Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-κB-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-κB. Herein, we describe the combined use of NF-κB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-κB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.