294 Flotillin-2 shapes CD8 + T cell anti-tumor immunity through T cell receptor spatial organization
Year: 2026
Type: conference-abstract
Abstract: Background T cell receptors (TCRs) are organized on the plasma membrane as individual molecules or nanoscale clusters. Although TCR nanoclusters are critical for T cell activation, their regulation and impact on CD8 + T cell function remain unclear. We recently showed that Flotillin-2 (Flot2), a membrane-associated scaffold protein, restricts TCR nanoclustering and antigen sensitivity in CD4+ T cells.1 Whereas Flot2-null CD4+ T cells exhibited augmented responses to anti-CD3/CD28 stimulation, Flot2-null CD8+ T cells signaled comparably to wild-type counterparts1 Here, we examined how Flot2 regulates TCR nanoclustering and function in CD8+ T cells.Methods Using published pan-cancer single-cell RNA-sequencing (scRNA-seq) datasets, 2–4 we examined FLOT2 expression in human tumors. Global, T cell-specific, OT-I, and Pmel-1 Flot2-deficient mouse models were tested in B16F10 melanoma models and analyzed using scRNA-seq and other methods. Antigen sensitivity was assessed by stimulating OT-I CD8+ T cells with altered OVA peptide ligand-pulsed wild-type or ICAM-1-deficient DCs, or under DC-free conditions. TCR nanoclustering and asymmetric cell division were analyzed by direct stochastic optical reconstruction microscopy (dSTORM) and confocal microscopy, respectively.Results Human pan-cancer scRNA-seq revealed that FLOT2 expression varied across T cell subsets and was enriched in exhausted T cells. High FLOT2 expression and FLOT2-related signatures were associated with poor overall survival across human cancers. scRNA-seq of murine melanoma showed that Flot2 deficiency enhanced stemness, proliferation, and metabolic fitness in tumor-infiltrating CD8 + T cells. Flot2-deficient mice showed reduced lung metastatic burden, and Flot2-deficient Pmel-1 CD8+ T cells produced more TNFα and IFNγ than control cells within the same tumor-bearing host. Mechanistically, Flot2-deficient naive and effector CD8+ T cells exhibited increased Nur77 expression and Lck phosphorylation, suggesting enhanced basal TCR signaling. Consistently, dSTORM showed increased TCR nanocluster number in naive Flot2-deficient CD8+ T cells. Functionally, Flot2-deficient OT-I cells showed increased activation and asymmetric division upon suboptimal antigenic stimulation. This enhancement was absent under DC-free stimulation and with ICAM-1-deficient DCs, indicating dependence on DC ICAM-1-mediated interactions. Finally, Flot2-silenced Pmel-1 CD8+ T cells improved tumor control after adoptive transfer.Conclusions These findings identify Flot2 as a negative regulator of TCR nanoclustering and antitumor function in CD8 + T cells. Flot2 deficiency enhances TCR nanoclustering in naive CD8+ T cells, lowering the threshold for productive activation to suboptimal antigenic stimulation in a DC ICAM-1-dependent manner, thus revealing a molecular dependency distinct from that of CD4+ T cells. Targeting Flot2 may tune TCR spatial organization and improve engineered anti-cancer T cell therapies.References Moon S, Zhao F, Uddin MN, et al. Flotillin-2 dampens T cell antigen sensitivity and functionality. JCI Insight 2024;9:e182328.Chen X, Shen Y, Choi S, et al. Concurrent loss of the Y chromosome in cancer and T cells impacts outcome. Nature 2025;642:1041–1050.Ibáñez-Molero S, Veldman J, Nieto JS, et al. Tumour-reactive heterotypic CD8 T cell clusters from clinical samples. Nature 2026;649:467–476.Xue R, Zhang Q, Cao Q, et al. Liver tumour immune microenvironment subtypes and neutrophil heterogeneity. Nature 2022;612:141–147.Ethics Approval This study used previously published, de-identified human cancer sequencing datasets, including datasets obtained from the original investigators upon request. No new human participants, human specimens, or identifiable private information were collected for this study. Therefore, additional ethics committee/IRB approval and informed consent were not required for this secondary analysis. Ethics approval and informed consent for the original studies were obtained by the investigators who generated the datasets, as described in the original publications. All animal experiments were conducted in accordance with an animal study protocol approved by the National Institute of Environmental Health Sciences (NIEHS) Animal Care and Use Committee.
Institution: National Institute of Environmental Health Sciences
Language: English
Topic: T-cell and B-cell Immunology
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225170864