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An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory

Nature Cell Biology volume 24, pages 1461–1474 (2022)Cite this article

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Abstract

The common view is that T lymphocytes activate telomerase to delay senescence. Here we show that some T cells (primarily naïve and central memory cells) elongated telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs) independently of telomerase action. Upon contact with these T cells, APCs degraded shelterin to donate telomeres, which were cleaved by the telomere trimming factor TZAP, and then transferred in extracellular vesicles at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled telomere fusion with T-cell chromosome ends lengthening them by an average of ~3,000 base pairs. Thus, there are antigen-specific populations of T cells whose ageing fate decisions are based on telomere vesicle transfer upon initial contact with APCs. These telomere-acquiring T cells are protected from senescence before clonal division begins, conferring long-lasting immune protection.

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Fig. 1: APCs donate telomeres to T cells.
Fig. 2: Synaptic TCRs are sufficient to extract telomere vesicles from APCs.
Fig. 3: TZAP is required for telomere transfer.
Fig. 4: APCs dismantle shelterin to donate telomeres.
Fig. 5: Defective recombinogenic potential in Rad51-deficient telomere vesicles.
Fig. 6: Generation of long-lasting immunity by telomere transfer.
Fig. 7: Vesicular Rad51 is required for the longevity effect of the telomere vesicles.
Fig. 8: Role of telomere vesicle transfer in immune defence.

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Data availability

Source data are provided with this paper. Further data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This study was supported by the Wellcome Trust (110229/Z/15/Z) and the Italian Ministry of Health (GR-2018 12365916) to A.L. Laboratory infrastructures were provided by Sentcell Ltd. M.L.D. was supported by the Wellcome Trust Principal Research Fellowship 100262Z/12/Z and the Kennedy Trust for Rheumatology Research. A.N.A. was supported by the Medical Research Council (MR/P00184X/1). M.K. is supported by the NIH (R37AI04477). A.L. is an Honorary Associate Professor of the University College London and the Chief Executive Officer of Sentcell Ltd. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Bruno Vaz, Clara D’Ambra, Salvatore Valvo, Claudia Vuotto.

Authors and Affiliations

  1. Sentcell UK Laboratories, IRCCS Fondazione Santa Lucia, Rome, Italy

    Alessio Lanna, Bruno Vaz & Clara D’Ambra

  2. Department of Experimental and Translational Medicine, Division of Medicine, University College London, London, UK

    Alessio Lanna, Arne N. Akbar & Derek W. Gilroy

  3. Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

    Salvatore Valvo & Michael L. Dustin

  4. Experimental Neuroscience, IRCCS Fondazione Santa Lucia, Rome, Italy

    Claudia Vuotto

  5. Institute of Translational Pharmacology, National Research Council, Rome, Italy

    Valerio Chiurchiù

  6. Laboratory of Resolution of Neuroinflammation, IRCCS Santa Lucia Foundation, Rome, Italy

    Valerio Chiurchiù

  7. Division of Infection and Immunity, University College London, London, UK

    Oliver Devine & Arne N. Akbar

  8. ISS, Core Facilities, Rome, Italy

    Massimo Sanchez

  9. NeuroImmunology Unit, IRCCS Fondazione Santa Lucia, Rome, Italy

    Giovanna Borsellino & Marco De Bardi

  10. Block.one, George Town, Cayman Islands

    Brendan Blumer

  11. Block.one, Hong Kong, Hong Kong

    Brendan Blumer

  12. Laboratory of Gene Regulation and Signal Transduction, University California, San Diego, CA, USA

    Michael Karin

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Contributions

A.L. conceived of, performed and directed the study, analysed and interpreted the data, provided funding and laboratory infrastructures, formed collaborations and wrote the paper; B.V., C.D. and C.V. designed and performed experiments and analysed individual datasets; S.V. performed lipid bilayer experiments; V.C. performed immune-phenotyping, cell-death (human) and donor cell (mouse) analysis and provided key samples; O.D. performed initial FISH and collected lymph nodes; M.S. performed fluorescence-activated vesicle sorting; M.D.B. performed APC subset isolation; A.N.A. provided initial infrastructures; G.B. performed APC subset re-analysis of telomere transfer under the guidance of A.L.; D.W.G. performed in vivo manipulations; M.L.D. provided lipid bilayer infrastructure, conceptual framework for synaptic vesicles, feedback and advice; B.B. inspired decentralized immunity concept and supported A.L.; M.K. inspired A.L., provided feedback and advice, and supported experimental revisions of the final version of the manuscript, which was read, commented on and approved by all authors. B.V., C.D., C.V., and S.V. contributed equally to this work.

