Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry
Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry
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DOI：
10.1016/s0022-1759(00)00321-5
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发表时间：
2001-01-01
影响因子：
2.2
通讯作者：
Jasani, B
中科院分区：
文献类型：
--
作者：
Clayton, A;Court, J;Jasani, B
我们提出了一种简单而强大的方法来分离和分析抗原呈递细胞（APC）释放的外泌体。外泌体是APC释放的小囊泡（40-90 nm），可能在体内具有免疫调节功能。此类外泌体源自 MHC II 类肽装载区室，因此表达高水平的 MHC LI 类。我们利用涂有 HLA DP、DQ、DR 特异性单克隆抗体的磁珠从细胞上清液中特异性分离外泌体。随后用缀合抗体对涂有外泌体的珠子进行染色，并通过行细胞术进行分析。通过该方法对外泌体进行表征表明，源自 B 淋巴细胞的外泌体表达丰富的 MHC I 类和 II 类分子。检测到的其他免疫学重要分子包括共刺激分子 B7.1 (CD80) 和 B7.2 (CD86)。还检测到粘附分子ICAM-1 (CD54)。这些外泌体还表达 B 细胞标记物 CD20 和补体抑制蛋白 CD59。溶酶体标志物 CD63 的表达存在差异，并且未检测到转铁蛋白受体 (CD71) 的表达。单核细胞衍生的树突状细胞（在GM-CSF/IL-4中培养7天）表现出不成熟的表型，并分泌具有相似表型的外泌体，具有丰富的MHC分子。 CD63 的表达始终很强，并且还存在 MHC I 类分子 CD1a。表明在脂质抗原的呈递中可能具有功能。 CD59 再次表达，表明 APC 外泌体在补体调节中可能发挥作用。没有检测到 CD71、CD40、CD14、CD20 或 CD83。提取方案的修改允许对不同条件下的外泌体分泌进行比较分析。用钙离子载体或佛波酯处理细胞导致外泌体释放明显增加，而磷脂酰肌醇3-激酶抑制剂渥曼青霉素则减少外泌体分泌。外泌体的免疫磁性分离和分析是一种用于分析 APC 外泌体的多功能且快速的工具，并且可能被证明是外泌体生物学研究的有价值的工具。 (C) 2001 Elsevier Science B.V. 保留所有权利。
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 immune-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class LI. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR 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 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 immune-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. (C) 2001 Elsevier Science B.V. All rights reserved.