arrows (in blue). P values are indicated on each graph. Source data is available in Figure 2—source data 1. (c) The histograms show representative intracellular staining of total H3, trimethylated H3K27 (H3K27me3) or trimethylated H3K4 (H3K4me3) from IL7 maintained (unstimulated) or TCR-stimulated (CD3/CD28) CD4+ T cells for 18 hr. Geometric mean fluorescence intensities (MFI) are shown in the histograms. (d) The graph shows ratios of H3, H3K27me3 and H3K4me3 MFIs from TCR-stimulated (CD3/CD28) CD4+ T cells compared to unstimulated CD4+ T cells. (e, f) OT2 (CD45.1) cells were adoptively transferred into WT CD45.2 hosts. The hosts were immunised with NP-OVA/alum and the transferred OT2 cells were analysed after 3 days. The histograms (left) show representative intracellular staining of H3K27me3 (e) and H3K4me3 (f). Graphs (right) show ratios of H3K27me3 and H3K4me3 MFIs in activated OT2 CD4+ T cells compared to non-activated host CD4+ T cells 3 days post-immunisation. (g) 3H radioactivity measured in RNA extracted from isolated CD4+ T cells stimulated through the TCR (CD3/CD28) in the presence of 3H-methionine for the indicated times. (h) 3H radioactivity measured in protein or RNA extracted from CD4+ T cells stimulated in parallel to (g) in the presence 3H-phenylalanine for 18 hr. (i) Levels of SAH from unstimulated (naïve cells), TCR-stimulated (CD3/CD28, 18 hr) CD4+ T cells or IL2 maintained Th1 cells ± methionine for 18 hr. (j) The histograms show representative intracellular staining of total H3, trimethylated H3K27 (H3K27me3) or trimethylated H3K4 (H3K4me3) from IL7 maintained (unstimulated) or TCR-stimulated (CD3/CD28) CD4+ T cells±methionine as indicated (18 hr). (k) Percentage of RNA with m6A modification, as determined by ELISA, in Th1 cells cultured with decreasing methionine concentrations for 5 hr (as indicated). (l) Percentage of RNA with m5C modification, as determined by ELISA, in Th1 cells cultured ±methionine for 5 hr. (Error bars are mean ± s.d of: (a–d, i–l) three biological replicates (e–f) six biological replicates. (g,h) 4 (RNA) biological replicates and five biological replicates (protein). MFIs are indicated in the histograms, points on the graphs indicate biological replicates. (b, e, l) t -test; (d,i,k) One-way ANOVA; P= *<0.05, **<0.01, ***<0.001, ****<0.0001; Flow cytometry gating strategies are provided in Supplementary file 1).

https://doi.org/10.7554/eLife.44210.004
Figure 2—source data 1

Spreadsheet containing the list of metabolite intensities derived from integrated peak areas of MS intensity from naïve CD4+ T cells (N1-3) and TCR-stimulated CD4+ T cells (S1-3).

https://doi.org/10.7554/eLife.44210.005
Proteomics expression of methionine pathway in T cells Quantitative proteomics data showing the abundance of key enzymes in the methionine cycle of the de novo.

(a) and the salvage pathways (b). Mean protein copy numbers, indicated in red, estimated using the proteomic ruler protocol and presented as log-transformed mean values are shown relative to their frequency in the total data set. The graphs show copy numbers from naïve CD4+ T cells of MAT2A (c), AHCY (d), MTR (e) and SRM/SMS (f), MTAP (g) and mtnA-D (h). (i) Quantitative proteomics data comparing the concentration of key enzymes in the methionine cycle of the de novo and the salvage pathways in naïve, TCR activated and Th1 effector CD4+ T cells. Concentration is calculated using the histone ruler and estimated mass of molecules per cell, as described in Wiśniewski et al. (2014). (Data are from three biological replicates and error bars are mean ± s.d.).

https://doi.org/10.7554/eLife.44210.006
Methyltransferase expression in T cells.

