1. Immunology and Inflammation
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Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses

  1. Ghada Alsaleh  Is a corresponding author
  2. Isabel Panse
  3. Leo Swadling
  4. Hanlin Zhang
  5. Felix Clemens Richter
  6. Alain Meyer
  7. Janet Lord
  8. Eleanor Barnes
  9. Paul Klenerman
  10. Christopher Green
  11. Anna Katharina Simon  Is a corresponding author
  1. The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, United Kingdom
  2. Division of Infection and Immunity, University College London, United Kingdom
  3. Fédération de médecine translationnelle Université de Strasbourg, France
  4. MRC-Versus Arthritis Centre for Musculoskeletal Ageing Research, Institute of Inflammation and Ageing, University of Birmingham, United Kingdom
  5. Peter Medawar Building for Pathogen Research,Nuffield Department of Medicine, University of Oxford, United Kingdom
  6. Translational Gastroenterology Unit, John Radcliffe Hospital, United Kingdom
  7. NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, United Kingdom
  8. Oxford Vaccine Group, Department of Paediatrics, University of Oxford, United Kingdom
Research Article
Cite this article as: eLife 2020;9:e57950 doi: 10.7554/eLife.57950
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 5 supplements
Autophagy is induced by vaccination in antigen-specific T cells and correlates with donor age.

PBMCs were isolated from blood samples of vaccinated healthy donors. LC3-II was measured in CD8+ cells using flow cytometry after 2 hr treatment with 10 nM bafilomycin A1 (BafA1) or vehicle. Autophagic flux was calculated as LC3-II mean fluorescence intensity (BafA1-Vehicle)/Vehicle. (a) Vaccine regimen for HCV and RSV trials. (b) Representative plots showing BafA1 in light blue and vehicle pink. (c) Quantification in HCV non-specific CD8+ T cells and HCV-specific CD8+ T cells detected by HCV pentamers from 10 vaccinees (includling duplicates) using different HCV vaccine regimens, priming with ChimAd and boosting with MVA or AD6 vectors. Autophagy was measured at the peak of the T cell response post prime vaccination, peak of the T cell response post boost vaccination and at the end of the study. (d) Autophagic flux was measured in CD8+ cells from young donors (N = 12,<65 years) and old donors (N = 21,>65 years) vaccinated with respiratory syncytial virus (RSV) 7 days after last boost, quantification calculated as mentioned above. Data represented as mean ± SEM. (e, f) Correlation of autophagic flux with total response to peptide pools specific T cell IFNγ response to RSV exposure measured by ELISpot in CD8+ cells from old donors (e) and young donors (f), donors as in (d). Linear regression with 95% confidence intervals from old and young donors. The goodness of fit was assessed by R2. The p value of the slope is calculated by F test.

Figure 1—source data 1

Autophagy is induced by vaccination in antigen-specific T cells and correlates with donor age.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Autophagy levels by flow cytometry-based assay and conventional LC3 western blot in Jurkat cell line and PBMCs.

(a–b) Human Jurkat T cell line was cultured for 24 hr and treated with or without Bafilomycin A1 for the last 2 hr. Cells were split into two aliquots, representative flow cytometry-based assay (a), representative western blot for LC3-II and GAPDH for the same sample (b). (c–d) PBMCs from young human donors were cultured with anti-CD3/CD28 for 3 days in the absence/presence of Bafilomycin A1 for the last 2 hr. Cells were split into two aliquots, representative flow cytometry-based assay (c), representative western blot for LC3-II and GAPDH for the same sample (d).

Figure 1—figure supplement 2
LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.

PBMCs were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hr, in the abence/presence of Bafilomycin A1 for the last 2 hr. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. Monocytes gated on CD14+ treated with IFNγ or LPS (a), B cells gated on CD19+ treated with anti-CD40L and anti-IgM (b), CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28 (c), CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28 (d).

Figure 1—figure supplement 2—source data 1

LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Regimen of immunizations and blood sampling for HCV trail.

HCV = Hepatitis C virus, ChAd = Chimpanzee Adenoviral Vector, MVA = Modified Ankara Virus vector.

Figure 1—figure supplement 4
Regimen of immunizations and blood sampling for RSV trail.

RSV = respiratory syncitial virus, ChAd = Chimpanzee Adenoviral Vector, MVA = Modified Ankara Virus vector, Unlike for HCV, the adults in the RSV study will have prior immune responses that have been boosted by natural exposure throughout life. In the context of RSV, we still use the term ‘prime’ to mean the first dose of vaccine. Similarly, the term ‘boost’ means the second dose of vaccine and not exposure.

