Ongoing classical IL-6 signaling is required to maintain a pathogenic Th17 response in vivo

Th17 precursors from WT and IL6ra-/- mice were co-transferred into Rag1-/- mice to induce colitis. RNA was isolated from CD4+ T cells pre-transfer and from colonic CD4+ T cells 4 weeks post- transfer Another aim is to compare gene expression of WT (IL-6) v WT (HDS) v IL6RaKO (HDS) in vitro polarized Thy1.1+ Th17p cells Overall design: WT (CD45.1) and Il6ra-/- (CD45.2) IL-17F/Thy1.1 CD4 T cells were cultured under Th17 conditions in vitro (2.5 ug/ml anti-CD3, 10 ug/ml anti-IFN-g, 10 ug/ml anti-IL-4, 2.5 ng/ml TGF-b, 20 ng/ml hyper-IL-6, with irradiated splenic APCs) then Th17p (Thy1.1+) were harvested (pre_WT and pre_KO) and co-transferred into Rag-/- mice to induce colitis. Four weeks after transfer, live CD4+ TCRb+ cells from the colon of recipient mice were sorted into CD45.1+ WT (post_WT) and CD45.2+ IL-6Ra KO (post_KO) groups for RNA-Seq analysis. 3 samples (WT IL-6, WT HDS63, KO HDS63) of T cells from spleen and lymph nodes with 2 replicates each (6 samples), plus two controls (WT Tn and KO Tn) also with two replicates (4 samples).