CD69 Downregulation Assay for Exosome Function

CD4+ T cells were isolated from PBMC by negative selection using AutoMACS as previously described (13 (link)). The purity of CD4+ T cells was always >95% as determined by flow cytometry. The CD69 down-regulation assay was performed as described by Muller et al. (14 (link)). Briefly, CD4+ T cells were activated with anti-CD3/anti-CD28 beads (Miltenyi, San Diego, CA, USA) at the 1:2 beads to cell ratio and IL-2 (150 U/mL, Peprotech) for 2 h. Exosomes (50 µL aliquots of SEC fraction #4) were added to activated T cells and incubated for 40 h in AIMV medium (Life Technologies, Pittsburgh, PA, USA) at 37°C. The percentages of CD69⁺ live T cells or MFI for CD69 expression levels were measured by flow cytometry after staining with CD69-FITC (BD Bioscience, San Jose, CA, USA), CD4-PE (Beckman Coulter) and 7-AAD (BD Bioscience). As controls, matching isotype Abs, non-activated T cells+PBS and activated T cells+normal control (NC) exosomes were tested in parallel.