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Background Failure of adoptive cell therapies (ACTs) is associated with insufficient persistence within the patient, inability to infiltrate tumor sites, and cell-intrinsic loss of functionality. One of the key factors responsible for T cell dysfunction is metabolic deregulation affecting their ability to find and kill cancer cell targets properly and effectively. Therefore, elucidating the metabolic pressures experienced in the TME could provide new therapeutic targets to improve ACTs. Reactive Oxidative Species (ROS) accumulation in the TME has detrimental effects on T cell function and anti-tumor response, although the precise targets of ROS are unclear. Previous data showing that mitochondrial ROS can have profound effects on the telomere status of cells. However, there is little evidence describing the role or oxidative stress on telomere health, or the importance of telomere function in immune cells.Methods In study we perform telomere-FISH assays to analyze TIL for DNA damage accumulation in human and mouse samples. We developed a novel transgenic mouse model harboring chemo-optogenetic FAPS-TAPS to generate singlet oxygen and consequent 8-oxo-guanine lesions specifically at telomeres specifically in T cells. 1 Results Telo-FISH analysis demonstrates an accumulation of telomeric DNA damage in TIL from B16 mouse tumors and human cancer samples shown by the presence of 53BP1 and ƔH2AX at telomeres. Our data show that mitochondria and telomeric ROS cause the accumulation of DNA damage at telomeres, as well as the development of telomere fragility. These cells ultimately become dysfunctional, showing a diminished capability for cytokine production. We tethered the antioxidant protein GPX1 to TRF1 to generate a telomere-guided ROS scavenger. Localizing the ROS scavenger GPX1 directly to telomeres reduced telomere fragility and improved the function of therapeutic T cells in solid tumor models. Our data suggest that dysfunctional T cells in cancer harbor damaged telomeres due to exposure to oxidative stress. Telomeric damage is sufficient to drive a dysfunctional state in newly activated T cells. Protecting telomeres by Localizing the ROS scavenger GPX1 directly to telomeres reduced telomere fragility in tumors and drive superior responses to adoptive cell therapies in solid tumor models ( figure 1).Conclusions Overall, this study utilizes a highly innovative mouse model to specifically address other mechanisms by which ROS can affect T cell function. This study highlights the importance of targeting ROS accumulation to improve the response to immunotherapies. This will allow us to further dissect other mechanisms that drive T cell dysfunction and unveil novel therapeutic targets.Reference Fouquerel E, Barnes RP, Uttam S, Watkins SC, Bruchez MP, Opresko PL. Targeted and persistent 8-oxoguanine base damage at telomeres promotes telomere loss and crisis. Mol Cell. 2019 Jul 11;75(1):117–130.e6. doi: 10.1016/j.molcel.2019.04.024. Epub 2019 May 14. PMID: 31101499; PMCID: PMC6625854Abstract 356 Figure 1Patient characteristics. (A) Tumor infiltrating lymphocytes from B16 tumors and spleen sorted on PD1-Tim3 expression. FISH analysis of Telomere (Telo) and DNA damage response elements 53BP1 and yH2AX. (B) Activated T cells expression TRF1-FAP get oxidative damage induced at telomeres (MG+L) by chemoptogenetic method. Control cells receive light alone (LIGHT). T cells are arrested and perform metaphase spreads for telomere fragility analysis. (C) Therapeutic T T cells co-expressing ROS scavenger GPX1 and TRF1 (telomere specific) transferred Into B16 tumor-bearing mice. Control T cells express control plasmid (CTL)