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. 2022 Jun 22;13:875825. doi: 10.3389/fimmu.2022.875825

Figure 1.

Figure 1

Characterization of Treg EVs isolated from Treg cells and in vitro suppressive function. (A) Nanoparticle analysis of Treg EVs demonstrates single peak distribution within a 20-200nm range consistent with functional EV subsets such as exosomes and microvesicles. (B) SEM imaging confirms EV particles within the range of the nanoparticle analysis. (C, D) Miltenyi MACSPlex exosome analysis shows Treg EVs are positive for common exosome markers of CD9, CD63, and CD81 while media EVs from added serum are only positive for CD81. Additional MACSPlex exosome assay results differentially define the Treg EV population as being positive for CD2, CD4, CD25, CD44, CD29, CD45 and HLA-DRDPDQ compared to the media EVs from supplemented serum components that express none of these markers. (E) In vitro treatment of pro-inflammatory myeloid cells (M1) with Treg EVs from ALS patient expanded Tregs reduces the IL-6 protein following overnight co-culture treatment. Suppression of IL-6 production with EV treatments normalized to max IL-6 output of M1 cells with activation and no treatment. (F) Escalating doses of Treg EVs from expanded Tregs differentially suppress Tresp proliferation compared to media EVs. Numbers shown as averages ± SEM with analysis via one-way ANOVA with Tukey’s post hoc testing (***p < 0.001). (A, B: representative data/images. C, D: Treg EVs n = 6 and media EVs n = 3. E, F: Treg EVs n = 7 and media only EVs n = 3).