Journal: bioRxiv

Article Title: Segmental flexibility of bispecific T-cell engagers regulates the dynamics of immune synapse formation

doi: 10.1101/2022.06.15.496334

Figure Lengend Snippet: ( A ) Representative TIRF images of immune synapse (top) and other adhesion phenotypes (bottom) formed by CD8 + human primary T-cells upon incubation with 5 pM TcE formats on SLBs for 10 min. Scale bars are 7 µm. ( B ) Time resolved analysis of TcE-mediated immune synapse formation frequency of CD8 + human primary T-cells on SLBs. Formats are shown on the left, Her2 densities on the SLB on top and individual values state the % of contact sites that were characterized as IS with bulls-eye actin structure. ( C ) Representative TIRF images of bulls-eye structures formed by CD8 + human primary T-cells on SLBs incubated with TcEs for 25 min. SLBs additionally present 400 molecules/ µm 2 CD45RABC-AlexaFluore647 to analyze exclusion from the contact site. Her2 densities on the SLB are shown on the bottom. Scale bars are 5 µm. ( D ) Quantification of CD45RABC exclusion as shown in C for CD8 + human primary T-cells incubated with 5 pM TcEs for 25 min. Each dot indicates an individual cell adhesion site plotted for the mean CD45RABC-AlexaFluor647 intensity within the actin ring divided by the mean intensity on the SLB. Shown as mean ± standard deviation with one-way ANOVA and Bonferroni post-hoc testing, ***p<0.0005. Results were pooled from two donors in independent experiments. Statistical significance is shown as comparison of the treatment condition with Format D to the other formats.

Article Snippet: Subsequently, cells were stained with anti-human ICAM-1-AlexaFluor647 (BioXcell, BE0020-2, 3 µM), anti-human CD58-AlexaFluor647 (BD Bioscience, 563567, 1:500 dilution) or anti-human CD80-AlexaFluor647 (BioLegend, 305216, 1:500 dilution) for 30 min at room temperature in culture medium containing 1% BSA.

Techniques: Incubation, Standard Deviation