DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties

Background. We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4(+) T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. Methods. Brown Nor-way (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4+ T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4(+) T cells pre-exposed to dnlKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. Results. CD4(+) T cells pre-exposed to dnIKK2-DCs were CD4(+)CD25(-/dim) and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10(5) ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). Conclusions. DnIKK2-Treg is a unique population of CD4(+)CD25(-) T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.