Abundance of (a) CMTR1 and RNMT cap methyltransferases (b) METTL3 m6A RNA methyltransferase and (c) NSUN2 m5C methyltransferase expression in naïve, TCR activated and effector Th1 CD4+ T cells (plotted as in 3 c). (e) DNA methyltransferases use SAM as a methyl donor to methylate CG residues. The concentration of the DNA methyltransferase complexes expressed in naïve CD4+ T cells (naïve), 24 hr TCR- stimulated (aCD3/aCD28, IL2/12) CD4+ T cells (TCR) and in vitro generated Th1 cells (Th1). (f) The polycomb repressor complex 2 (PRC2) uses SAM to methylate lysine residues for example K27 on histone tails. Concentration of the PRC2 components calculated from proteomics data from naïve CD4+ T cells (naïve), 24 hr TCR- stimulated CD4+ T cells (TCR) and in vitro generated Th1 cells (Th1). (g) Flow cytometry plots showing EZH2 staining in naïve CD4+ T cells, CD4+ T cells stimulated through the TCR (aCD3/aCD28, IL2/12) for 18 hr and in vitro generated Th1 cells. MFI are shown on the graph. (h) Concentrations of histone methyltransferases calculated from proteomics data of naïve CD4+ T cells (naïve), 24 hr TCR- stimulated CD4+ T cells (TCR) and in vitro generated Th1 cells (Th1). Histone methyl modifications are indicated. (Error bars are mean ± s.d. Data are from three biological replicates.).

https://doi.org/10.7554/eLife.44210.007
Acute methionine restriction on methionine cycle proteome Quantitative ‘single-shot’ proteomics was performed on in vitro generated IL2 maintained Th1 cells cultured for 5 hr in 100 μM or 1 μM methionine.

(a) The graph shows the protein copy numbers in Th1 cultured with 1 μM methionine plotted against those in 100 μM methionine. Pearson correlation is indicated. (b) Proteins that were significantly differentially expressed (= <0.05, with a 1.5 fold cut-off) are listed by gene name. They are ranked as most decreased by acute methionine deprivation to those that are increased. (c-g) The graphs show mean copy numbers (estimated using the proteomic ruler protocol) of (c) MAT2A, (d) AHCY, (e) SMS/SRM, (f) MTAP and (g) mtnA. (h-j) The graphs show mean copy numbers of (h) RNA cap methyltransferases RNMT and CMTR1; (i) DNA methyltransferase complex components UHRF1, DNMT1 and DNMt3a and (j) histone methyltransferases SUV39H1, SETD3 and EZH2. (Error bars are mean ± s.d. Data are from three biological replicates.).

https://doi.org/10.7554/eLife.44210.008
Impact of methionine restriction on c-myc expression and mTORC1 activity.

(a, b) CD4+ T cells from GFP-MycKI mice were activated with CD3/CD28 antibodies ± methionine for 5 hr. (a) Flow cytometry plots show CD69 expression (left) and GFP-Myc expression (right) of IL7 maintained control CD4+ T cells (grey), CD4+ T cells activated with (black) or without (red) methionine. The MFI are shown in (b). (c-e) In vitro generated Th1 CD4+ T cells were cultured for 3 days prior to acute methionine deprivation (1 hr). (c) Histograms show ribosomal protein S6 phosphorylation (pS6) in Th1 cells in methionine replete media (100 μM, MET)±rapamycin (Rap, 20 nM); (d) methionine free media (no MET), no amino acids (no AA) (e) or methionine free media supplemented with SAM (100 μM, no MET +SAM). The corresponding MFI are shown in (f). (a, c-e Data are representative of 3 biological replicates. (b,f) Points indicate individual biological replicates. Error bars are mean ± s.d. One-way ANOVA; (d) t-test; P = *<0.05, **<0.01, ***<0.001, ****<0.0001).

https://doi.org/10.7554/eLife.44210.009
Antigen receptor and cytokine signalling regulate methionine bioavailability through SLC7A5 expression.

