Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses
- The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, United Kingdom
- Division of Infection and Immunity, University College London, United Kingdom
- Fédération de médecine translationnelle Université de Strasbourg, France
- MRC-Versus Arthritis Centre for Musculoskeletal Ageing Research, Institute of Inflammation and Ageing, University of Birmingham, United Kingdom
- Peter Medawar Building for Pathogen Research,Nuffield Department of Medicine, University of Oxford, United Kingdom
- Translational Gastroenterology Unit, John Radcliffe Hospital, United Kingdom
- NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, United Kingdom
- Oxford Vaccine Group, Department of Paediatrics, University of Oxford, United Kingdom
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. …
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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), …
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 …
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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 …
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.
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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
Figure 2
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 …
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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 …
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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 …
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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 …
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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
Figure 4
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 (S…
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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 …
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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+ …
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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 …
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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 | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Atg7flox | PMID:15866887 | MGI:3587769 | Dr. Masaaki Komatsu (Juntendo University) |
| Genetic reagent (M. musculus) | TFEBflox | PMID:23604321 | Prof. Andrea Ballabio (Telethon Institute of Genetics and Medicine (TIGEM)). | |
| Cell line (H. sapiens) | Jurkat E6.1 | ATCC | RRID:CVCL_0367 | |
| Antibody | Rabbit anti-GAPDH | Millipore | MAB374, RRID:AB_2107445 | WB (1:5,000) |
| Antibody | Rabbit anti-LC3 | Sigma | L8918, RRID:AB_1079382 | WB (1:1500) |
| Antibody | Mouse anti-eIFA5 | BD Biosciences | 611976, RRID:AB_399397 | WB (1:3000) |
| Antibody | Rabbit anti-hypusine | Millipore | ABS1064, RRID:AB_2631138 | WB (1:1500) |
| Antibody | Rabbit anti-TFEB | Bethyl | A303-673A, RID:AB_11204751 | WB (1:1500) |
| Antibody | Mouse anti-Actin | Cell Signaling | 3700, RRID:AB_2242334 | WB (1:10,000) |
| Antibody | IRDye 800CW Donkey Anti-Rabbit IgG (H+L) | LI-COR | 926–32213, RRID:AB_621848 | WB (1:10,000) |
| Antibody | IRDye 680RD Donkey Anti-Mouse IgG (H+L) | LI-COR | 926–68022, RRID:AB_10715072 | WB (1:10,000) |
| Antibody | BV605 anti-human CD14 | BioLegend | 301834, Clone: M5E2 | FACS (1:200) |
| Antibody | PE anti-human Granzyme B | eBioscience | 12-8899-41, Clone: GB11 | FACS (1:100) |
| Antibody | PE/Cy7 anti-human CD8a | BioLegend | 344712, Clone: SK1 | FACS (1:100) |
| Antibody | PE/Cy5 anti-human CD19 | BioLegend | 302210, Clone:HIB19 | FACS (1:100) |
