Fig. 4: Extracellular vesicles are the major source of AMPase activity in human CD8 T cell culture supernatants.

a, b CD8 T cells were stimulated with αCD3/αCD28 and kept in culture for 4 days. Analysis of AMPase activity in a cell culture supernatants of stimulated or unstimulated total CD8 T cells and b supernatants and EVs of sorted CD8 CD73⁺ or CD8 CD73⁻ T cells after differential centrifugation. The conversion of eAMP to eADO was measured by HPLC. c–e Analysis of CD73 and EV markers on EVs derived from stimulated cell culture supernatants of CD73⁺ or CD73⁻ CD8 T cells by c western blot, d flow cytometry, and e electron microscopy with immunogold labeling. Recombinant CD73 or cell lysate from stimulated CD8 T cells served as positive controls. f EVs were isolated from CD8 T cells stimulated with αCD3/αCD28 for 3 days in the presence or absence of GW4869 (10 µM), and particle concentration and size were measured by NTA. g Microscopy analysis of CD73, CD9, and CD81 expression in sorted CD73⁺ or CD73⁻ CD8 T cells after 4 days of stimulation with αCD3/αCD28. GW4869 (10 µM) was added at day 0. h Microscopy analysis of CD73 and CD9 expression in CD8 T cells before and after activation. Pearson coefficient was determined to quantify the co-localization of CD73 and CD9. Data were analyzed from seven (d4) or eight (d0, d1) high power fields (center line: median, box limits: 25th to 75th percentiles, whiskers: min to max). Donors analyzed: a, b three donors in three independent experiments; c six donors in three independent experiments; d–f three donors; g, h samples from three donors processed independently. Each figure panel shows a representative donor/experiment. Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare Pearson coefficients of co-localization in h.