Exosomes with Immune Modulatory Features Are Present in Human Breast Milk 1 Charlotte Admyre, 2 * Sara M. Johansson,* Khaleda Rahman Qazi,* Jan-Jonas File ´n, † Riitta Lahesmaa, † Mikael Norman, ‡ Etienne P. A. Neve, § Annika Scheynius,* and Susanne Gabrielsson* Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30 –100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN- production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3  CD4  CD25  T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses. The Journal of Immunology, 2007, 179: 1969 –1978. B reast milk contains a potent mixture of diverse compo- nents such as milk fat globules (MFG), 3 immune com- petent cells, and soluble proteins like IgA, cytokines, and antimicrobial peptides (1) and is considered to provide protection against early infections in the infant (2). It has further been sug- gested that breast milk might have a role in tolerance induction (3) and may protect infants from allergy development (4); however, this is controversial (5). Exosomes are MHC class I- and class II-bearing nanovesicles, 30–100 nm in size, produced from different cell types including dendritic cells (6), macrophages, and lymphocytes (7, 8) as well as epithelial (9) and tumor cells (10). They have been shown to be present in physiological fluids such as bronchoalveolar lavage (11), human plasma (12), malignant effusions (13), and urine (14) and on the surface of follicular dendritic cells (15). Although exo- somes have been implicated in cell-to-cell signaling, their physi- ological role in vivo is largely unknown. They have been found to be involved in both immune stimulation and tolerization, depend- ing on the cell origin (7, 16), and several studies have suggested the potential use of exosomes in immunotherapy (17, 18). Two phase one clinical trials using exosomes in cancer therapy have recently been published showing promising results (19, 20). In the present study we investigated whether human breast milk contains exosomes that might contribute to immune regulatory functions in the infant. We isolated vesicles from colostrum and mature milk by differential ultracentrifugation and sucrose gradient fractionation and characterized them both morphologically by electron microscopy (EM) and phenotypically by flow cytometry, Western blot and mass spectrometry (MS). We found that these vesicles display similar shapes, sizes, and densities as those of previously described exosomes. They also expressed molecules that are characteristic of exosomes such as MHC, tetraspanins and heat shock proteins. Furthermore, functional analyses showed that the vesicle preparations inhibited anti-CD3-induced IL-2 and IFN- pro- duction in vitro and increased the number of Foxp3 + CD4 + CD25 + T regulatory cells. Taken together, our results indicate that exosomes with immunomodulatory features are present in human breast milk. This novel finding might shed light on how breast milk can modulate the development of the infant’s immune system. Materials and Methods Subjects and sampling of milk and peripheral blood Colostrum (within 4 days after delivery) and mature milk (within 1– 6 mo after delivery) were collected by healthy mothers (Table I) using a manual breast pump in sterile tubes. Smoking or medication during pregnancy, known autoimmune disorder, diabetes, or allergic asthma excluded women from participation in this study. All mothers had vaginal deliveries at full term of healthy, normal birth weight infants. The colostrum samples were *Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden; † Turku Centre for Biotechnology, Turku, Finland; ‡ Department Clinical Science, Intervention and Technology, Division of Pediatrics, Karolinska Institutet and University Hospital, Stockholm, Sweden; and § Ludwig Institute for Cancer Research, Stockholm Branch, Stockholm, Sweden Received for publication July 31, 2006. Accepted for publication May 25, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by Swedish Research Council Grants 15243-01, 7924- 18A, and 71P-14158 and by the Centre for Allergy Research at the Karolinska In- stitute, the Hesselman Foundation, the Jeansson Foundation, the European Science Foundation, the Åke Wiberg Foundation, the Magnus Bergvall Foundation, the Swed- ish Cancer and Allergy Foundation, and the Swedish Asthma and Allergy Associa- tion’s Research Foundation. 2 Address correspondence and reprint requests to Dr. Charlotte Admyre, Department of Medicine, Clinical Allergy Research Unit L2:04, Karolinska Institutet and Uni- versity Hospital, Stockholm, Sweden. E-mail address: Charlotte.Admyre@ki.se 3 Abbreviations used in this paper: MFG, milk fat globule; ACN, acetonitrile; CBA, cytometric bead array; EM, electron microscopy; Hsc70, heat shock cognate protein 70; LC, liquid chromatography; MDDC, monocyte-derived dendritic cell; MS, mass spectrometry; MS/MS, tandem MS; MUC-1, mucin-1; SFC, spot-forming cell. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org