Fig. 5. ADAM10, TSG101, CD81, and BST2 are key proteins participating in the biogenesis of CD40L⁺ tSV.

a–e Flow cytometry analyses showing the transfer of tSV markers to BSLBs expressed as percent of the maximum transfer (Tmax%) observed in BSLBs co-cultured with CD4-edited controls. BSLBs were reconstituted with ICAM1 (200 molec./µm²), CD40 (20 molec./µm²) and anti-CD3ε Fab (0–2000 molec./µm²). Please refer to Supplementary Fig. 1a for a representative gating strategy showing the identification of single BSLBs and T cells by flow cytometry. a Heatmaps showing Tmax% for CD40L, b TCRαβ, c CD63, d CD81, and e BST2 in BSLB incubated with different CRISPR/Cas9-edited cells (gRNA gene targets shown in italic). f TIRFM quantification of normalized medians for the integrated fluorescence intensities of CD40L within synapses formed by CRISPR/Cas9-edited cells. Circles represent the integrated fluorescence median per donor and per CRISPR/cas9 target normalized to its internal control (teal). CD81- (violet circles), ADAM10- (orange circles), BST2- (dark red circles), and TSG101-edited cells (green circles) were imaged with TIRFM. g Representative TIRFM images (g) showing the levels of centrally clustered CD40L in synapses of CRISPR/Cas9-edited cells imaged as depicted in h and compared with CD4-edited controls. Scale bar = 5 µm. MFI = Mean Fluorescence Intensity, arb. units = arbitrary fluorescence units. IRM = internal reflection microscopy. Data are shown as means ± SEM from n = 10 (a–e), and 7 (f) biologically independent samples (donors) across five independent experiments. In a–e, normality was determined using Shapiro–Wilk test, and statistical significance was determined by parametric two-tailed Multiple t test with Holm-Šídák correction (α = 0.05) for the multiple comparisons of Tmax% across different anti-CD3ε Fab densities and between CRISPR/Cas9-edited cells and CD4-edited controls (a–e), and by one-way ANOVA with mixed-effect analysis and Fisher’s LSD test (95% CI) to compare separately each CRISPR/Cas9-edited cells to controls (f). Values represent calculated P values for each comparison to control.