FIGURE 1.

Characterisation of human naive CD4⁺ T cells stimulated by TCR engagement, and of their secreted MVs (MV.Act). (A) Left panel: FACS plot showing human PBMC‐derived CD4⁺ naïve T cells 5 days after TCR stimulation by anti‐CD3/anti‐CD28 beads. Cells were labelled with CFSE before stimulation. Mid panel: Representative CFSE dilution profiles of activated and nonactivated T cells, day 5. Right panel: Quantitative analysis of T cell proliferation 5 days after TCR stimulation. (B) FACS plot showing gated CD4⁺‐activated T cells stained with anti‐CD45RO/CD45RA. CD45RA ^lo CD45RO⁺ and CD45RA⁺CD45RO ^lo phenotypes correspond to activated and nonactivated T cells, respectively. Quantitative analysis of CD45RA ^lo CD45RO⁺ cells in activated T cells and nonactivated T cells are shown. (C) Representative FACS plot gated CD4⁺ CD45RA ^lo CD45RO⁺ showing subpopulation distribution of TCR‐stimulated T cells, day 5. Quantitative analysis of phenotypic T cell subsets: central memory (T_CM) ‐ CD45RA ^lo CD45RO⁺CCR7⁺CD27⁺, effector memory (T_EM) ‐ CD45RA ^lo CD45RO⁺CCR7⁻CD27⁻, effector (T_EFF) ‐ CD45RA ^lo CD45RO⁺CCR7⁻CD27⁺. TCM was the predominant T cell phenotype. (D) Flow cytometric representative plots showing CFSE‐labelled MV.Act and MV.NAct. Red, blue and green circles indicate calibration beads (0.5 µm, 0.9 µm and 3.0 µm, respectively). A CFSE⁺ population of a size traditionally associated with MVs is indicated. (E) Nanoparticle Tracking Analysis (NTA) of MV.Act. Upper panel: Particle diameter distribution (peak, 260 nm). (F) Electron microphotographs of activated T cells shedding MVs and isolated MV.Act. (G) Western blot analysis of TSG101, GRP94, synthenin‐I and mitofilin in activated T cells and MV.Act. (H) ImageStream^X imaging flow cytometry of MV.Act from CFSE‐labelled T cells (green). Both single‐channel images and merged images of Annexin V (yellow) and CD3/CD4 (red)‐stained cells are shown. A schematic depicting CFSE‐labelled, Annexin V‐positive, MVs expressing CD3 and CD4 is shown (data shown in panels A and B were analysed with the Student's t‐test for two independent groups, data shown in panel C were analysed using one‐way ANOVA, and data shown in panel D were analysed using the Mann–Whitney test *p < .05)