Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry

We present a simple yet powerful method for the isolation and analysis of e xosomes released by antigen-presenting cells (APC). Exosomes are small vesi cles (40-90 nm) released by APC, and may have an immune-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compart ments and, as such, express high levels of MHC Class LI. We have utilised m agnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, D R for the specific isolation of exosomes from cell-fret supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by Row cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detecte d included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The ad hesion molecule ICAM-1 (CD54) was also detected. These exosomes also expres sed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no det ectable expression of transferrin receptor (CD71). Monocyte derived dendrit ic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phe notype, and secreted exosomes with a similar phenotype, with abundant MHC m olecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present. suggesting a possible function in t he presentation of lipid antigens. Again CD59 was expressed suggesting a po ssible role for APC exosomes in complement regulation. There was no detecta ble CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditio ns. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-k inase inhibitor, wortmannin, reduced exosome secretion. The immune-magnetic isolation and analysis of exosomes is a Versatile and rapid tool for the a nalysis of APC exosomes, and may prove a valuable tool for the study of exo some biology. (C) 2001 Elsevier Science B.V. All rights reserved.