Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Department of Neurology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Cytotherapy. 2023 Feb;25(2):138-147. doi: 10.1016/j.jcyt.2022.09.006. Epub 2022 Oct 14.
BACKGROUND AIMS: Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay. METHODS: The bona fide MSC antigen CD73 possesses ecto-5'-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5'-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5'-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity. RESULTS: The authors showed that the ecto-5'-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5'-nucleotidase activity was resistant to EV-destroying detergent treatment. CONCLUSIONS: Ecto-5'-nucleotidase activity neither reflects the potency of the authors' MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5'-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors' MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.
背景目的：源自人间质基质细胞（MSC）的细胞外囊泡（EVs）在体外和体内的多种测定中均显示出免疫调节活性。在以前的工作中，作者比较了独立的 MSC-EV 制剂在多供体混合淋巴细胞反应（mdMLR）测定和优化的类固醇难治性急性移植物抗宿主病（aGVHD）小鼠模型中的免疫调节潜力。作者观察到，只有一部分 MSC-EV 制剂具有免疫调节能力，并证明只有具有 mdMLR 免疫调节活性的 MSC-EV 制剂才能在体内抑制 aGVHD 症状，反之亦然。由于 mdMLR 测定复杂且依赖于不同供体的原代人细胞，作者试图建立一种更容易标准化且符合成为效力测定标准的测定方法。 方法：真正的 MSC 抗原 CD73 具有外切 5'-核苷酸酶活性，可将促炎细胞外腺苷一磷酸切割成抗炎腺苷和游离磷酸盐。为了测试 MSC-EV 制剂的外切 5'-核苷酸酶活性是否反映了它们的免疫调节潜力，作者采用了一种酶测定法，以定量方式监测 CD73 的外切 5'-核苷酸酶活性，并比较了具有或缺乏 mdMLR 免疫调节活性的特征良好的 MSC-EV 制剂的活性。 结果：作者表明，MSC-EV 制剂的外切 5'-核苷酸酶活性与它们在 mdMLR 测定中调节 T 细胞反应的能力无关，因此与它们在优化的小鼠 aGVHD 模型中改善疾病症状的效力无关。此外，外切 5'-核苷酸酶活性耐受 EV 破坏洗涤剂处理。 结论：外切 5'-核苷酸酶活性既不能反映作者的 MSC-EV 制剂的效力，也不能提供有关各自 EV 完整性的任何信息。因此，外切 5'-核苷酸酶酶活性不能指示作者的 MSC-EV 产品的免疫调节效力。MSC-EV 产品的适当效力测定的开发仍然具有挑战性。
Extracell Vesicles Circ Nucl Acids. 2024-2-6
Front Immunol. 2024
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