Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221002, Jiangsu, China.
Cell Biochem Biophys. 2012 Dec;64(3):233-40. doi: 10.1007/s12013-012-9389-3.
T helper (Th) 17 cells are difficult to isolate which hinders experimental studies with these cells. Here, we report a novel method to obtain sortable, engineered mouse Th17 cells. First, we developed lentiviral vector (XZ12) containing RORγt gene and mouse ΔNGFR gene complemented with IL17A promoter (pXZ12-RORγt). As control, we used vector pXZ12 containing mouse ΔNGFR gene complemented with IL17A promoter. Mouse CD4(+)CD25(-) T cells were transduced with pXZ12-RORγt or pXZ12 vectors and cultured in the presence of transforming growth factor (TGF)-β or interleukin (IL)-6. Then, we isolated Th17 cells using anti-ΔNGFR magnetic beads. The cytokine production profiles of isolated Th17 cells were assessed by qPCR, while cell functional capabilities tested in an experimental model of mouse autoimmune encephalomyelitis (EAE). We observed that overexpression of RORγt in the presence of TGF-β and IL-6 is highly efficient to produce Th17 cells. After sorting, the purity of IL-17A(+) population was over 90 %. The phenotype of pXZ12-RORγt transduced cells in the presence of TGF-β and IL-6 was similar to natural Th17 cells, in contrast to cells overexpressing RORγt alone which were deficient for IL-21. The engineered Th17 cells intensified EAE in C57BL6 mice indicating that these cells were phenotypically and functionally similar to natural Th17 cells. In conclusion, the engineered Th17 cells described here can be a useful tool to advance studies on Th17 cells.
辅助性 T 细胞 17（Th17）细胞难以分离，这阻碍了对这些细胞的实验研究。在这里，我们报告了一种获得可分选的、工程化的小鼠 Th17 细胞的新方法。首先，我们开发了包含 RORγt 基因和补充有 IL17A 启动子（pXZ12-RORγt）的小鼠 ΔNGFR 基因的慢病毒载体（XZ12）。作为对照，我们使用了含有补充有 IL17A 启动子的小鼠 ΔNGFR 基因的载体 pXZ12。用 pXZ12-RORγt 或 pXZ12 载体转导小鼠 CD4(+)CD25(-)T 细胞，并在转化生长因子（TGF）-β或白细胞介素（IL）-6 的存在下培养。然后，我们使用抗 ΔNGFR 磁珠分离 Th17 细胞。通过 qPCR 评估分离的 Th17 细胞的细胞因子产生谱，同时在实验性自身免疫性脑脊髓炎（EAE）模型中测试细胞功能能力。我们观察到，在 TGF-β和 IL-6 的存在下过表达 RORγt 可高效产生 Th17 细胞。分选后，IL-17A(+)细胞群的纯度超过 90%。在 TGF-β和 IL-6 的存在下，pXZ12-RORγt 转导细胞的表型与天然 Th17 细胞相似，而单独过表达 RORγt 的细胞则缺乏 IL-21。工程化的 Th17 细胞在 C57BL6 小鼠中加重 EAE，表明这些细胞在表型和功能上与天然 Th17 细胞相似。总之，本文描述的工程化 Th17 细胞可以成为研究 Th17 细胞的有用工具。
