{
  "abstract": "Background T cell exhaustion (TEX) limits the efficacy of adoptive therapies for solid tumors, driven by factors in the tumor microenvironment (TME) such as hypoxia, nutrient deprivation, and inhibitory signaling. 1 2 Although immune checkpoint blockade (ICB) has shown limited success in restoring function in exhausted T cells, the underlying mechanisms of ICB-resistant TEX are unclear. In lymphocytes, the DNA damage response (DDR) kinases ATR, DNA-PK, and ATM play unique roles in proliferation and activation, implicating DDR modulation as a factor in T cell function. This study identifies DNA damage accumulation due to prolonged replication stress as a contributor to TEX, offering a novel therapeutic avenue for improvement of immunotherapy outcomes.Methods Human CD8+ T cells were isolated using negative selection (Mojo) on PBMCs isolated from healthy donors. T cells were stimulated either once (acute stimulation) or every two days (chronic stimulation) using CD2/3/28 antibody cocktail (Stemcell Technologies) as described previously to simulate TEX. 3 After 6-10 days of in vitro exhaustion, T cells were evaluated using mass spectrometry (TMT and phospho-TMT), flow cytometry, immunofluorescence, western blotting, and comet assay. Lentiviral transduction was used for anti-CD19-28z CAR and DDR protein overexpression. CRISPR was performed using nucleofection (Lonza). Cytotoxicity assays were performed using a MetaXpress HT.ai automated confocal microscope with a 40x water immersion objective to monitor RFP-labeled tumor cell (Nalm6) number over time. All experiments are performed using at least three unique human donors (n=3).Results Flow cytometry, immunofluorescence, and western blotting show markers of replication stress are upregulated during chronic stimulation (i.e. gamma-H2AX, pATR, pRPA32, pChek1). Neutral comet assay demonstrates increased unrepaired DNA breaks. Additionally, we show exogenous DNA damage accelerates TEX. Proteomic analysis reveals modulation of the DDR during TEX and highlights targets for DDR modulation in T cells ( figure 1). Cytotoxicity assays reveal targeted modulation of DDR enhances T cell cytotoxicity, persistence, and survival (figure 2). We also show that mice harboring a genetic mutation in the DDR protein 53BP1 have enhanced response to ICB therapy in HNSCC model, demonstrating the potential for DDR modulation to improve response to immunotherapies.Conclusions This study demonstrates the role of the DDR in T cell function. We show that modulation of the DDR in human T cells confers survival and cytotoxicity benefits. Overall, this study highlights the importance of understanding DNA repair biology during T cell function and provides a basis for the improvement of immunotherapies against solid tumors by manipulating DNA repair.References Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol, Aug 2025;15(8):486–499. doi: 10.1038/nri3862Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol, Nov 2020;20(11):651–668. doi: 10.1038/s41577-020-0306-5Dunsford LS, Thoirs RH, Rathbone E, Patakas A. ‘A human in vitro t cell exhaustion model for assessing immuno-oncology therapies’, in methods in pharmacology and toxicology. Humana Press Inc., 2020:89–101. doi: 10.1007/978-1-0716-0171-6_6Abstract 236 Figure 1Illustration of the DNA damage response overlayed with Log2FC as measured using TMTAbstract 236 Figure 2Schematic of cytotoxicity assay (top). Results from cytoxicity assay for two DDR targets modified in T cells as compared to control. Percent tumor remaining is normalized to 100% at each replate (bottom)",
  "authors": [
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Jessica L Kelliher"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Mousumi Patra"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Sydnye L Shuttleworth"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Daniel Fil"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Katherine Cruse"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "T Wonder NO Meridia"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Michael A Bauer"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Katherine Wallis"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Conway, AR, USA"
      ],
      "name": "Thomas A Williams"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Lora J Rogers"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Fen Xia"
    },
    {
      "affiliations": [
        "University of Arkansas for Medical Sciences, Little Rock, AR, USA"
      ],
      "name": "Brian Koss"
    }
  ],
  "title": "236 Manipulating the DNA damage response to combat T cell exhaustion and improve immunotherapy response",
  "uid": "26fa0c4d-15e6-5728-a3de-b5f0ea97782b"
}