**Background:** Biological aging is associated with increased susceptibility to infections, poor vaccine responses, and chronic inflammation, partly due to CD4+ T cell dysfunction. Mitochondrial dysfunction, characterized by altered metabolism, increased oxidative stress, and impaired redox signaling, is thought to underlie these age-related T cell defects. This study investigates whether exogenous delivery of functional mitochondria (mito-transfer) into CD4+ T cells from old mice can reverse aging-associated mitochondrial dysfunction and improve T cell function.
**Methods:** Mitochondria were isolated from mouse embryonic fibroblasts (MEFs) and delivered into CD4+ T cells from old C57BL/6 mice (18-24 months) via centrifugation (mito-centrifugation). Transfer efficiency was optimized using cardiolipin quantification and confirmed by flow cytometry (Mitotracker Deep Red, HK-GFP), confocal microscopy, and transmission electron microscopy (TEM). Mitochondrial function was assessed by measuring mitoROS (MitoSOX Red, EPR with Mito-Tempo-H), cytoROS (CellROX), total thiols, and glutathione. Proteomic analysis (LC-MS/MS) and pathway analysis (IPA, GO) evaluated changes in mitochondrial and TCR signaling proteins. Metabolic function was measured using Seahorse extracellular flux analysis (OCR, ECAR, ATP production). T cell activation was assessed by phosphorylation kinetics of TCR signalosome proteins (p-SLP76, p-PLCg1, p-ZAP70, p-LCK), expression of CD25 and CD69, IL-2 production (ELISpot, intracellular staining, Luminex), and proliferation (EdU incorporation). In vivo, naïve CD4+ T cells from old mice (with or without mito-transfer) were adoptively transferred into Rag1-KO mice, which were then infected with influenza A virus (IAV) or Mycobacterium tuberculosis (M.tb). Morbidity (weight loss) and mortality were monitored for IAV; bacterial burden (CFU) in lungs and spleen was assessed for M.tb.
**Key Results:** Mito-transfer achieved 100% efficiency using 5 nmol cardiolipin-equivalent mitochondria. TEM showed increased mitochondrial area (from 0.92±0.06 to 1.24±0.06 µm²), number (from 7.03±0.35 to 8.46±0.46), and % area per cell (from 4.38±0.24 to 5.88±0.28) in old CD4+ T cells after transfer. Mito-transfer significantly reduced mitoROS levels (MitoSOX MFI) to levels comparable to young T cells, without affecting cytoROS. Proteomics revealed upregulation of mitochondrial pathways, including ETC proteins (e.g., NDUS2, SDHA, COX15, QCR7, ATPK). Western blot confirmed increased UQCRC2 and ATP5A expression. Seahorse analysis showed increased basal OCR (from ~40 to ~60 pmol/min), maximal respiration, spare respiratory capacity, proton leak, and ATP production (total ATP production rate increased by ~50%). Glycolysis (ECAR, glycoPER) and glucose uptake (2-NBDG) also increased. TCR signaling kinetics improved, with significantly higher phosphorylation of p-SLP76 and p-PLCg1 at 15-60 min post-stimulation. Mito-transfer increased CD25+CD69+ cells by ~50%, IL-2-producing cells by ~50%, and EdU+ proliferating cells by 2-fold. In vivo, Rag1-KO mice receiving mito-transferred naïve CD4+ T cells showed delayed IAV-induced morbidity (weight loss) and reduced mortality (median survival 13 days vs 9 days for controls). In M.tb infection, mito-transferred cells significantly reduced bacterial burden in the spleen (p=0.016 vs non-manipulated) and lungs (p=0.004 vs PBS).
**Clinical Implications:** This proof-of-concept study demonstrates that extracellular delivery of functional mitochondria can reverse aging-associated CD4+ T cell dysfunction by improving mitochondrial metabolism, reducing oxidative stress, enhancing TCR signaling, and boosting cytokine production and proliferation. The in vivo protection against viral (IAV) and bacterial (M.tb) infections in a mouse model suggests that mito-transfer could be a novel therapeutic strategy to rejuvenate aged immune responses. The ability to achieve 100% transfer efficiency using centrifugation and the potential for autologous or allogeneic lymphocyte manipulation make this approach clinically translatable. However, further studies are needed to elucidate the precise mechanisms, assess long-term safety, and evaluate efficacy in human cells and other disease contexts (e.g., cancer immunotherapy).