Intercellular telomere transfer extends T cell lifespan

Background

Mammalian telomeres are TTAGGG repeats that mark chromosome ends. At each cell division, telomeres shorten due to the uncomplete replication of DNA ends. When telomere length shortens to a critical point, cells stop proliferating and enter a state known as replicative senescence (Campisi and d’Adda di Fagagna, 2007). Stem and cancer cells can circumvent this problem by upregulating telomerase, a ribonucleoprotein able to elongate telomeres by synthesizing new telomeric repeats. Some cancer cells adopt an alternative lengthening of telomeres (ALT) mechanism based on homologous-recombination (Doksani, 2019). While telomerase-dependent and -indepent phatways have been extensively studied, the possibility that telomeres are transferred between cells to counteract telomere shortening had never been explored—until now. In their preprint, Lanna and colleagues suggest that T lymphocytes, key cells in the immune system, elongate their telomeres by acquiring those of antigen presenting cells (APC).

From Vaz et al, 2020 (CC-BY-NC-ND 4.0 International license).

 

Key observation: At the immunological synapse, T cell telomeres elongate while APC telomeres shorten.

The authors mixed human primary T cells with autologous APCs and specific antigens to obtain immunological synapses. Then they measured telomere length by either in situ hybridisation (IF-FISH) in fixed cells (still involved in the synapses) or by telomere restriction fragment (TRF) or qPCR analyses in the two different cell populations (sorted based on their cellular markers). They observed that synapse formation was followed by telomere elongation in T cells to the expense of APC telomeres.

The mechanism was still unclear; however, it seemed to differ from the ‘canonical’ ones. Telomerase-dependent elongation mechanisms were excluded by reproducing the results in TERT (the catalytic subunit of telomerase) knockout T cells. Moreover, from the observation that telomeres still elongate in T cells upon immunological synapse formation, despite chemical inhibition of DNA synthesis (by 48h treatment with aphidicolin or thymidine), the authors excluded ALT-based elongation mechanisms.

 

The proposed model, step by step:

• Upon formation of the immunological synapse, APCs downregulate the telomere protection machinery, upregulate TZAP-mediated telomere trimming and release telomere vesicles.

The authors observed that activated APCs release telomeric DNA into the extracellular space. After labelling APC DNA with the thymidine analogue BrdU, the presence of extracellular DNA was tested by BrdU IP in cell-free supernatant. Not only was DNA present in the extracellular space, but this was telomeric DNA, as revealed by dot-blot using telomeric probes. Moreover, DNase degraded the extracellular telomeric DNA only upon pre-treatment with detergents, thus suggesting that telomeric DNA is contained in lipidic vesicles. This was further confirmed by transmission electron microscopy (TEM) and immunogold-based telomeric detection.

Activated APCs downregulated the telomere protection machinery (shelterin complex) and upregulated the telomeric zinc-finger associated protein TZAP, which promotes trimming of telomeric DNA ends (Li, 2017). Consistent with TZAP binding to telomeres only in the presence of low shelterin levels (Li, 2017), overexpression of shelterin reduced telomere vesicle release. Conversely, depletion of shelterin components induced the release of telomere-containing vesicles. TZAP was contained in the telomeric vesicles and required for their formation.

 

• APC telomeric DNA is integrated into T cell chromosome ends

To monitor the presence of APC telomeric DNA at T cell chromosome ends, the authors incubated T cells with vesicles containing BrdU-labelled APC telomeres. From the analysis of T cells metaphase spreads the authors concluded that 10% of the telomeric signal, detected by IF-FISH, is also BrdU positive, thus suggesting co-localization with APC DNA. A similar result was obtained with APCs live-labelled with fluorescent telomeric probes. From the observation that T cell chromosomes with APC-derived telomeres are destroyed upon incubation with T7 endonuclease (an enzyme that cleaves DNA mismatches and non-β DNA structures including recombination intermediates), the authors concluded that APC telomeres are integrated within T cell chromosomes.

 

• The recombination protein RAD51 associates with APC telomeres and facilitates integration of APC telomeric repeats at T cell chromosome ends.

The authors observed that telomere vesicles contained several DNA damage proteins, including the recombination protein RAD51. Activated APCs depleted for RAD51 formed telomere vesicles with the same efficiency as control cells, as monitored by flow cytometry. However, those telomers had shorter single-stranded overhangs compared to controls. They co-localized with the T cell telomeres to a lower extent and failed to promote T cell telomere elongation, as monitored by quantitative (Q)-FISH.

 

• APC telomeres support T cell expansion in vitro and in vivo.

Upon activation, T cells expanded exponentially for ~10 days and then reached a plateau phase, corresponding to an increase in senescence markers, as monitored by beta-galactosidase staining. Incubating proliferating T cells with telomere vesicles (both autologous and allogenic from mismatched human and mouse APCs) reduced the percentage of senescent cells and sustained T cell expansion, thus suggesting that APCs support T cell proliferation in vitro.

To test whether the same process occurs in mice, the authors exploited the OT-II OVA system: they introduced into mice live-stained T cells expressing a transgenic T cell receptor for the chicken ovalbumin (obtained from the OT-II mice) and ovalbumin-pulsed APCs with fluorescently labelled telomeres. One day later, they observed that antigen-specific T cells had acquired fluorescent telomeres from APCs. Additionally, they demonstrated that administration of APC telomere vesicles sustains the expansion of a subset of T cells upon vaccination of the mice with ovalbumin, thus recapitulating the proposed model in vivo.

 

Why I chose this preprint

I was extremely curious about this preprint since few days after it was posted my Twitter was full of notifications citing it–and it is not even exactly my field! I love the novelty and the provoking model suggested and the possible implications this finding could have, in particular in ageing-related diseases and cancer immunotherapy.

 

Questions for the author

The authors prove that resting T cells are still able to integrate APC telomeres. Does it imply that T cells not involved in the same immunological synapse, and possibly other cell types, could uptake those vesicles as well? Do T cells control this process somehow? More philosophically, it would be interesting to know whether other cell types could undergo the same telomere exchange.

I am curious about the mechanism of telomeric integration. It would be interesting to monitor DNA damage induction at telomeres in both the cell types and eventually monitor the composition of the telomere overhangs contained in the telomere vesicles. The authors propose that RAD51 role is to protect the ssDNA overhangs, which are important for the ‘fusions’ of APC with T cell recipient telomeres. Is that the only role for RAD51? Does a recombination intermediate form (as suggested by the T7 endonuclease assay)? Does the homologous recombination machinery in the recipient T cells have any role in the process? Could this imply a possible use of DNA repair inhibitors in modulation of autoimmunity?

At last, could senescent T cells be “reactivated” by up-taking APC telomeres?

 

References:

• Campisi, J., d’Adda di Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol(2007).
• Doksani, Y. The Response to DNA Damage at Telomeric Repeats and Its Consequences for Telomere Function. Genes (Basel) (2019).
• Li, J. S. Z. et al. TZAP: a telomere-associated protein involved in telomere length control. Science (2017)

 

Tags: senescence, t lymphocytes, telomeres

Posted on: 23 October 2020

doi: https://doi.org/10.1242/prelights.25432

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