(a) Uptake of 3H-methionine in purified CD4+ T cells±TCR activation using CD3/CD28 antibodies for 3 or 18 hr. (b) 3H-methionine uptake in 5 day in vitro expanded Th1 cells switched for final 18 hr into indicated concentrations of IL2. (c) The graphs show copy numbers of potential methionine transporters from proteomics data sets of naïve CD4+ T cells, 24 hr TCR- stimulated CD4+ T cells and effector Th1 cells. (nd = not detected) (d) Uptake of 3H-methionine in IL2 maintained Th1 cells in the presence or absence of BCH, ALA, LYS, MeAIB or MET (all 5 mM). (e) Uptake of 3H-methionine (left panel) or 14C glutamine (right panel) in IL2 maintained Th1 cells in presence or absence of sodium in the uptake buffer. (f) 14C-glutamine uptake in IL2 maintained Th1 cells in the presence or absence of GLN, ALA and BCH (all 5 mM). (g) 3H-phenylalanine uptake in IL2 maintained Th1 cells in the presence or absence of BCH, ALA, LYS, LEU or MET (all 5 mM). (h–i) 3H radioactivity of TCA precipitated protein (h) or RNA (i) from CD4+ T cells stimulated through the TCR (CD3/CD28) for 6 hr in the presence of 3H-methionine ± the System L inhibitor BCH. (j) SAH levels as determined by ELISA in CD4+ T cells stimulated through the TCR (CD3/CD28)±the System L inhibitor BCH for 18 hr. (k) Percentage of RNA with m6A modification, as determined by ELISA, in Th1 cells cultured in 20 μM MET ±BCH for 5 hr. (l) Uptake of 3H-methionine in TCR stimulated (CD3/CD28, 18 hr) CD4+ T cells from Slc7a5fl/fl or Cd4-Cre+::Slc7a5fl/fl mice, compared to unstimulated CD4+ T cells maintained in IL7,±System L transporter inhibitor BCH (5 mM). (m) SAH levels in CD4+ T cells from Slc7a5fl/fl or Cd4-Cre+::Slc7a5fl/fl mice stimulated through the TCR (CD3/CD28) for 18 hr, compared to naive cells. ((a,b- d, f, g, l, m) ANOVA, (e,h–k) t-test; P= *<0.05, **<0.01, ***<0.001, ****<0.0001. Uptakes performed in triplicate. Error bars are s.d. from minimum three biological replicates. Points indicate individual biological replicates.).

