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Group 2 innate lymphoid cells (ILC2s) are recently reported to play a more critical role in allergic diseases. We previously identified that mesenchymal stromal cells (MSCs) elicited therapeutic effects on allergic airway inflammation. Small extracellular vesicles (sEV) derived from MSCs possess striking advantages including low immunogenicity and high biosafety, and is extremely promising cell-free therapeutic agents. However, the effects of MSC-sEV on ILC2s are still unclear. Additionally, scalable isolation protocols are required for the mass production of homogenous MSC-sEV especially in clinical application. We previously reported that induced pluripotent stem cells-derived MSCs were the ideal cellular source for the large preparation of MSC-sEV. Here we developed a standardized scalable protocol of anion-exchange chromatography for isolation of MSC-sEV, and investigated the effects of MSC-sEV on ILC2 function from patients with allergic rhinitis and in a mouse ILC2-dominant asthma model. The characterization of MSC-sEV was successfully demonstrated in terms of size, morphology and specific markers. Using flow cytometry and human Cytokine Antibody Array, MSC-sEV but not fibroblasts-sEV (Fb-sEV) were found to significantly inhibit the function of human ILC2s. Similarly, systemic administration of MSC-sEV but not Fb-sEV exhibited an inhibition of ILC2 levels, inflammatory cell infiltration and mucus production in the lung, a reduction in levels of T helper 2 cytokines, and alleviation of airway hyperresponsiveness in a mouse model of asthma. Using RNA sequencing, miR-146a-5p was selected as the candidate to mediate the above effects of MSC-sEV. We next revealed the uptake of ILC2s to MSC-sEV, and that transfer of miR-146a-5p in MSC-sEV to ILC2s in part contributed to the effects of MSC-sEV on ILC2s in vitro and in a mouse model. In conclusion, we demonstrated that MSC-sEV were able to prevent ILC2-dominant allergic airway inflammation at least partially through miR-146a-5p, suggesting that MSC-sEV could be a novel cell-free strategy for the treatment of allergic diseases.