Flotillin-2 suppresses CD8⁺T cell anti-tumor function by restricting T cell receptor nanoclustering and activation

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, 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 (Flot2CKO), 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 alphaCD3 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-bet hi and CD8 hi effector precursors. In vivo, both Flot2 knockout and Flot2CKO 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.