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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 Norway (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 dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation., Results: CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 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 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 CD4CD25 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.

Background: It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells., Methods: Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer., Results: Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti-third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of naïve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti-IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+ CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a naïve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into naïve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a naïve MLR against donor antigens., Conclusions: Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+ CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.

Background: Immature dendritic cells (DC), characterized by low expression of both major histocompatibility complex class II antigens and co-stimulatory molecules, can be instrumental in the induction of peripheral tolerance. Because nuclear factor (NF)-kappa B is central to DC maturation, the authors engineered DC with an adenoviral vector (Adv) encoding for a kinase-defective dominant negative form of IKK2 (dnIKK2) to block NF-kappa B activation and inhibit DC maturation., Methods: DC were obtained by culturing bone marrow from Brown Norway (BN) rats with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 11 days. To block NF-kappa B activation, at day 9, cells were transfected with AdV-dnIKK2. At day 11, cells were used as stimulators in primary mixed leukocyte reaction (MLR) with naive Lewis rat lymphocytes as responders. CD4+ T cells were purified from primary MLR and tested in secondary MLR with allogeneic mature DC and in co-culture MLR with naive lymphocytes. The tolerogenic potential of dnIKK2-DC was evaluated in vivo in a model of rat kidney allotransplantation., Results: DnIKK2-DC were immature and lacked any allostimulatory activity. T cells preexposed to allogeneic dnIKK2-DC were hyporesponsive to a secondary stimulation with mature DC and acquired potent regulatory properties, inhibiting naive T-cell proliferation toward allogeneic stimuli. Pretransplant infusion of allogeneic donor dnIKK2-DC prolonged the survival of a kidney allograft from the same allogeneic donor, without the need for immunosuppressive therapy., Conclusions: Allogeneic DC, rendered immature by dnIKK2 transfection, induce in vitro differentiation of naive T cells into CD4+ T-regulatory cells, effective at low ratios with target cells, rendering them applicable for cellular therapy of immune-mediated abnormalities and for preventing transplant rejection.