title: Novel approach to generate genetically engineered, sortable, ΔNGFR-tagged mouse Th17 cells.
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. 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.
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221002, Jiangsu, China.
authors: Chong
authors: C
authors: Chen
authors: Huanxin
authors: H
authors: Zhang
authors: Zhengxiang
authors: Z
authors: Han
authors: Jiang
authors: J
authors: Cao
authors: Jianjun
authors: J
authors: Zhang
authors: Wei
authors: W
authors: Chen
authors: Lingyu
authors: L
authors: Zeng
authors: Kailin
authors: K
authors: Xu
mesh_terms: D000818:Animals; D060846:Cell Engineering / Q000379:methods*; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000473:pathology; D005240:Feasibility Studies; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D018189:Immunomagnetic Separation; D020381:Interleukin-17 / Q000235:genetics / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D015850:Interleukin-6 / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020800:Receptor, Nerve Growth Factor / Q000235:genetics*; D058504:Th17 Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: C098680:Il17a protein, mouse; D020381:Interleukin-17; D053645:Interleukin-2 Receptor alpha Subunit; D015850:Interleukin-6; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3; D020800:Receptor, Nerve Growth Factor; D016212:Transforming Growth Factor beta