Hypothesis

Senescent immune cells release extracellular vesicles (EVs) enriched in oxidized mitochondrial DNA (mtDNA) that act as a damage‑associated molecular pattern (DAMP). When these EVs are taken up by parenchymal or stromal cells, the mtDNA activates the cytosolic cGAS‑STING pathway, triggering type‑I interferon production and a secondary senescence program. This EV‑mediated mtDNA transfer constitutes a core mechanistic link through which the immune system actively drives systemic aging, distinct from the mere accumulation of senescent cells.

Mechanistic Rationale

1. EV cargo specificity – Senescent T‑cell EVs contain higher ratios of oxidized mtDNA and mitochondrial RNA compared with EVs from young immune cells, as shown by mass‑spectrometry‑based oxidation profiling (see oxidation signatures in 1).
2. cGAS‑STING activation – Oxidized mtDNA is a potent ligand for cGAS; its delivery via EVs has been demonstrated to stimulate IFN‑β production in fibroblasts (see functional assays in 2).
3. Paracrine senescence – STING signaling induces NF‑κB and IRF3 pathways, upregulating p16^INK4a^ and SASP factors in recipient cells, thereby spreading senescence without direct cell‑cell contact.
4. Feedback amplification – Newly senescent parenchymal cells secrete cytokines that further activate immune cells, increasing EV output and creating a self‑reinforcing loop (consistent with the feed‑forward model in 2).
5. Independence from senescent cell load – Because the pathogenic signal resides in the EV cargo, reducing EV release should attenuate tissue aging even if the number of senescent immune cells remains unchanged.

Testable Predictions

• Prediction 1: Genetic or pharmacological inhibition of EV biogenesis specifically in senescent CD8^+ T cells will lower circulating oxidized mtDNA levels by >40% in aged mice.
• Prediction 2: Mice with T‑cell‑specific EV blockade will exhibit reduced cGAS‑STING activation (measured by phospho‑TBK1 and IFN‑β) in liver and lung parenchyma.
• Prediction 3: Despite comparable senescent immune cell frequencies (p16^+^ CD8^+ T cells), EV‑blocked mice will show improved metabolic homeostasis (lower serum ALT/AST, better glucose tolerance) and extended median lifespan (~15% increase) relative to littermate controls.
• Prediction 4: Administering purified oxidized mtDNA‑enriched EVs from aged immune cells to young mice will recapitulate tissue‑specific cGAS‑STING activation and premature senescence markers, an effect abrogated by STING knockout.

Experimental Approach

1. Model: Use Cd8a‑Cre^ERT2^ crossed with Rosa26‑LSL‑Rab27a^fl/fl^ mice to inducibly knock out Rab27a (essential for EV exocytosis) in CD8^+ T cells after senescence induction via sub‑lethal irradiation.
2. Readouts:
   • Isolate plasma EVs; quantify oxidized mtDNA by qPCR after DNA oxidation damage assay.
   • Assess cGAS‑STING pathway activation in target tissues via Western blot for phospho‑TBK1, IRF3 nucleation, and IFN‑β ELISA.
   • Measure senescence markers (p16, SA‑β‑gal) in hepatocytes and lung epithelial cells.
   • Perform metabolic profiling (glucose tolerance test, serum lipids) and survival analysis.
3. Controls: Littermate Rab27a^fl/fl^ without Cre, and a parallel group treated with senolytic (dasatinib + quercetin) to compare effects of EV blockade versus senescent cell clearance.

Significance

If validated, this hypothesis reframes immune aging as a communicable disease driven by EV‑borne oxidized mtDNA, suggesting that targeting EV biogenesis or mtDNA oxidation in specific immune subsets could decouple immune senescence from tissue degeneration, offering a therapeutic avenue distinct from senolytics.