title: [Generation of engineering Th17 cells and its function evaluation].
abstract: OBJECTIVE
  To generate engineering Th17 cells from mice CD4(+)CD25(-) naïve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Th17 cells.
  
  
  METHODS
  Recombinant lentivirus carrying mouse RORγt (pXZ9-RORγt) and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4(+)CD25(-) naïve T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-β plus IL-6 and divided into five groups: pXZ9-RORγt (group A), pXZ9 + TGF-β + IL-6 (group B), pXZ9-RORγt + TGF-β + IL-6 (group C), pXZ9 (group D) and control (group E). Production efficiency of engineering Th17 cells was referred as the percentage of IL-17A producing cells. Cytokine production profiles of these cells were assayed by realtime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis (EAE).
  
  
  RESULTS
  (1) High-title lentivirus was prepared and was succeeded to transduce CD4(+)CD25(-) naïve T cells. Forced expression of RORγt (group A) resulted in (40.25 ± 5.46)% CD4(+)CD25(-) naïve T cells converted into engineering Th17 cells and the convert efficiency increased to (60.59 ± 8.15)% in addition of TGF-β and IL-6 (group C), or decreased to (14.36 ± 5.27)% when presence of TGF-β and IL-6 only (group B). (2) IL-17A, IL-17F and IL-21 production of pXZ9-RORγt infected cells combined with TGF-β and IL-6 were most similar to natural Th17 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. (3) Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized cells and polarized of RORγt transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models.
  
  
  CONCLUSIONS
  High yield of engineering Th17 cells was prepared from CD4(+)CD25(-) naïve T cells by over expression RORγt plus TGF-β and IL-6 polarization. These engineering Th17 cells were similar to the natural Th17 cells in phenotypes and functional identification.
authors.affiliation: The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors.forename: Huan-xin
authors.initials: HX
authors.lastname: Zhang
authors.forename: Chong
authors.initials: C
authors.lastname: Chen
authors.forename: Ling-yu
authors.initials: LY
authors.lastname: Zeng
authors.forename: Yin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kai-lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005821:Genetic Techniques; D020381:Interleukin-17 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3 / Q000235:genetics*; D058504:Th17 Cells / Q000166:cytology* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D020381:Interleukin-17; D015850:Interleukin-6; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3; D016212:Transforming Growth Factor beta