Abstract
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. Μouse 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.
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Acknowledgments
This project was supported by the Nature Sciences Foundation of China (Grant 30971281 to Kailin Xu, grant 81000210 to Chong Chen and grant 30901753 to Zhengxiang Han) and China Ministry of Education (Grant NCET-09-0166 to Lingyu Zeng).
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None of the authors has any financial arrangement or involvement with commercial organizations producing competing products.
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Chong Chen, Huanxin Zhang, and Zhengxiang Han contributed equally to this study.
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Chen, C., Zhang, H., Han, Z. et al. Novel Approach to Generate Genetically Engineered, Sortable, ΔNGFR-Tagged Mouse Th17 Cells. Cell Biochem Biophys 64, 233–240 (2012). https://doi.org/10.1007/s12013-012-9389-3
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DOI: https://doi.org/10.1007/s12013-012-9389-3