Figure 1—figure supplement 5
Correlation of age with total and peptide-pool specific T cell IFNγ response to RSV exposure measured by ELISpot in CD8+ cells, donors as in Figure 1e.

Linear regression with 95% confidence intervals from old and young donors. The goodness of fit was assessed by R2. The p value of the slope is calculated by F test.

Figure 1—figure supplement 5—source data 1

Correlation of age with total and peptide-pool specific T cell IFNγ response to RSV.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig1-figsupp5-data1-v2.xlsx
Autophagy is required for CD8+ T cell function.

(a–c) Splenocytes from control mice (Ctrl: CD4-cre;Atg7+/+) and Atg7 knockout mice (Atg7Δcd4: CD4-cre;Atg7-/-) were cultured with anti-CD3/CD28 for 4 days and IFNγ assessed by ELISA in tissue culture supernatant (a), intracellular IFNγ by flow cytometry (b), intracellular perforin by flow cytometry (c), all gated on CD8+ T cells. (d–i) PBMCs from human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM Hydroxychloroquine (HcQ), 10 µM BSI-0206965 (SbI), 10 µM Resveratrol (Res) and IFNγ assessed by ELISA in tissue culture supernatant (d, g), intracellular IFNγ by flow cytometry (e, h), intracellular perforin by flow cytometry (f, i), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (d–i).

Figure 2—source data 1

Autophagy is required for CD8+ T cell function.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig2-data1-v2.xlsx
Figure 3 with 2 supplements
Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in old donors.

(a) Spermidine (Spd), spermine (Spm), and putrescine (Put) content of PBMCs collected from healthy donors were measured by GC-MS. Linear regression with 95% confidence intervals. The goodness of fit was assessed by R (Lurie et al., 2020). The p value of the slope is calculated by F test. (b–f) PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine alone or with 10 µM Hydroxychloroquine (HcQ), 10 µM SBI-0206965 (SbI), and autophagic flux measured by flow cytometry (b), IFNγ assessed by ELISA in tissue culture supernatant (c), intracellular IFNγ by flow cytometry (d), intracellular perforin by flow cytometry (e), intracellular granzyme B (f), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (b–f).

Figure 3—source data 1

Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in old donors.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Spermine does not improve function of CD8+ T cell from old donors.

PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine or 10 µM spermine and intracellular IFNγ assessed by flow cytometry (a), intracellular perforin by flow cytometry (b), intracellular granzyme B (d), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (a–c).

Figure 3—figure supplement 1—source data 1

Spermine does not improve function of CD8+ T cell from old donors.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Spermidine reduces mitochondrial mass in CD8+ T cell from old donors.

PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine and quantified for mitochondrial mass by flow cytometry using MitoTracker Green (MTG) (a) or nonylacridine orange (NAO) (b). Mitochondrial membrane potential was assessed by flow cytometry using TMRM dye (c) and mitochondrial ROS (mtROS) by mitoSOX staining (d). All gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. MFI normalized to Old untreated group. Statistics by paired t-test for (a–d).

Figure 3—figure supplement 2—source data 1

Spermidine reduces mitochondrial mass in CD8+ T cell from old donors.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig3-figsupp2-data1-v2.xlsx
Endogenous spermidine maintains levels of autophagy and T cell function.

(a–d) PBMCs cells from young human donors (<65 years) were activated with anti-CD3/CD28 for 7 days and treated with spermidine synthesis inhibitor 1 mM DFMO alone or together with 10 µM spermidine (Spd). Autophagic flux (a) was assessed each day and IFNγ (b), Perforin (c), Granzyme B (d) were measured by flow cytometry in CD8+ cells on day 4. (e–f) PBMCs cells from young human donors (<65 years) were cultured with anti-CD3/CD28 for 4 days and streated with 10 µM spermidine. (e) Intracellular IFNγ, (f) intracellular perforin, (g) and intracellular granzyme B were measured in CD8+ cells by flow cytometry. Data represented as mean ± SEM.

Figure 4—source data 1

Endogenous spermidine maintains levels of autophagy and T cell function.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig4-data1-v2.xlsx
Figure 5 with 2 supplements
Spermidine’s mode of action is via eIF5A and TFEB in human CD8+ T cells.

