Retroviral Overexpression of Th17 Regulators

Retrovirus-mediated gene overexpression of Sox5 or Stat3 in Th17-polarized cells was performed by a RetroNectin-bound virus infection method^{30 (link),54 (link)}. In brief, we constructed the retrovirus vectors which expressed Sox5 and Stat3 genes. Then 48-well plates were coated with RetroNectin (25 µg/ml) and anti-CD3 mAb (BioXCell) overnight at 4 °C. Medium containing retrovirus was added to the RetroNectin-coated plate and the plate was centrifuged for 2 h at 2000×g at 32 °C. After washing with PBS, Th17-polarized cells were added to the retrovirus-bound RetroNectin/anti-CD3 mAb-coated plates in the presence of anti-CD28 mAb (2 µg/ml, BioXCell, D665) and were cultured for 48 h at 37 °C. The cells were then harvested for the analysis of target gene expression.