Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

31-POS Board 15 PROBING CAR OLIGOMERIZATION AT THE NK CELL IMMUNOLOGICAL SYNAPSE BY FLIM-FRET Rio Koshiba 1 ; Cornelia Monzel 1 ; 1 University of Stuttgart, 2nd Physics Institute, Stuttgart, Germany Fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer (FLIM-FRET) is employed to study the spatiotemporal organization and dynamic interactions of Chimeric Antigen Receptors (CARs) in Natural Killer (NK) cells at the immunological synapse (IS). Time-correlated single-photon counting (TCSPC) data were acquired on a confocal microscope setup from NK cells expressing mNeonGreen- and mScarlet3-tagged CAR constructs as donor and acceptor, respectively. To analyze receptor oligomerization, a custom biexponential pattern-fitting pipeline was developed: donor-only reference lifetimes were first extracted from median decay curves, and donor-acceptor co-expressing cells were subsequently fitted to yield the FRET fraction as a quantitative measure of receptor clustering within the contact area. NK cells were co-cultured with tumor target cells (UM17A) to investigate the formation of B7-H3-CAR binding to B7-H3 antigens in the cytotoxic-competent synapse. This was compared to a NK cell adhesion site free of B7-H3 antigens as a control. Comparing the FRET fraction between both conditions revealed a distinct and reproducible increase in the FRET-active fraction under synapse-forming conditions relative to the control, consistent with active receptor recruitment during immunological synapse formation. Notably, the FRET fraction remained largely independent of the donor-to-acceptor expression ratio across individual cells. Our data suggest a pre-oligomerization of the CAR that is further enhanced upon synapse engagement. To conclude, FLIM-FRET measurements of Chimeric Antigen Receptors offer quantitative insights into how immunological synapse formation modulates CAR distribution and oligomerization, with implications for engineering more effective CAR-NK cell therapeutics.

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