Flotillin-2 dampens T cell antigen-sensitivity and functionality
T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and LCK upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2-null CD4+ T cells follow a similar route of activation as wild-type CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4+ T cells.  Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections. Overall design: scRNA-seq was performed on Flot2WT or Flot2CD4 naive CD4+ T cells following a 3 hour stimulation with varying concentrations (0, 0.25, or 1 µg/ml) of plate-bound anti-CD3. A fixed dose of soluble anti-CD28 (1 µg/ml) was provided under the conditions of 0.25 or 1 µg/ml of plate-bound anti-CD3. Cells stimulated with 0, 0.25, or 1 µg/ml of plate-bound anti-CD3 were hash-tagged with barcode sequence 'ACCCACCAGTAAGAC', 'GGTCGAGAGCATTCA', or 'CTTGCCGCATGTCAT', respectively, and then combined.