Corresponding author

Correspondence to Alessio Lanna.

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A.L. is the founder of Sentcell and Electra Life Sciences Ltd. The remaining authors declare no present competing interests.

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Nature Cell Biology thanks Clotilde Théry and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Telomere elongation in the absence of DNA synthesis.

(a) Representative IF-FISH and (b, top) pooled data showing telomere elongation in nonsenescent CD4 + T cells and concomitant telomere shortening in APCs after forming the synapse. APCs from human donors were pre-loaded with cytomegalovirus lysates and allowed to interact with autologous nonsenescent CD4+ T cells in 3:1 ratio for the indicated time points. Conjugates were fixed and analysed by IF-FISH. Data are from n = 3 donors (three independent experiments). Scale bar, 10 µm. T = 0, initial time at which conjugates are observed (20 min). Images were z-stacked, and the raw telomere integrated fluorescence signals (AU, arbitrary units) are shown. One-hundred two conjugates were analysed. (b, bottom) Analysis of telomere length by qPCR in APCs and nonsenescent CD4 + T cells after forming conjugates. (c-d) Primary human nonsenescent CD4+ T cells were transfected by nucleofection with sgCtrl or sgTERT CRISPR constructs, activated with anti-CD3 plus anti-CD28 and elimination of telomerase was confirmed by (c) qPCR and (d) TRAP assay. (e) Telomerase positive (transfected with sgCTRL) and negative (transfected with sgTERT) nonsenescent T cells were exposed to APCs in the presence of antigen pool, and telomere content was quantified by Flow-FISH using TelC telomere probe. Absolute telomere length by Flow-FISH was determined from Mean Fluorescence Intensity (MFI) values using a standard curve formed by cryopreserved samples with known telomere length as determined by TRF. (f) Telomere content was measured by flow-FISH using either TelC or TelG telomere PNA specific probes coupled to anti-BrdU detection (to monitor telomere elongation vs DNA synthesis in T cells) in telomerase negative nonsenescent CD4+ T cells (CRISPR KO sgTERT) and control T cells (sgCtrl) stimulated with antigen pool for 48 h. (g) Telomere length by flow-FISH demonstrating telomere elongation in primary human nonsenescent CD4+ T cells treated with DNA polymerase inhibitors aphidicolin and thymidine prior to exposure to APCs for 48 h. Data are from n = 3 donors throughout. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

Source data

Extended Data Fig. 2 Generation of live APCs with fluorescent telomeres.

(a) PNA-telomere probes were dissolved 1:50 in telomere loading buffer (80 mM KCl, 10 mM K2PO4, 4 mM NaCl, pH 7.2) and gently introduced on adherent APCs by rolling large glass beads (size: 425-600 µm) for 2 min on cell surface to create temporary pores on the APC membranes and allow telomere TelC PNA probe entry into the APCs while preserving their viability (no cell fixative) needed for subsequent synapse studies with T cells. Arrow indicates beads. The beads were completely removed by washing two times with PBS prior to assays. (b) Absence of any residual glass beads from TelC PNA probe labelled APCs was confirmed by FESEM. Scale bar, 2 µm. Representative of n = 3 experiments (three donors). (c) Identical telomere detection by FISH (fixed cells) or glass bead-mediated telomere PNA probe delivery (live cells) in APCs. Nuclei were counterstained by DAPI (blue). Representative of n = 4 experiments (four donors). Scale bar, 20 µm. (d) Manders co-localization scores of experiments as in (c). Negative control, APCs with unlabelled telomeres. (e) Immunofluorescence staining of POT1 and telomere TelC PNA probe live delivery on primary human APCs (CD3-depleted PBMCs). POT1 recruitment to telomeres was detected directly ex vivo. Scale bar, 10 µm. (f) Manders co-localization scores of experiments as in (e). Results are from n = 3 donors (two experiments). Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

Source data

Extended Data Fig. 3 APCs donate telomere vesicles.