https://doi.org/10.7554/eLife.44210.010

Tables

Key resources table
Reagent type (species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (M. musculus)JAX C57BL/6J, WTJAX C57BL/6J Mice, Charles River UKstrain code 632
Genetic reagent (M. musculus)Cd4Cre Slc7a5fl/flSinclair et al., 2013
Genetic reagent (M. musculus)GFP-MycKIHuang et al., 2008;
Preston et al., 2015
In these mice, a fusion protein of Myc and enhanced green fluorescent protein (GFP‐Myc) is expressed from the endogenous Myc locus (GFP‐MycKI)
Genetic reagent (M. musculus)OT2Barnden et al., 1998maintained in house on a CD45.1 (LY5.1) background
AntibodyAnti-CD3 (armenian hamster, monoclonal)ThermoFisherCat # 14-0031-82, RRID:AB_467049T cell activation: 1 μg/ml; Th1 differentiation: 2 μg/ml
AntibodyAnti-CD28 (syrian hamster, monoclonal)ThermoFisherCat # 16-0281-82, RRID:AB_468921T cell activation: 2 μg/ml; Th1 differentiation: 3 μg/ml
AntibodyAnti-CD4 (rat, monoclonal)BiolegendCat # 100530, RRID:AB_389325cell surface staining 1:200
AntibodyAnti-TCRb (armenian hamster, monoclonal)BiolegendCat # 109220, RRID:AB_893624cell surface staining 1:200
AntibodyAnti-CD62L (rat, monoclonal)BiolegendCat # 104412, RRID:AB_313099cell surface staining 1:200
AntibodyAnti-TCR V alpha 2 (rat, monoclonal)BiolegendCat # 127808, RRID:AB_1134183cell surface staining 1:200
AntibodyAnti-CD44 (rat, monoclonal)BiolegendCat # 103030, RRID:AB_830787cell 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-CD69 (armenian hamster, monoclonal)BiolegendCat # 104522, RRID:AB_2260065cell surface staining 1:200
AntibodyAnti-INFg (rat, monoclonal)BiolegendCat # 505810, RRID:AB_315404intracellular cytokine staining 1:50
AntibodyAnti-phospho-S6 (rabbit, monoclonal)Cell Signaling TechnologiesCat #
2211, RRID:AB_331679
intracellular
staining 1:50
AntibodyAnti-Histone H3 (rabbit, monoclonal)Cell Signaling TechnologiesCat # 82241Sintracellular staining 1:50
AntibodyAnti-Histone H3K4me3 (rabbit, monoclonal)Cell Signaling TechnologiesCat # 12064intracellular
staining 1:50
AntibodyAnti-H3K27me3 (rabbit, monoclonal)Cell Signaling TechnologiesCat # 12158intracellular
staining 1:50
AntibodyAnti-rabbit A647Cell Signaling TechnologiesCat #
4414, RRID:
AB_10693544
intracellular
staining 1:500
AntibodyAnti-mouse CD16/CD32 Fc Block, (rat, monoclonal)BD BiosciencesCat #
553141, RRID:
AB_394656
Fc block 1:70
Peptide, recombinant proteinIL2Novartis, UKProleukinTh1 differentiation:
20 ng/ml
Peptide, recombinant proteinIL12RnD Systems, UKCat # 419 MLTh1 differentiation:
10 ng/ml
Peptide, recombinant proteinNP-OVABioSearch technolgies, UKCat # N-5051Immunisation
100 μg per mouse
Commercial assay or kitEasySep CD8 positive isolation kitSTEMCELL Technologies, UKCat # 19853
Commercial assay or kitSAH ElisaAxis-ShieldCat # FHCY100
Commercial assay or kitm6A methylationEpigentekCat # P-9005
Commercial assay or kitm5C methylationEpigentekCat # P-9009
Commercial assay or kitRneasy minikitQiagenCat # 74104
Chemical compound, drugImject AlumPierce, UKCat # 77161Immunisation
adjuvant
Chemical compound, drugOPPJena BioscienceCat # NU-93120 μM,
10 min incubation
Chemical compound, drugClick EdUCarbosynthCat # NE0870110 μM,
45 min incubation
Chemical compound, drugClick EUThermoFisherCat # E10345500 μM,
30 min incubation
Chemical compound, drugAlexa 647 azideThermoFisherCat # A102775 μM, in Click
reaction buffer
Chemical compound, drugActinomycin DSigmaCat # A14105 μg/ml, 45 min
Chemical compound, drugCyclohexamideSigmaCat # C7698100 μg/ml, 30 min
Chemical compound, drugRapamycinThermoFisherCat # PHZ123520 nM
Chemical compound, drug[3H] L-methioninePerkinElmerCat # NET061 × 001MC1 μci/ml uptake buffer
Chemical compound, drug[3H] L-phenylalaninePerkinElmerCat # NET1122001MC0.5 μci/ml uptake
buffer
Chemical compound, drug[14C] L-glutaminePerkinElmerCat # NEC451050UC0.1 μci/ml uptake
buffer
Chemical
compound, drug
silicone oilSigmaCat # 175633layering buffer
for uptake assay
Chemical compound, drugdibutyl pthalateSigmaCat # 524980layering buffer
for uptake assay
Chemical compound, drugOptiphase HiSafe 3PerkinElmerCat # 1200.437scintillant
Software, algorithmFlowJo softwareTreestarversions 9 and 10

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files, or have been submitted to the PRIDE ProteomeXchange consortium under Project IDs PXD012052,PXD012053 and PXD012058.

The following data sets were generated
  1. 1
  2. 2
  3. 3
    PRIDE
    1. V Sinclair Linda
    2. JM Howden Andrew
    3. Brenes Alejandro
    (2019)
    ID PXD012052. TCR activated CD4 proteome.

Additional files

Supplementary file 1

Flow cytometry plots showing representative gating strategies for flow data shown in Figures 1 and 2.

https://doi.org/10.7554/eLife.44210.011
Transparent reporting form
https://doi.org/10.7554/eLife.44210.012

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