(a–d) Human T cell line Jurkat was cultured for 24 hr with 100 µM GC7, then eIF5A and hypusinated eIF5A were measured by WB (a). Jurkat cell line was stimulated with increasing concentrations of GC7 and cell lysates blotted for LC3B (b). (c–d) PBMCs from young human donors were cultured with anti-CD3/CD28 for 7 days and treated with GC7. The protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by Western blot on day 4 (c) and autophagic flux was determined as in Figure 1 (d). PBMCs from young human donors were cultured with anti-CD3/CD28 for 4 days and treated with spermidine synthesis inhibitor DFMO alone or together with 10 µM spermidine and the protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by wWestern blot (e), representative images (left) and quantified (right). PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine and the protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by wWestern blot (f), representative images (left) and quantified (right). Target band intensity was normalized to eIF5A (for Hyp) or GAPDH (for TFEB). Data represented as mean ± SEM.

Figure 5—source data 1

Dpermidine’s mode of action is via eIF5A and TFEB in human CD8+ T cells.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Spermidine does not improve eIF5A and TFEB in young donors.

PBMCs from young human donors were cultured with anti-CD3/CD28 for 4 days and treated with 10 µM spermidine for 4 days. The protein levels of TFEB and eIF5A hypusination were measured in sorted CD8+ cells by wWestern blot, representative images (left) and quantified (right). Target band intensity was normalized to eIF5A (for Hyp) or GAPDH (for TFEB). Data represented as mean ± SEM.

Figure 5—figure supplement 1—source data 1

Spermidine does not improve eIF5A and TFEB in young donors.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
TFEB is required for CD8+ T cell function.

Splenic T cells from wildtype C57BL/6 mice (WT) or tamoxifen-inducible Tfeb knockout mice (KO: CAG-Cre;Esr1+;Tfeb-/-) were cultured with anti-CD3/CD28 and 4-Hydroxytamoxifen (4-OHT) for 4 days. The protein levels of TFEB and GAPDH were measured in sorted CD8+ cells by wWestern blot (a) representative images (left) and quantified (right). Target band intensity was normalized to GAPDH. Intracellular IFNγ by flow cytometry (b), intracellular perforin by flow cytometry (c), intracellular granzyme B (d). All cells were gated on CD8+ T cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by unpaired t-test.

Figure 5—figure supplement 2—source data 1

TFEB is required for CD8+ T cell function.

https://cdn.elifesciences.org/articles/57950/elife-57950-fig5-figsupp2-data1-v2.xlsx

