FIGURE 1.

Characterization of human CD4⁺ T helper cell-derived extracellular vesicles. A, FlowJo proliferation tool was used to calculate the percentage of divided cells (left panel) and the proliferation index (right panel; n = 6) of CD4⁺ T cell subsets (as indicated) analyzed for Cell Trace Violet fluorescence by flow cytometry upon 72 h of aCD3-aCD28 stimulation. B, a representative NTA-size distribution of CD4⁺ T cell-derived EVs. C, whisker plots showing EV size (mode, n = 6) determined by NTA for the indicated subset derived EVs. D, representative image by transmission electron microscopy of CD4⁺ T cell-derived EVs. E, Western blot from protein lysates from either CD4⁺ T cells or derived EVs for the indicated proteins. F, FACS analysis of purified EVs released by either unstimulated or stimulated CD4⁺ T cells previously stained with SYTO RNASelect green fluorescent. Isolated EVs were subsequently stained with Pacific Blue Cell Trace for assessing membrane integrity. G, bioanalyzer qualitative analysis of CD4⁺ T cell intracellular (left panel) and EV-associated (right panel) RNA. One representative sample is reported. H, correlation of Log10 relative (to miRNA global mean) Quantity values (evaluated by RT-qPCR) for 122 miRNAs (each circle is a single miRNA) coexpressed (Ct < 35) in extracellular vesicles isolated from an expanded Treg cell line conditioned medium by ultracentrifugation (final speed, 100000 × g) versus ExoSpin protocol (mean of 4 samples/group). The p value and Pearson r value are reported.