Figure 3

Circulating EVs of seropositive patients and HCs differ in number, cell sources, and phenotypes. Plasma EVs (A) count (above) and frequency of different sizes (below) in anti-CCP⁻RF⁻, anti-CCP⁺RF^+/−, and anti-CCP^hiRF^hi RA patients and HCs; (B) Left (above), representative dot plot of EVs in FSC-A of an anti-CCP⁺RF^+/− patient and a HC, with the intensity of Pacific blue beads of different sizes. Right (above), representative microphotographs of EVs from a HC and an anti-CCP⁺RF^+/− patient (below) using STEM. (C) Left: frequency of circulating EVs from different cellular sources: platelets (CD41a), leukocytes (CD45), endothelium (CD205), erythrocytes (CD235a), and other sources (EV negative for the evaluated molecules) from anti-CCP⁻RF⁻, anti-CCP⁺RF^+/−, and anti-CCP^hiRF^hi RA patients and HCs. Right: frequency of circulating EVs from different leukocyte sources in anti-CCP⁻RF⁻, anti-CCP⁺RF^+/−, and anti-CCP^hiRF^hi RA patients and HCs. (D) Frequency of circulating EVs positive to IgG + IgM-, IgG-IgM+, IgG+IgM+, C1q+, HMGB1+, and CP+ from anti-CCP⁻RF⁻, anti-CCP⁺RF^+/−, and anti-CCP^hiRF^hi RA patients and HCs. Comparisons among the study groups were made by performing the Kruskal–Wallis test and Dunn’s post-hoc test. (E) Collective t-SNE of EVs (1 × 10⁵) derived from five samples from each group analyzed and plotted (n = 20). Every dot represents a single EV, and the color indicates ArcSinh5-transformed expression values for each given marker analyzed and calculated over EVs from all samples varying from blue for lower expression to red for higher expression.