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)Atg7floxPMID:15866887MGI:3587769Dr. Masaaki Komatsu (Juntendo University)
Genetic reagent (M. musculus)TFEBfloxPMID:23604321Prof. Andrea Ballabio (Telethon Institute of Genetics and Medicine (TIGEM)).
Cell line (H. sapiens)Jurkat E6.1ATCCRRID:CVCL_0367
AntibodyRabbit anti-GAPDHMilliporeMAB374, RRID:AB_2107445WB (1:5,000)
AntibodyRabbit anti-LC3SigmaL8918, RRID:AB_1079382WB (1:1500)
AntibodyMouse anti-eIFA5BD Biosciences611976, RRID:AB_399397WB (1:3000)
AntibodyRabbit anti-hypusineMilliporeABS1064, RRID:AB_2631138WB (1:1500)
AntibodyRabbit anti-TFEBBethylA303-673A, RID:AB_11204751WB (1:1500)
AntibodyMouse anti-ActinCell Signaling3700, RRID:AB_2242334WB (1:10,000)
AntibodyIRDye 800CW Donkey Anti-Rabbit IgG (H+L)LI-COR926–32213, RRID:AB_621848WB (1:10,000)
AntibodyIRDye 680RD Donkey Anti-Mouse IgG (H+L)LI-COR926–68022, RRID:AB_10715072WB (1:10,000)
AntibodyBV605 anti-human CD14BioLegend301834, Clone: M5E2FACS (1:200)
AntibodyPE anti-human Granzyme BeBioscience12-8899-41, Clone: GB11FACS (1:100)
AntibodyPE/Cy7 anti-human CD8aBioLegend344712, Clone: SK1FACS (1:100)
AntibodyPE/Cy5 anti-human CD19BioLegend302210, Clone:HIB19FACS (1:100)
AntibodyBV711anti-human CD19BioLegend302245, Clone:HIB19FACS (1:200)
AntibodyAPC anti-human CD3BioLegend300312, Clone:HIT3aFACS (1:200)
AntibodyAPC anti-human PerforinBioLegend353311, Clone:B-D48FACS (1:100)
AntibodyAPC anti-human CD4BioLegend317416, Clone:OKT4FACS (1:200)
AntibodyAlexa Fluor 700 anti-human IFNγBioLegend506515, Clone:B27FACS (1:100)
AntibodyPE/Cy7 anti-mouse CD8a AntibodyBioLegend100722, RRID:AB_312761FACS (1:200)
AntibodyBV605 anti-mouse CD4 AntibodyBioLegend100451, Clone: GK1.5FACS (1:200)
AntibodyAlexa Fluor 700 anti-mouse IFNγBioLegend505823, Clone: XMG1.2FACS (1:100)
AntibodyAPC anti-mouse PerforinBioLegend154303, Clone: S16009AFACS (1:100)
AntibodyPE anti-mouse Granzyme BBioLegend372207, Clone: QA16A02FACS (1:100)
AntibodyAPC anti-human CD19BioLegend302212, RRID:AB_314242FACS (1:200)
AntibodyAlexa Fluor 700 anti-mouse CD8aBioLegend100730, RRID:AB_493703FACS (1:100)
AntibodyPE-labeled MHC class I pentamer (HLA-A*02:01, HCV peptide KLSGLGINAV)ProImmune(HLA-A*02:01, HCV peptide KLSGLGINAV)FACS (1:50)
Commercial assay or kitFlowCellect Autophagy LC3 Antibody-based Detection KitMerck MilliporeFCCH100171FACS (1:20)
Commercial assay or kitLIVE/DEAD Fixable Near-IR Dead Cell Stain KitInvitrogen
ThermoFisher
L10119FACS (1:1000)
Commercial assay or kitLIVE/DEAD Fixable Aqua Dead Cell Stain KitInvitrogen
ThermoFisher
L34957FACS (1:1000)
Commercial assay or kitCellTrace Violet Cell Proliferation KitInvitrogen,ThermoFisherC34557FACS (1:1000)
Commercial assay or kitMitoTracker GreenInvitrogen,ThermoFisherM7514FACS (150 nM)
Commercial assay or kitMitoSoxInvitrogen,ThermoFisherM36008FACS (5 uM)
Commercial assay or kitCD3/CD28 activation DynabeadsThermo Fisher11161D
Commercial assay or kitHuman IFN-γ ELISpotPLUS kit (ALP)Mabtech3420-4APT-2
Commercial assay or kitIFNγ ELISA KitLife Technologies Ltd88-7316-22
Commercial assay or kitEasySep Human CD8+ T Cell Isolation KitStemcell17953
Commercial assay or kitBCA AssayThermo Fisher23227100 μl/sample
Chemical compound, drugSpermidineCayman Chemical1491810 μM
Chemical compound, drugSpermineCayman Chemical1804110 μM
Chemical compound, drugHydroxychloroquine SulfateStratech Scientific LtdB4874-APE-10mM10 μM
Chemical compound, drugBafilomycin A1Cayman Chemical1103810 μM
Chemical compound, drugResveratrolStratech Scientific LtdA4182-APE10 μM
Chemical compound, drugBSI-0206965Stratech Scientific LtdA8715-APE10 μM
Chemical compound, drugAICRStratech Scientific LtdA8184-APE10 μM
Chemical compound, drugNAOThermo Fisher ScientificA1372100 nM
Chemical compound, drugTRMRThermo Fisher ScientificT668FACS (100 nM)
Chemical compound, drugDifluoromethylornithine (DFMO)Enzo Life SciencesALX-270–283 M0101 mM
Chemical compound, drugGC7Millipore259545–10 MG10 μM
Chemical compound, drug4-HydroxytamoxifenSigmaH7904100 nM
Chemical compound, drugLPSSanta Cruzsc-353510 µg/ml
Chemical compound, drugAanti-IgMJackson Immuno Research109-005-0435 µg/ml
Chemical compound, drugAanti-CD40LEnzo Life scienceALX-522–015 C010100 ng/ml,
Peptide, recombinant proteinHuman IFNγEnzo Life scienceENZ-PRT141-010020 ng/ml
Software, algorithmImage Studio LiteLI-COR
Software, algorithmPrismGraphPad

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