Kalouskova, B. | Search bioRxiv

bioRxiv2026

A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex

Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.

biophysics↗

bioRxiv2021

Crystal structure of the human NKR-P1 bound to its lymphocyte ligand LLT1 reveals receptor clustering in the immune synapse

Human NKR-P1 (CD161, KLRB1) and its ligand LLT1 (CLEC2D) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohns disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated with glioblastoma, non-Hodgkins lymphoma, breast, and prostate cancer. However, the lack of structural data on the formation of the NKR-P1:LLT1 complex limits our understanding of this signaling. We thus solved the crystal structures of NKR-P1 and the NKR-P1:LLT1 complex. NKR-P1 forms a homodimer with an unexpected arrangement that enables LLT1 binding in two modes, bridging two LLT1 molecules, thereby forming interaction clusters suggestive of an inhibitory immune synapse. Moreover, observing the formation of these clusters by SEC-SAXS analysis in solution and by dSTORM super-resolution microscopy on the cell surface, and following their role in receptor signaling using in vitro cytotoxicity assay with freshly isolated NK cells, we show how NKR-P1:LLT1 clustering allows these proteins to overcome the weak affinity of C-type lectin-like receptors to their ligands. Furthermore, only the ligation of both primary and secondary LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. Therefore, our findings show how inhibitory receptor cross-linking and clustering work together to trigger signal transduction upon cellular contact in the immune synapse.

molecular biology↗