Reduced expression of mitochondrial transcription factor A (TFAM) in CD8? T cells drives senescence-associated inflammatory lung injury during respiratory viral infection

Aging leads to dysregulated immune responses that worsen the severity of respiratory viral infections. As part of this dysfunction, CD8? T cells exhibit altered inflammatory regulation, contributing both to viral clearance and to adverse lung injury. However, the cell-intrinsic mechanisms driving age-associated shifts in CD8? T cell function remain poorly understood. Analysis of mouse and human single-cell datasets reveals that reduced expression of mitochondrial transcription factor A (TFAM) is a conserved feature of aging and acute viral illness, correlating with heightened inflammatory programs in CD8? T cells. To define the functional consequences of this decline, we generated a CD8? T cell–specific TFAM haploinsufficient mouse model (TFAM?/?) that recapitulates the physiological reduction of TFAM observed in aged human CD8? T cells. We show that reduced TFAM expression is sufficient to disrupt mitochondrial integrity, activate mitochondrial DNA stress pathways, and prime CD8? T cells toward a senescence-like state at homeostasis. During influenza infection, TFAM-insufficient CD8? T cells mount an exaggerated early hyper-cytotoxic effector response and rapidly acquire exhaustion features. Despite adequate viral clearance, TFAM?/? mice exhibit sustained inflammatory lung injury. Together, these findings demonstrate that aging-like TFAM insufficiency reprograms CD8? T cell behavior through mitochondrial dysfunction, driving early inflammatory hyperactivation, premature exhaustion, and persistent lung immunopathology. Overall design: Wild-type (WT) C57BL/6, TFAMfl/fl, and CD8a-Cre mice purchased from the Jackson Laboratory and bred in-house. In this Cre-expressing strain, unlike the proximal Cd8a promoter, the E8I enhancer is not active during the double-positive (DP) stage of thymocyte development. Its activity is restricted until the cells have committed to the CD8 single-positive (SP) lineage, allowing Cre-mediated deletion to occur only in mature CD8 T cells and avoids any 'off-target' recombination in the CD4 lineage. An equal number of male and female mice, aged 8-10 weeks, were included in the study. All animal experiments were performed in accordance with the guidelines of the University of Florida Institutional Animal Care and Use Committee. Mice were provided ad libitum access to food and water. CD8a-Cre mice were crossed with TFAMfl/fl mice to achieve a heterozygous TFAM deletion in CD8a-expressing cells, resulting in TFAM+/- mice. For Influenza A virus (strain A/Puerto Rico/8/1934 H1N1; PR8), Mice were anesthetized with a 4% (v/v) isoflurane and intranasally inoculated with 250 PFUs of IAV in 100 µL PBS. Mock-infected mice received 100 µL PBS. Following euthanasia by CO2 exposure and cervical dislocation (D7 and D14), the lungs were perfused, and BALF was collected at by instillation and aspiration of the lung with 1 ml of sterile ice-cold PBS through a 20-gauge tracheal catheter (BD Biosciences), and lungs were aseptically collected and processed for downstream analyses.