(a) Telomere transfer through the immune synapse, confocal imaging. Scale bar, 5 µm. Representative of n = 3 donors. (b, top left) Telomere vesicle release triggered by 18 h antigen-specific contacts of APCs and nonsenescent CD4+ T cells. (b, bottom left) Alu release in the same T cells. (n = 6 donors b, right). (c, left and middle) Side scatter (SSC) threshold and calibration beads used in FAVS-based vesicle purifications, gated on singlets (Extended Data Fig. 3c, left panel). Extended Data Fig. 3c, middle panel, size of the beads. (c, right) PKH67 lipid staining of all vesicles produced by primary human APCs upon 18 h ionomycin activation. Unstained PKH67, threshold control. Gating strategy for individual vesicles <100 nm up to 300 nm. Larger particles (>300 nm) due to aggregates were excluded. (d, left) PKH67 staining and (d, middle and right) presence of telomere vesicles in ~10% of the total APC single particle vesicle fraction. (n = 9 donors; d, far right). (e) Telomeric DNA from telomere vesicles was purified and confirmed by qPCR (n = 3 donors form three independent experiments). (f) Presence of a small population (~1%) of vesicle free telomeres released by APCs during FAVS. Representative of n = 9 experiments. (g) Dot-blot analysis of telomeric DNA isolated from different fraction of vesicles isolated from sequential centrifugation of APC supernatants under native or denaturing conditions. APC genomic DNA (gDNA), loading control. Representative of n = 3 donors. (h) Super-resolution Zeiss Airyscan microscopy of FAVS-purified Tel+ vesicles. A representative experiment from n = 3 independent experiments (three donors) is shown. Scale bar, 200 nm. (i) TEM analysis of Tel− vs Tel+ vesicles. Examples for small−sized vesicles (top) and larger vesicles (bottom) are shown for both Tel- and Tel+. Quantifications of telomeric DNA in telomere vesicles distributed by size (right). Representative images (left) and pooled vesicle data (right) (n = 10; from three independent experiments). (j) Further examples of T cell chromosomes with APC telomeres. Metaphase experiments of T cells with APC telomeres were performed n = 5 times. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

Source data

Extended Data Fig. 4 APC telomeres at T cell chromosome ends.

(a) Representative metaphase spreads showing T cell chromosomes with APC derived telomeres. Scale bar, 0.85 µm. Representative of n = 3 donors. (b) Metaphase spreads generated as in (a) were treated with 1 unit T7 endonuclease for 30 min at 37 °C. The number of T cell chromosomes with APC telomeres before (black values) and after (red values) T7 endonuclease treatment is shown. Pooled from n = 3 experiments (three donors). (c) Presence of APC-derived telomeres in purified nonsenescent T cell plasma membranes from (106) vs T cell nuclei from the same cells 24 h after transfer of fluorescent telomere enriched supernatants derived from APCs activated with ionomycin. The T cell plasma membranes were assessed by confocal imagining on LEICA SP2. APC telomeres were only observed in the nuclear fraction but not in the T cell plasma membrane after the 24 hours incubation. Pooled data from n = 3 independent experiments (three donors). (d) Quantification of APC telomere signal after T cell chromatin immunoprecipitation. Primary human nonsenescent CD3+ T cells (107) were activated by anti-CD3 plus anti-CD28 overnight in in the presence or absence of fluorescent telomere enriched supernatants. The nuclei were digested after lysis with MNase-based Thermofisher Pierce Agarose Chip kit. Chromatin derived from digestion was immunoprecipitated with polyclonal anti-POT1 (1:100) or control rabbit IgG antibodies. The Cy3-fluorescence of APC-derived telomeres was quantified with a microplate reader. Control IP, T cell extracts precipitated with irrelevant IgG. Presence of APC-derived telomeres was confirmed by adding DNase directly to the POT1 IP for 10 min at room temperature prior to fluorescence reading. Pooled results from n = 3 donors. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

Source data

Extended Data Fig. 5 Telomere transfer does not cause APC death.

(a) Analysis of cell death of APCs after 18 h stimulation with or without ionomycin (0.5 µg/mL), or with hydrogen peroxide (H2O2; 500 µM) as death positive control. Cell death was analysed 18 h later by FITC Annexin V/PI Apoptosis staining with flow cytometry. Pooled data from n = 7 independent biological experiments are shown. (b) FESEM micrographs (10,000x) of resting APCs or activated APCs upon treatment with ionomycin or H2O2 for 18 h. Scale bar 1 µm. Pooled data from n = 12 micrographs (3-5 APCs per micrograph at 10,000x magnification) depicting %APCs with structural alterations (blebbing or membrane damage) are shown. Note that ionomycin treatment does not induce membrane blebbing. APCs treated with H2O2 (500 µM) served as positive control throughout experiments. Each dot is an individual cell from n = 3 independent experiments (three donors) (c, left). APCs were coupled to nonsenescent CD4+ T cells in the presence or in the absence of the antigen pool for 18 h then analysed by Annexin/PI with flow cytometry. (c, right). Pooled results from n = 3 independent experiments (three donors). (d) APCs were separated into their main subsets of DCs, Monocytes and B cells by FACS sorting then 106 cells/subset were live labelled with TelC PNA probes and PKH67 lipid dye, stimulated with ionomycin for 18 h, followed by FAVS analysis of APC subset supernatants. Note that hypo or non-proliferative myeloid cells are the major telomere donors. (d, right) Cumulative data from n = 3-4 donors are shown (three experiments). (e) The sorting strategies and related purities to derive DCs (99.1%), Monocytes (99.3) and B cells (98.8%) for experiments in (d). Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

