Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.
PLoS One. 2013;8(3):e60461. doi: 10.1371/journal.pone.0060461. Epub 2013 Mar 26.
Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis.
间充质干细胞 (MSC) 优先迁移到受损组织，并且由于其免疫调节和营养特性，有助于组织修复。尽管 MSC 表达膜辅助蛋白 (CD46)、补体衰变加速因子 (CD55) 和保护素 (CD59) 等分子，从而免受补体介导的裂解，但 MSC 在移植后会被过敏毒素募集和激活，这可能导致 MSC 死亡并限制治疗效果。我们之前已经证明，通过逆转录病毒转导 MSC 表达 HCMV-US 蛋白会导致 HLA-I 表达降低，从而导致 MSC 植入水平更高。在这里，我们研究了工程 MSC 表达 US2 (MSC-US2)、US3 (MSC-US3)、US6 (MSC-US6) 或 US11 (MSC-US11) HCMV 蛋白是否可以改变补体识别，从而更好地保护 MSC 免受补体攻击和裂解。通过流式细胞术评估中位荧光强度比 (MFI)，发现 HCMV-US 蛋白以不同水平增加了 MSC CD59 的表达。在 MSC-US2、MSC-US3 和 MSC-US6 中观察到 CD59 表达显著增加，但在 MSC-US11 中没有观察到。只有 MSC-US2 显示出 CD46 的表达增加，而 US2 和 US3 蛋白都能够增加表达该分子的 MSC 的百分比。无论表达哪种 HCMV 蛋白，都不会改变 CD55 的 MFI；然而，表达 US6、US11 和 US2 都增加了表达该分子的 MSC 的百分比。由于 US2 蛋白在上调所有三种补体调节蛋白方面最有效，因此我们使用功能性补体介导的细胞毒性测定来研究 MSC-US2 是否免受补体介导的裂解。我们证明，与未转导的 MSC 相比，US2 蛋白的过表达使补体裂解减少了 59.10±12.89%。这是我们所知的第一个描述 HCMV-US 蛋白在补体逃避中的作用的报告，我们的数据表明，MSC 上 US2 蛋白的过表达可以作为保护这些细胞免受补体裂解的一种策略。
