{
  "abstract": "Background Despite the high initial responses to chimeric antigen receptor T (CART) cell therapy, most patients relapse within the first 1-2 years. Patient-derived T cells are used to generate CD19-targeting CART cells that are costimulated with either 4-1BB (BBζ) or CD28 (28ζ) and are prone to transition into dysfunctional states. Previously, we developed an in vitro model with cycles of antigen-specific CART cell activation followed by rest periods, mimicking cancer relapse, to study the development and mechanisms of CART cell dysfunction.1 We identified UACA and NEFM genes to be downregulated in recurrently activated/rested BBζ. Therefore, we overexpressed NEFM and UACA individually or in combination (dual) in BBζ or 28ζ and examined CART immunophenotype and effector functions.Methods Briefly , UACA and/or NEFM were ectopically expressed in BBζ or 28ζ (n=4 healthy donors) through lentiviral transduction. CART cells along with controls were cocultured with target cells. After coculture, exhaustion and senescence markers on CART cells were measured by flow cytometry. The amount of cycling CART cells was measured by EdU assay. CART efficacy was measured by antigen-specific cytotoxicity and proliferation assays.Results The amount of cycling CD19-targeting CART cells 24 hours after coculture with target cells increased upon overexpressionfrom all donors (). Target cell killing increased in NEFM-, UACA- and dual-overexpressing BBζ and 28ζ across multiple effector-to-target ratios (BBζ: p=0.0077 to <0.001; 28ζ: p=0.0485 to <0.001). T cell exhaustion markers, including LAG3 (BBζ: p<0.001; 28ζ: p=0.003), TIM-3 (BBζ: p=0.001; 28ζ: p=0.003), and PD1 (BBζ: p=<0.001; 28ζ: p=0.044), were all significantly downregulated in NEFM-, UACA- and dual-overexpressed CART cells compared to control CART cells. Finally, we overexpressed these genes in 4-1BB-costimulated TSHR-targeting CART cells. NEFM- and UACA-overexpressing TSHR-CART cells also showed decreased T cell exhaustion marker expression and improved CART cell killing and proliferation.Conclusions Our previous RNA-seq analysis indicated downregulation of UACA and NEFM in activated/rested BBζ. In this study, we overexpressed these two genes in CART cells that were costimulated with either 4-1BB or CD28. Ultimately, our study indicates that UACA and NEFM overexpression appears to confer improved cytotoxicity and proliferative ability in multiple CART cell types, independent of costimulatory domain or antigen target. The increased expression of these genes also led to a decreased exhaustive immunophenotype. Further examination of the role of these two genes could shed light into pathways that are key for CART cell dysfunction.Acknowledgements This study was partly funded by Regenerative Medicine Minnesota RMM 072523 TR 004 (IC), Mayo Clinic Division of Hematology (IC), Eagles 5th District Cancer Telethon Funds for Cancer Research (IC), Mayo Clinic Center for Individualized Medicine (SSK)Reference Can I, Siegler EL, Sirpilla OL, et al. Differential susceptibility and role for senescence in CART cells based on costimulatory domains. Molecular Cancer. 2025;24(1):172. doi:10.1186/s12943-025-02371-1Ethics Approval The use of recombinant DNA in the laboratory was approved by the Mayo Clinic Institutional Biosafety Committee (IBC), IBC number HIP00000252.43. Animal studies were performed according to the Institutional Animal Care and Use Committee (IACUC) at the Mayo Clinic animal facilities (IACUC # A00001767-16-R22).",
  "authors": [
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Ismail Can"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Elizabeth Siegler"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Grace DeFranco"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Jennifer M Feigin"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Sophia Goldberg"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Omar L Gutierrez Ruiz"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Truc Huynh"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Brooke Kimball"
    },
    {
      "affiliations": [
        "Mayo Clinic, Jacksonville, FL, USA"
      ],
      "name": "Imene Hamaidi"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Long Mai"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Claudia Manriquez Roman"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Ekene Ogbodo"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Olivia Sirpilla"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Dominic Skeele"
    },
    {
      "affiliations": [
        "Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, USA"
      ],
      "name": "Carli M Stewart"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Hong Xia"
    },
    {
      "affiliations": [
        "Izmir Institute of Technology, Izmir, NA, Turkey"
      ],
      "name": "Fatih Sezer"
    },
    {
      "affiliations": [
        "Izmir Institute of Technology, Izmir, NA, Turkey"
      ],
      "name": "Berke Alkan"
    },
    {
      "affiliations": [
        "Izmir Institute of Technology, Izmir, NA, Turkey"
      ],
      "name": "H Atakan Ekiz"
    },
    {
      "affiliations": [
        "Mayo Clinic, Rochester, MN, USA"
      ],
      "name": "Saad S Kenderian"
    }
  ],
  "title": "206 The role of NEFM and UACA genes in CART cell effector functions",
  "uid": "395b3e25-6ec5-5fdd-a7ba-e7e6c8eba6a5"
}