Oksvold Morten P, Kullmann Anette, Forfang Lise, Kierulf Bente, Li Mu, Brech Andreas, Vlassov Alexander V, Smeland Erlend B, Neurauter Axl, Pedersen Ketil W
Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
Life Technologies AS, Oslo, Norway.
Clin Ther. 2014 Jun 1;36(6):847-862.e1. doi: 10.1016/j.clinthera.2014.05.010.
Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation.
Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81.
Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity.
Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.
外泌体是培养的所有细胞类型分泌的小（30至100纳米）囊泡，存在于大多数体液中。来自健康供体的平均1毫升血清中约含有10¹²个外泌体。根据疾病不同，外泌体数量会有所波动。血液和所有其他体液中外泌体的浓度极高。几种B细胞表面抗原（CD19、CD20、CD22、CD23、CD24、CD37、CD40和HLA - DR）以及常见白细胞抗原CD45在血液系统恶性肿瘤的免疫治疗方面具有研究意义。外泌体分离的既定标准是超速离心。然而，这种方法无法区分外泌体亚群和其他纳米囊泡。本研究的主要目的是使用一种简便可靠的免疫磁分离方法，根据这些表面标志物对从各种类型B细胞淋巴瘤细胞系释放后的外泌体的CD81⁺和CD63⁺亚群进行表征。
使用蛋白质印迹法、流式细胞术和电子显微镜，将总预富集细胞外囊泡（EV）库与使用针对外泌体标志物CD63和CD81的抗体偶联磁珠进行特异性分离后的各囊泡组分进行比较。
基于磁珠的分离是研究和比较从B细胞淋巴瘤细胞释放的外泌体亚群的便捷方法。数据表明，特异性分离的囊泡与总预富集的EV库不同。CD19、CD20、CD24、CD37和HLA - DR在外泌体上表达，但CD22、CD23、CD40和CD45不表达，不同B细胞淋巴瘤细胞系释放的外泌体之间存在很大异质性。有趣的是，这些源自B细胞淋巴瘤的EV能够使淋巴瘤细胞免受利妥昔单抗诱导的补体依赖性细胞毒性作用。
含有CD19、CD20、CD24、CD37和HLA - DR的外泌体分布可能会干扰针对这些抗原的免疫治疗，这对于优化治疗至关重要。使用免疫磁分离平台能够轻松分离和表征外泌体亚群，以便进一步研究外泌体生物学，了解其治疗和诊断应用潜力。
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