Figure 3.

OVA-Ts-EVs modulate vesicle-mediated interaction of Raji B cells and Jurkat T cells at the immune synapse. (A) CD81-GFP-transfected Raji B cells were pulsed with SEE superantigen for 30 min at 37 °C and then cultured with Jurkat T cells in the presence of OVA-Ts-EVs. Twenty four hours later, cells were stained with viability dye and fluoresceinated antibodies against CD19, and analyzed with flow cytometry (n = 3). Relative changes in the percentage of CD19^negGFP^pos Jurkat T cells caused by SEE-stimulation of Raji B cells were calculated as follows: (percentage of CD19^negGFP^pos events in SEE-stimulated sample)/(percentage of CD19^negGFP^pos events in unstimulated sample); and shown in the graph. (B) CD81-GFP-transfected Raji B cells were left untreated (upper panel) or were treated with OVA-Ts-EVs for 4 h at 37 °C (lower panel), both populations were then pulsed with SEE for 30 min at 37 °C, and mixed with CMAC-stained Jurkat T cells (1 × 10⁵ cells) in a ratio 1:1. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. (C) CD81-GFP-transfected Raji B cells were treated with OVA-Ts-EVs for 4 h at 37 °C, pulsed with SEE for 30 min at 37 °C, and cultured with CMAC-stained Jurkat T cells (1 × 10⁵ cells) in a ratio 1:1 for 24 h on standard culture plate. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. Data are expressed as mean ± SD. Two-tailed Student t-test; * p < 0.05.