{
  "abstract": "Background Spatial organization of the T cell receptor (TCR) on the plasma membrane is thought to regulate T cell activation threshold. Flotillin-2 (Flot2), a membrane scaffolding protein, regulates TCR nanoclusters and function in CD4 + T cells,1 but its role in CD8+ T cells is unclear. Here, we investigated the role of Flot2 in CD8+ T cell responses, with a focus on anti-tumor function and therapeutic potential in adoptive cell transfer models.Methods To assess Flot2 function in CD8 + T cells, we used Flot2 global knockout, T cell-specific knockout (Flot2CD4), Flot2-deficient OT-I (OVA-specific) and Pmel-1 (gp100-specific) mice. Tonic TCR signaling was measured by flow cytometry. Naïve OT-I CD8+ T cells were stimulated with wild-type dendritic cells (DCs) pulsed with altered peptide ligands (APLs) of graded affinity and dose, or under DC-free conditions using peptide alone or plate-bound αCD3 antibodies, then assessed for activation. TCR nanoclusters were analyzed by dSTORM super-resolution imaging. Asymmetric cell division was measured by CD8 or T-bet polarity. Anti-tumor function was examined in B16F10 and MC38 tumor models and adoptive transfer of Flot2-silenced Pmel-1 CD8+ T cells.Results At steady state, Flot2-deficient naïve and effector CD8 + T cells exhibited increased expression of Nur77 and elevated phosphorylation of Lck, suggesting enhanced tonic TCR signaling. Consistent with a lowered TCR activation threshold, Flot2-deficient OT-I CD8+ T cells also displayed increased expression of Nur77 and CD69 upon in vitro stimulation by DCs pulsed with either high-dose, low-affinity or low-dose, high-affinity APLs. This effect was absent under DC-free conditions, indicating that Flot2 limits T cell activation in a DC-dependent manner. Super-resolution imaging demonstrated a significant increase in TCR nanocluster number in Flot2-deficient OT-I CD8+ T cells, suggesting a mechanism for the enhanced sensitivity. Furthermore, Flot2-deficient OT-I CD8+ T cells exhibited increased asymmetric division upon weak antigen stimulation, generating T-bethiand CD8hi effector precursors. In vivo, both Flot2 knockout and Flot2CD4 mice demonstrated improved tumor control in B16F10 and MC38 models, with increased intratumoral proliferation of effector CD8+ T cells. Adoptive transfer of Flot2-silenced Pmel-1 CD8+ T cells into B16F10 tumor-bearing wild-type hosts also led to superior tumor control compared to controls.Conclusions These findings identify Flot2 as a negative regulator of CD8 + T cell activation and anti-tumor function, potentially by limiting surface TCR nanoclusters and thereby raising the activation threshold. Targeting Flot2 may offer a strategy to modulate TCR spatial organization and to enhance engineered TCR and CAR T cell therapies.Reference Moon S, Zhao F, Uddin MN, Tucker CJ, Karmaus PW, Fessler MB. Flotillin-2 dampens T cell antigen sensitivity and functionality. JCI Insight 2024;9(24):e182328.",
  "authors": [
    {
      "affiliations": [
        "The National Institute of Environmental Health Sciences, Durham, NC, USA"
      ],
      "name": "Sookjin Moon"
    },
    {
      "affiliations": [
        "The National Institute of Environmental Health Sciences, Durham, NC, USA"
      ],
      "name": "Fei Zhao"
    },
    {
      "affiliations": [
        "The National Institute of Environmental Health Sciences, Durham, NC, USA"
      ],
      "name": "Peer WF Karmaus"
    },
    {
      "affiliations": [
        "The National Institute of Environmental Health Sciences, Durham, NC, USA"
      ],
      "name": "Michael B Fessler"
    }
  ],
  "title": "830 Flotillin-2 suppresses CD8+ T cell anti-tumor function by restricting T cell receptor nanoclustering and activation",
  "uid": "2868b431-257a-5470-8e08-8fe7eddd15d1"
}