Source data

Extended Data Fig. 6 Generation of artificial shelterin APCs.

(a, left) IF-FISH demonstrating recruitment of artificial shelterin factors (POT1 and TRF2) to telomeres in primary human APCs transduced with the lentiviral vectors (mock vector and TRF2/POT1 vectors, see methods) and activated with ionomycin for 18 h, 96 h post transduction. The Pearson’s co-localization score for artificial POT1 and TRF2 with APC telomeres are shown. Scale bar, 5 µm. (a, right) Validation of shelterin overexpression (TRF2 and POT1) by immunoblotting in primary human APCs. Numbers indicate shelterin overexpression efficiency. H2B, loading control. (b) Immunoblot analysis of TRF2 and POT1 following indicated siRNA treatment in primary human APCs. H2B, loading control. The numbers indicate knock-down efficiency. Shelterin knock down APCs were generated by siTRF2 plus siPOT1 transfection from resting primary human APCs. Seventy-two hours later telomere release was analyzed as above described in the absence of ionomycin activation. Results are representative of n = 3 independent experiments (three donors) throughout.

Source data

Extended Data Fig. 7 Telomere vesicle effects do not require telomerase.

(a) Expansion of human T cells by heterologous telomere vesicles. Nonsenescent CD4+ T cells were activated with anti-CD3 and anti-CD28 and cultured ten days with or without 1,000 telomere vesicles (Tel+) or telomere depleted vesicles (Tel-) purified by FAVS. The vesicles were derived from donor mismatched human (h) or mouse (m) APCs, as indicated, upon ionomycin activation. Different biological cultures are shown (n = 6 no vesicle; n = 18 Tel neg; n = 12 Tel pos human, n = 6 Tel pos; n = 3 free Tel human). (b) Confirmation of CRISPR-based telomerase enhancement in nonsenescent CD4+ T cells by TRAP assay (top) and immunoblots (bottom). (c) Population doublings (n = 3 donors) of nonsenescent CD4+ T cells cultured as indicated for 30 days. (d) Telomere positive and negative nonsenescent CD4+ T cells were activated with anti-CD3 and anti-CD28 for 10 days in the presence of 1,000 telomere vesicles, telomere depleted vesicles or left without any vesicles; n = 3 experiments (three donors) throughout cultures. (e) Defective proliferation in primary human nonsenescent CD4+ T cells supplemented with siTZAP telomere vesicles that do not express TZAP compared to those expressing TZAP. Results from n = 4 independent experiments (four donors). (f, top). Telomere vesicles produced by TZAP-artificial APCs were purified by FAVS following ionomycin activation for subsequent stimulation of T cells. (f, bottom) Proliferative expansion of T cells with TZAP + vesicles was tested as in (a). N = 13 (Ctrl); n = 16 (TZAP + ), n = 10 (no vesicle); n = 14 (Tel pos). (g) Reduced load of ultra-short telomeres (<3 kb) in nonsenescent CD4+ T cells activated by anti-CD3 plus anti-CD28 for 48 h followed by transfer of 1,000 FAVS-purified telomere vesicles (Tel pos) or vector-based telomerase enhancement assessed by U-STELA. Controls, T cells with mock vector or 1,000 telomere depleted vesicles. Results from n = 5 (tel neg); n = 8 (tel pos; mock vector) or n = 6 (TERT-OE). Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

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Extended Data Fig. 8 Signaling and phenotypic changes of T cells with APC telomeres.

