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

Abstract

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40–90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow 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 detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Introduction

Peptide loading of MHC Class II molecules within antigen presenting cells (APC) and other cells occurs in endocytic compartments called MIICs (MHC class II enriched compartments) (Geuze, 1994). Functionally different sub classes of MIICs have been described (Kleijmeer et al., 1997), and some of these contain multiple internal vesicles of less than 0.1 μm in diameter, generated from inward budding of the limiting membrane of MIICs (Fernandez-Borja et al., 1999). The fusion of these multivesicular bodies (MVBs) with the plasma membrane leads to the release of the internal vesicles into the extracellular space. The released vesicles, termed exosomes, have generated considerable interest, as they may have a range of physiological functions, in diverse cell types.

Exosomes were initially described during the late stages of erythrocyte differentiation, where their release was postulated as functioning in shedding redundant plasma membrane proteins, such as the transferrin receptor (CD71) (Johnstone et al., 1991). Other reports have described the release of exosome-like vesicles from a range of cells, such as platelets (Heijnen et al., 1999), T cells (Peters et al., 1991) and antigen-presenting cells (APC) such as dendritic cells and B cells (Raposo et al., 1996; Zitvogel et al., 1998). The composition and function of APC derived exosomes have been extensively studied. B-Lymphocyte-derived exosomes comprise abundant MHC Class II molecules, and they were demonstrated to be functional in antigen presentation, leading to the induction of antigen-specific, MHC Class II restricted T cell proliferation in vitro (Raposo et al., 1996).

A potential therapeutic use for exosomes in immunotherapy has been recently demonstrated. Murine dendritic cells (DC) pulsed with tumour-specific peptide, were administered to mice with established P815 tumours. At day 60, around 20% of mice were tumour free. The administration of exosomes, derived from the DC, however, resulted in a more effective response, with tumour eradication in 60% of mice. The tumour clearance was an MHC Class I restricted response as specific CTL activities were detected in the spleen of cured animals (Zitvogel et al., 1998). Exosomes may therefore herald a new approach to antigen presenting therapies.

Previously, exosomes have been prepared through a process of serial centrifugation of culture supernatant, with a final exosome pelleting step by centrifugation at around 100 000×g for 1 h or longer. In addition, exosomes have been further purified by centrifugation through flotation on linear sucrose gradients, with equilibrium occurring at around 1.14 g/ml (Raposo et al., 1996). These preparations demonstrate a relatively homogeneous population of vesicles, under electron microscopy, with a size range of around 40–90 nm in diameter. Immuno-gold labelling, under the electron microscope, has demonstrated the localisation of several molecules on the exosome surface, and these results have been confirmed by immuno-blotting and peptide mass mapping. Through these techniques, the list of molecules present on exosomes is growing rapidly, and includes Class II, Class I, CD63, CD81, MAC-1, B7.2, and milk-fat globule-EGF Factor VIII (MGF-E8) (Raposo et al., 1996; Escola et al., 1998; Zitvogel et al., 1998; Thery et al., 1999). Both the preparative and analytical methods utilized in these studies, however, are complex and time consuming. Moreover they may well be influenced by exosomes and contaminating proteins contained within foetal bovine serum, where as much as 10% of exosomes in the preparations may be of bovine origin (Thery et al., 1999).

Here we present a simple and rapid method, suitable for the routine isolation and analysis of exosomes, based upon immuno-magnetic extraction of exosomes bearing human MHC Class II. The subsequent analysis of bead-exosome complexes by flow cytometry facilitates a rapid semi-quantitative characterisation of the exosome phenotype. Furthermore, we demonstrate the principle that unsaturated exosome-bead complexes can be used to examine alterations in exosome secretion, and therefore assist in evaluating mechanisms of exosome regulation. We believe that this method will be a valuable tool in the study of exosome biology.