JNK/AP-1–mediated death in freshly isolated epidermal Langerhans cells, regulation of Bcl transcripts, and reversal of JNK/AP-1–induced DC death by Bcl family members. (A) Inhibition of JNK/AP-1 activation restores CD40-mediated survival in IκBαAA-transfected primary skin LCs. Epidermal cells containing 10% to 20% CD1a⁺ LCs were transfected with pIRES2-EGFP or IκBαAA-pIRES2-EGFP. Twelve hours after transfection, JNK inhibitor SP600125 (1 μM) was added where indicated; 30 minutes later, cells were exposed to CD40L or were left unstimulated. Cells were cultured for 24 hours and then were labeled with CD1a-PE and propidium iodide (PI). Viable transfected EGFP⁺/CD1a⁺/FSC^high DCs that excluded PI were enumerated by FACS. Data are shown as x-fold alteration of survival (mean ± SD of 4 independent experiments) relative to the survival of vector-transfected LCs cultured in medium only. (B) DCs were transfected with pIRES2-EGFP or IκBαAA-pIRES2-EGFP, FACS-sorted, and cultured in the absence or presence of CD40L or JNK inhibitor SP600125 for 6 hours. RNA was extracted and processed for microarray analysis. Relative expression of Bcl-2 and Bcl-xL transcripts is shown. (C) Induced Bcl-2 expression restores CD40-mediated survival in NF-κB–deficient, HPC-derived DCs. DCs were cotransfected with Bcl-2 or empty vector (control) together with pIRES2-EGFP (vector) or IκBαAA-pIRES2-EGFP (IκBαAA). CD1a⁺/EGFP⁺ DCs were FACS-sorted and exposed to the indicated stimuli for 3 days. (A,C) Data are shown as x-fold alteration of survival (mean ± SD of 3 independent experiments) relative to the survival of vector-transfected DCs cultured in medium only.