Transcriptional regulation of the Th17 immune response by IKKα.pptVIP

Li Li, Qingguo Ruan, Brendan Hilliard, Jennifer DeVirgiliis, Michael Karin, and Youhai H. Chen 1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 2Department of Pharmacology, University of California, San Diego, San Diego, CA 92093 Transcriptional regulation of the Th17 immune response by IKKα INTRODUCTIONS IKKα NF-κB: Rel (cRel), p65(RelA, NF- κB3), RelB, p50(NF- κB1), p52(NF- κB2) I κB: I κBα, I κB β , I κB γ, I κB ε, Bcl3, p100, p105 MATERIALS AND METHODS Mice and cell transfer IkkαAA mutant B6 mice Rag1-/- B6 mice bone marrow chimeric mice（ B6 recipient mice were irradiated and then intravenously injected with 107 bone marrow cells or CD4+ T cells collected from either WT or IkkαAA mice. ） Cell isolation, cell culture, and reagents. Naive CD4+CD25-CD44low CD62L+ T cells （isolated by FACS） CD4+ T cells （isolated by MACS） anti-IKKα，anti-RelA,，anti-RelB，anti–c-Rel ，anti-β-actin ，anti-CD3 ，anti-CD28 ，anti-IκBα， anti-pRelA536，anti-p100/p52，anti-pSer10H3 MOG 38–50 peptide Th cell differentiation CD4+ T cells were isolated using autoMACS automatic cell sorter Cells were cultured with IL-2, anti-CD28, anti-CD3 under the following inducing conditions： Th0- (no more addition of cytokines or antibodies) Th1- (IL-12，anti–IL-4) Th17- (anti–IL-4，anti–IFN-γ， IL-6，TGF-β1).， 5 d later，cells were washed and restimulated with anti-CD3 and anti-CD28 for 12 h， stained with anti– IL-17A and anti–IFN-γ， and examined by flow cytometry. EAE induction and evaluation. Mice first received a subcutaneous immunization with MOG emulsified in CFA and an intravenous injection of pertussis toxin，second injection of pertussis toxin 48 h later. Mice were examined daily for clinical signs of EAE and scored as follows: 0， no disease； 1； tai