| Antibody | BV711anti-human CD19 | BioLegend | 302245, Clone:HIB19 | FACS (1:200) |
| Antibody | APC anti-human CD3 | BioLegend | 300312, Clone:HIT3a | FACS (1:200) |
| Antibody | APC anti-human Perforin | BioLegend | 353311, Clone:B-D48 | FACS (1:100) |
| Antibody | APC anti-human CD4 | BioLegend | 317416, Clone:OKT4 | FACS (1:200) |
| Antibody | Alexa Fluor 700 anti-human IFNγ | BioLegend | 506515, Clone:B27 | FACS (1:100) |
| Antibody | PE/Cy7 anti-mouse CD8a Antibody | BioLegend | 100722, RRID:AB_312761 | FACS (1:200) |
| Antibody | BV605 anti-mouse CD4 Antibody | BioLegend | 100451, Clone: GK1.5 | FACS (1:200) |
| Antibody | Alexa Fluor 700 anti-mouse IFNγ | BioLegend | 505823, Clone: XMG1.2 | FACS (1:100) |
| Antibody | APC anti-mouse Perforin | BioLegend | 154303, Clone: S16009A | FACS (1:100) |
| Antibody | PE anti-mouse Granzyme B | BioLegend | 372207, Clone: QA16A02 | FACS (1:100) |
| Antibody | APC anti-human CD19 | BioLegend | 302212, RRID:AB_314242 | FACS (1:200) |
| Antibody | Alexa Fluor 700 anti-mouse CD8a | BioLegend | 100730, RRID:AB_493703 | FACS (1:100) |
| Antibody | PE-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 kit | FlowCellect Autophagy LC3 Antibody-based Detection Kit | Merck Millipore | FCCH100171 | FACS (1:20) |
| Commercial assay or kit | LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit | Invitrogen ThermoFisher | L10119 | FACS (1:1000) |
| Commercial assay or kit | LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Invitrogen ThermoFisher | L34957 | FACS (1:1000) |
| Commercial assay or kit | CellTrace Violet Cell Proliferation Kit | Invitrogen,ThermoFisher | C34557 | FACS (1:1000) |
| Commercial assay or kit | MitoTracker Green | Invitrogen,ThermoFisher | M7514 | FACS (150 nM) |
| Commercial assay or kit | MitoSox | Invitrogen,ThermoFisher | M36008 | FACS (5 uM) |
| Commercial assay or kit | CD3/CD28 activation Dynabeads | Thermo Fisher | 11161D | |
| Commercial assay or kit | Human IFN-γ ELISpotPLUS kit (ALP) | Mabtech | 3420-4APT-2 | |
| Commercial assay or kit | IFNγ ELISA Kit | Life Technologies Ltd | 88-7316-22 | |
| Commercial assay or kit | EasySep Human CD8+ T Cell Isolation Kit | Stemcell | 17953 | |
| Commercial assay or kit | BCA Assay | Thermo Fisher | 23227 | 100 μl/sample |
| Chemical compound, drug | Spermidine | Cayman Chemical | 14918 | 10 μM |
| Chemical compound, drug | Spermine | Cayman Chemical | 18041 | 10 μM |
| Chemical compound, drug | Hydroxychloroquine Sulfate | Stratech Scientific Ltd | B4874-APE-10mM | 10 μM |
| Chemical compound, drug | Bafilomycin A1 | Cayman Chemical | 11038 | 10 μM |
| Chemical compound, drug | Resveratrol | Stratech Scientific Ltd | A4182-APE | 10 μM |
| Chemical compound, drug | BSI-0206965 | Stratech Scientific Ltd | A8715-APE | 10 μM |
| Chemical compound, drug | AICR | Stratech Scientific Ltd | A8184-APE | 10 μM |
| Chemical compound, drug | NAO | Thermo Fisher Scientific | A1372 | 100 nM |
| Chemical compound, drug | TRMR | Thermo Fisher Scientific | T668 | FACS (100 nM) |
| Chemical compound, drug | Difluoromethylornithine (DFMO) | Enzo Life Sciences | ALX-270–283 M010 | 1 mM |
| Chemical compound, drug | GC7 | Millipore | 259545–10 MG | 10 μM |
| Chemical compound, drug | 4-Hydroxytamoxifen | Sigma | H7904 | 100 nM |
| Chemical compound, drug | LPS | Santa Cruz | sc-3535 | 10 µg/ml |
| Chemical compound, drug | Aanti-IgM | Jackson Immuno Research | 109-005-043 | 5 µg/ml |
| Chemical compound, drug | Aanti-CD40L | Enzo Life science | ALX-522–015 C010 | 100 ng/ml, |
| Peptide, recombinant protein | Human IFNγ | Enzo Life science | ENZ-PRT141-0100 | 20 ng/ml |
| Software, algorithm | Image Studio Lite | LI-COR | ||
| Software, algorithm | Prism | GraphPad |
