Thielen Astrid J F, van Baarsen Iris M, Jongsma Marlieke L, Zeerleder Sacha, Spaapen Robbert M, Wouters Diana
Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Hematology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, Meibegdreef 9, 1105 AZ Amsterdam, The Netherlands.
J Immunol Methods. 2018 May;456:15-22. doi: 10.1016/j.jim.2018.02.004. Epub 2018 Feb 12.
To prevent unwanted complement activation and subsequent damage, complement activation must be tightly regulated on healthy host cells. Dysregulation of the complement system contributes to the pathology of diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. To investigate complement regulator deficiencies, primary patient cells may be used, but access to patient cells may be limited and cells are heterogeneous between different patients. To inhibit regulator function on healthy host cells, blocking antibodies can be used, though it may be difficult to exclude antibody-mediated effects. To circumvent these issues, we created single and combined complement regulator human knockout cells to be able to in vitro investigate complement activation and regulation on human cells.
CRISPR/Cas9 was used to knockout (KO) complement regulatory proteins CD46, CD55 and/or CD59 in human HAP1 cells. Single cell derived cell lines were profiled by Sanger sequencing and flow cytometry. To confirm the lack of complement regulatory function, the cells were exposed to complement in normal human serum and subsequently C3 and C4 deposition on the cell surface were detected by using flow cytometry.
We created single KO cell lines that completely lacked CD46, CD55 or CD59. We additionally generated double CD46/CD55, CD46/CD59 and CD55/CD59 KOs and triple CD46/CD55/CD59 KOs. Upon classical pathway activation, deletion of CD46 resulted in increased C3 and C4 deposition, while deleting CD55 mainly resulted to increased C3 deposition, confirming their reported function in complement regulation. Upon alternative pathway activation, C3 deposition was only observed on the triple CD46/CD55/CD59 KO cells and not on any of the other cell lines, suggesting that human cells are resistant to spontaneous complement activation and suggesting a role for CD59 in C3 regulation.
The generation of complement regulator KO cell lines provides a relevant tool for future in vitro investigations of complement activation and regulation on human cells. Furthermore, these cell lines may also be helpful to evaluate therapeutic complement inhibitors and may shed light on novel roles of complement regulatory proteins as we here observed for CD59.
为防止不必要的补体激活及随后的损伤，补体激活必须在健康宿主细胞上受到严格调控。补体系统失调会导致诸如阵发性夜间血红蛋白尿和非典型溶血尿毒综合征等疾病的病理过程。为研究补体调节蛋白缺陷，可使用患者原代细胞，但获取患者细胞可能受限，且不同患者的细胞存在异质性。为抑制健康宿主细胞上调节蛋白的功能，可使用阻断抗体，不过可能难以排除抗体介导的效应。为规避这些问题，我们创建了单一及联合补体调节蛋白人源敲除细胞，以便能够在体外研究人细胞上的补体激活与调节。
利用CRISPR/Cas9敲除人HAP1细胞中的补体调节蛋白CD46、CD55和/或CD59。通过桑格测序和流式细胞术对单细胞衍生的细胞系进行分析。为确认补体调节功能缺失，将细胞暴露于正常人血清中的补体，随后使用流式细胞术检测细胞表面C3和C4的沉积情况。
我们创建了完全缺乏CD46、CD55或CD59的单一敲除细胞系。我们还构建了双敲除细胞系CD46/CD55、CD46/CD59和CD55/CD59，以及三敲除细胞系CD46/CD55/CD59。在经典途径激活后，CD46的缺失导致C3和C4沉积增加，而CD55的缺失主要导致C3沉积增加，证实了它们在补体调节中的已知功能。在替代途径激活后，仅在三敲除细胞系CD46/CD55/CD59上观察到C3沉积，而在其他任何细胞系上均未观察到，这表明人细胞对自发补体激活具有抗性，并提示CD59在C3调节中发挥作用。
补体调节蛋白敲除细胞系的构建为未来体外研究人细胞上的补体激活与调节提供了一个相关工具。此外，这些细胞系可能也有助于评估治疗性补体抑制剂，并可能揭示补体调节蛋白的新作用，正如我们在此观察到的CD59的情况。
