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, 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 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-bethi and CD8hi 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. Citation Format: Sookjin Moon, Fei Zhao, Peer Karmaus, Michael Fessler. Flotillin-2 suppresses CD8⁺T cell anti-tumor function by restricting T cell receptor nanoclustering and activation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr LB136.
Moon et al. (Fri,) studied this question.