Feedback-regulated NF-κB and AP-1 transcriptional activities are selectively induced in DCs on cognate T-cell contact and CD40 triggering. (A-B) Profiling of transcriptional activity. DCs were cotransfected on d11.5 with reporter constructs for the indicated transcription factors or plasmids containing the TK promoter together with pIRES2-EGFP. EGFP⁺/CD1a⁺ DCs were FACS-sorted and cultured alone and in the presence of allogeneic (A) or autologous (B) T cells. Allogeneic CD8⁺ and CD4⁺ T cells used in panel A were isolated from MNCs of the same donor. The DC/T-cell ratio was 1:100. DCs were cultured in the presence of CD40L(A) or were pulsed with TSST-1 or cultured in the presence of T cells or blocking anti-CD40L mAbs (B). Cell-free supernatants were harvested after 36 hours, and hPAP-dependent luminescence was measured. Results are expressed as mean relative light unit (rlu) (y-axis), as obtained in quadruplicate cultures. rlu values produced by mock-transfected DCs and by DCs transfected with promoterless plasmids were negligible. Reporter activity of the TK control plasmid was similar under all conditions tested. Error bars indicate SD. Results are representative of at least 3 independent determinations. (C) IκBαAA augments basal and CD40L-induced c-Jun phosphorylation but not total c-Jun levels. FACS-purified IκBαAA or control-transfected DCs were stimulated with CD40L for 30 minutes or left untreated. Cells were lysed, and extracts prepared from equal cell numbers were separated by SDS-PAGE. After protein transfer, nitrocellulose membranes were probed with the indicated antibodies. Finally, membranes were stripped and reprobed with anti–actin antibodies to ensure equal loading and transfer of cellular proteins. (D-E) IκBαAA augments basal and CD40L-induced JNK and c-Jun phosphorylation. DCs were transfected with IκBαAA or control vector and were stimulated for the indicated time periods with CD40L. Cells were washed, fixed, and permeabilized for intracellular staining with mAbs specific for p-c-Jun (D) or p-JNK (E). Data are presented as x-fold induction of phosphorylation of indicated proteins by CD40 triggering over nonstimulated control-transfected cells. Mean ± SD of 3 independent experiments are shown. (F-G) IκBαAA impairs NF-κB function and up-regulates AP-1–mediated transcriptional activity. DCs were cotransfected with reporter constructs for AP-1 or NF-κB or TK promoter–containing plasmids together with pIRES2-EGFP (vector; left column in F-G) or IκBαAA-pIRES2-EGFP (IκBαAA; right column in F-G). EGFP⁺/CD1a⁺ DCs were FACS-sorted and cultured in the absence (F) or presence (G) of CD40L. The JNK inhibitor SP600125 (1 μM) was added (bottom row in F-G). rlu (mean ± SD, y-axis) was obtained in quadruplicate cultures. Results are representative of at least 3 independent determinations.