(a) Representative immunofluorescence (IF) staining of sestrin 1 in primary human T cells cultured with or without telomere vesicles derived from APCs previously transfected with either siCtrl or siRad51 RNAs then transferred to primary human nonsenescent CD4+ T cells activated by anti-CD3 plus anti-CD28 for ten days. Telomere depleted vesicles (telomere neg) as background control. Representative of 3 donors. (b) Data shown are pooled from n = 3 donors, with each dot being an individual T cell. (c) Primary human nonsenescent CD4 + T cells (105) were activated with anti-CD3 (0.5 µg/mL) and recombinant human IL-2 (10 ng/mL) for 10 days in the presence of 250 FAVS-purified telomere vesicles derived from either human or mouse APCs prior to multiparametric flow cytometry. Control T cells were activated without any vesicle or with 250 telomere depleted vesicles obtained by FAVS. Representative plots and (d) pooled data from n = 5 independent experiments are shown. Numbers indicate mean fluorescence intensity (MFI) value from a representative experiment. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.

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Extended Data Fig. 9 Naive and central memory T cells are the major telomere acquiring cells from APCs.

(a) Analysis of telomere transfer by flow FISH flow cytometry upon conjugation of APCs live-labelled with TelC PNA telomere probes and total primary human CD3+ T cells for 24 hours. Each dot is an individual donor from n = 4 independent biological experiments. Control, APCs loaded with antigen pool and stimulated with T cells but without telomere labelling throughout experiments (no APC telomere). No antigen (pool) control is also shown confirming antigen dependency. (b) Naïve and central memory T cells are the major APC telomere acquiring cells. Purified primary human CD4+ T cell populations (CD28+ CD45RA+ naïve purity 98.7%; CD28+ CD45RA− central memory (CM) purity 95%; CD28− CD45RA− senescent effector memory (EM) 97.5%; senescent CD28− CD45RA+ EMRA purity 94%) were treated as in (a) and telomere transferred was measured by flow FISH with TelC probe. Pooled results from n = 3 (CM and EM) and n = 4 (naïve and EMRA) independent individual donors. Note that since primary human CD4+ T cells first lose expression of CD27 followed by that of CD28, CD28− CD4+ T cells are highly differentiated cells, many of which are considered senescent8,31,32,33,34,42. The opposite regulation occurs in primary human CD8 T cells, where the CD27- population is considered highly differentiated/senescent since the cells first expression of CD28 followed by that of CD279,32. The reason for this is not known. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M.

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Extended Data Fig. 10 Existence of T cells with telomeres of APC origin in mice.

(a) Quantification of APC telomeres at mouse T cell chromosomes upon in vivo APC labelling. Two representative examples are shown. As control, mouse OTII CD4+ T cells were analysed by IF-FISH in the absence of telomere vesicles (no vesicle control). Scale bar, 2 µm. FACS plot, Edu incorporation control in donor APC prior to telomere transfer. Representative of n = 3 mice. (b) Naïve CD45.2 OTII CD4+ T cells were incubated with congenic TelC labelled APCs in the presence of OVA (3 μM) for 18 hours then sorted into CD45.2 OTII CD4+ Tel + (T cells with APC telomeres) versus CD45.2 OTII CD4+ Tel- (T cells without APC telomeres) based on telomere transfer prior to transfer into CD45.1 recipients and vaccination with OVA (30 μg). Effector responses were assessed 5 days post-transfer; for memory responses mice were re-vaccinated with OVA (30 μg) forty days after the first vaccination and observed after additional fifty days. Note that the in vitro efficacy of telomere transfer is much lower than that observed in vivo, possibly due to the well-recognized lower efficiency of in vitro APC-T cell conjugates versus their physiological counterparts in vivo. (c) Phenotype of donors CD45.2 OTII CD4+ T cells as in Fig. 6h. and (d) the same markers for experiments as in Fig. 6i-j. (e-f) Percentage of CD45.2 OTII CD4+ Tel+ vs CD45.2 OTII CD4+ Tel- in the blood of recipient mice vaccinated with OVA during effector and memory responses. Each dot is an individual animal (Tel neg n = 9; Tel pos n = 7 animals, e; and n = 5 per group in f). Statistical Tests are provided in the Supplementary Table 1. Each dot is an individual mouse. Error bars indicate SEM.

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Supplementary information

Reporting Summary

Supplementary Table

This file contains an Excel workbook with multiple tabs. Supplementary Table 1. Statistical tests. Supplementary Table 2. Antibody list. Supplementary Table 3. Primer sequences. Supplementary Table 4. Clinical score criteria.

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Lanna, A., Vaz, B., D’Ambra, C. et al. An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory. Nat Cell Biol 24, 1461–1474 (2022). https://doi.org/10.1038/s41556-022-00991-z

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