Figure 4. SSLB as a platform to study 3D TCR tension.

a Schematic showing the workflow of using SSLBs to study TCR mechanics through high resolution confocal microscopy and high throughput flow cytometry. b Representative images showing the spatial distribution of DOTS, TCR, and tension signal at the middle layer of the T cell-SSLB junction. c Plot comparing the Cy3B/Atto647N ratios on SSLB surfaces and SSLB-T cell junctions with and without locking strand. Data from each group was acquired from >25 cells d 3D reconstructed image showing the 3D distribution of DOTS and tension signals at the junction. e Flow cytometry fluorescence dot plot (Atto647 vs CFSE) showing T cells, SSLBs (green dash line), and conjugates events (purple dash line). Forward vs side scatter plot was used to gate out SSLB monomers (green dash line) and T cell-SSLB conjugates at 1:1 binding stoichiometry (purple dash line). f Atto647N fluorescence histograms of SSLBs (green) and 1:1 T cell/SSLB conjugate (purple) events. g Atto647N fluorescence histograms of SSLBs and 1:1 T cell-SSLB conjugates when locking strand was absent. h Atto647N fluorescence histogram of SSLBs and 1:1 T cell/SSLB conjugates where SSLBs were modified DOTS presenting antigens with different potencies. i Plot quantifying the Atto647N mean fluorescence intensity (MFI) difference between 1:1 T cell/SSLB conjugates and SSLBs. ***P < 0.001 ****P < 0.0001. Scale bars = 5 μm.