Background Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge. Objective To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure. Methods Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling. Results The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q2 = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles. Conclusion These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

Exosomes and the invariant NKT (iNKT) immune cell ligand a-galactosylceramide (aGC) may offer novel tools for cancer immunotherapy. In this study, we investigated whether exosomes loaded with aGC can activate iNKT cells and potentiate a cancer-specific adaptive immune response. aGC loaded exosomes readily activated iNKT cells both in vitro and in vivo. Exosomes loaded with aGC plus the model antigen ovalbumin (OVA) induced potent NK and gd T-cell innate immune responses, and they also synergistically amplified T-and B-cell responses that were OVA specific. In contrast to soluble aGC, which anergizes iNKT cells, we found that aGC/OVA-loaded exosomes did not induce iNKT cell anergy but were more potent than soluble aGC þ OVA in inducing adaptive immune responses. In an OVA-expressing mouse model of melanoma, treatment of tumor-bearing mice with aGC/OVA-loaded exosomes decreased tumor growth, increased antigen-specific CD8 þ T-cell tumor infiltration, and increased median survival, relative to control mice immunized with soluble aGC þ OVA alone. Notably, an additional injection of aGC/OVA-loaded exosomes further augmented the treatment effects. Our findings show that exosomes loaded with protein antigen and aGC will activate adaptive immunity in the absence of triggering iNKT-cell anergy, supporting their application in the design of a broad variety of cancer immunotherapy trials. Cancer Res; 73(13); 3865-76. Ó2013 AACR.

Extracellular vesicles (EV), including exosomes and microvesicles (MV), represent a rapidly expanding field of research with diagnostic and therapeutic applications. Although many aspects of EV function remain to be revealed and broad investigations are warranted, most published findings focus on only one vesicle category or a non-separated mix of EVs. In this paper, we investigated both MVs and exosomes from Ovalbumin (OVA)-pulsed dendritic cells for their immunostimulatory potential side-by-side in vivo. Only exosomes induced antigen-specific CD8+ T-cells, and were more efficient than MVs in eliciting antigen-specific IgG production. Further, mainly exosome-primed mouse splenocytes showed significant ex vivo interferon gamma production in response to antigen restimulation. Exosomes carried high levels of OVA, while OVA in MVs was barely detectable, which could explain the more potent antigen-specific response induced by exosomes. Moreover, exosomes induced increased germinal center B cell proportions, whereas MVs had no such effect. Immunisation with both vesicle types combined showed neither inhibitory nor synergistic effects. We conclude that DC-derived MVs and exosomes differ in their capacity to incorporate antigen and induce immune responses. The results are of importance for understanding the role of EVs in vivo, and for future design of vesicle-based immunotherapies and vaccines.

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