Mitochondrial DNA Release from Aged T Cells Drives Systemic Senescence via cGAS-STING Signaling: A Testable Mechanism Linking Immunometabolic Dysfunction to Tissue Aging

Hypothesis

Aged T cells release mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING pathway. This triggers a senescence-associated secretory phenotype (SASP) in the T cells themselves and in neighboring stromal cells, fueling inflammaging and tissue senescence. Inhibiting mtDNA release or cGAS-STING signaling in T cells will break this loop, reducing systemic senescence and improving tissue function independent of senolytics.

Mechanistic Rationale

• Aged T cells accumulate mitochondrial damage and exhibit defective mitophagy, leading to cytosolic mtDNA [2].
• Cytosolic mtDNA binds cGAS, producing cyclic GMP-AMP that activates STING, driving IRF3‑NF‑κB signaling and SASP cytokine production [4].
• SASP factors (IL-6, TNFα, CCL2) induce paracrine senescence in epithelial, endothelial, and fibroblast cells, expanding the senescent burden [3].
• STING activation also primes the NLRP3 inflammasome, amplifying IL-1β release and reinforcing inflammaging [1].
• Thymic involution limits naïve T cell renewal, increasing reliance on these dysfunctional memory T cells [5], thus sustaining the loop.

Experimental Design

Model: 20‑month‑old C57BL/6 mice. Interventions:

• Group A: T‑cell‑specific knockout of cGAS (using Cd4‑Cre cGAS^fl/fl).
• Group B: T‑cell‑specific overexpression of mitochondrial transcription factor A (TFAM) to enhance mtDNA packaging and limit release.
• Group C: Wild‑type littermates receiving vehicle.
• Group D: Wild‑type mice treated with a senolytic (dasatinib + quercetin) as positive control. Readouts (4 weeks post‑intervention):
• Flow cytometry for p16^high^ CD4^+^ T cells and SASP cytokine production.
• Plasma IL-6, TNFα, IL-1β levels (ELISA).
• Tissue senescence markers (SA‑β‑gal, p21) in liver, kidney, and muscle.
• Functional assays: grip strength, treadmill endurance, frailty index.
• Mitochondrial ROS and mtDNA copy number in sorted T cells.

Predicted Outcomes

If the hypothesis is correct, Groups A and B will show:

• Significant reduction in SASP‑producing T cells compared with Group C.
• Lower circulating inflammaging cytokines, approaching levels seen in young mice.
• Decreased senescence burden in parenchyma, comparable to senolytic‑treated Group D.
• Improved physical performance and reduced frailty. No change in Group C; Group D will improve via senolysis but not affect T‑cell mtDNA release.

Potential Pitfalls and Alternatives

• Compensatory pathways (e.g., AIM2 inflammasome) may sustain inflammation; measure AIM2 activation.
• T‑cell‑specific manipulations could affect immunity to infection; monitor viral clearance after LCMV challenge.
• Off‑target effects of Cre driver; include Cd4‑Cre negative controls.

Falsifiability: If T‑cell‑restricted cGAS loss or TFAM overexpression fails to reduce SASP cytokine production, tissue senescence, or functional decline despite confirmed target engagement, the hypothesis is falsified.