{
  "abstract": "Background Radiation therapy (RT) is a cornerstone treatment for glioblastoma. In several cancers, RT induces anti-tumor immunity, but in glioblastoma, tumors often recur within the irradiated margins, indicating that RT fails to generate a durable immune response. One potential explanation is the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a factor associated with poor prognosis in many cancers including glioblastoma. Although initially characterized as a metalloprotease inhibitor, TIMP-1 also functions as an immunomodulatory cytokine that can suppress T cell responses via its receptor CD63. Our preliminary data indicate that RT increases TIMP-1 secretion, leading us to hypothesize that the TIMP-1/CD63 axis is a novel immune evasion mechanism in irradiated glioblastoma.Methods SB28 murine glioblastoma cells were irradiated with 0, 2, 6, 8, 10 and 20Gy. Supernatants were collected 24 hrs post-RT, and TIMP-1 levels were measured by ELISA. CD8 + T cells from healthy C57Bl/6N spleens were activated with anti-CD3/anti-CD28, and co-cultured for five days with previously irradiated SB28 cells (0, 6 and 10Gy). Expression of CD63 and exhaustion markers (PD-1 and TOX) on CD8+ T cells was assessed by flow cytometry. In vivo, C57Bl/6N mice were intracranially injected with GL261 murine glioblastoma cells and received five daily fractions of 6Gy starting on day 10. On day 18, tumor-infiltrating CD8+ T cells were analyzed by flow cytometry to assess CD63 expression (n=5/group), and CD63+ cells were sorted for single-cell RNA sequencing (scRNAseq).Results Irradiation of SB28 cells promoted a dose-dependent secretion of TIMP-1. CD8+ T cells co-cultured with irradiated SB28 cells showed increased CD63 expression, with CD63 +CD8+ T cells exhibiting higher levels of exhaustion markers compared to CD63-CD8+ T cells. In vivo, CD63 expression on tumor-infiltrating CD8+ T cells was increased in irradiated glioblastoma compared to control tumors. ScRNAseq of CD63+ cells revealed 20 distinct clusters, including both tumor and immune cells. Among immune populations, several displayed immunosuppressive phenotypes, including exhausted CD8+ T cells (Pdcd1, Tox, Lag3), CD4+ regulatory T cells (Foxp3, Il2ra, Ikzf2, Ctla4), and mature regulatory dendritic cells (Birc3, Ccr7, Cd40, Cd274).Conclusions Our results demonstrate that RT induces TIMP-1 secretion by glioblastoma cells and increases expression of its receptor CD63 on dysfunctional CD8 + T cells. The enrichment of CD63 in immunosuppressive populations suggests that CD63 may serve as a novel immunoregulatory marker in the irradiated glioblastoma tumor microenvironment. Ongoing studies will determine whether CD63 is a novel actionable target to restore anti-tumor immunity in irradiated glioblastoma.Ethics Approval Animal studies were approved by the IACUC #2019-0042.",
  "authors": [
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Maria Cecilia Lira"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Mara De Martino"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Camille Daviaud"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Ryan Getler"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Liron Yoffe"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Claire I Vanpouille-Box"
    }
  ],
  "title": "902 CD63 as a marker of T cell dysfunction and immune evasion in irradiated glioblastoma",
  "uid": "df220580-cd3f-5c4e-aa43-73c37195053c"
}