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 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.
Aiello
Sistiana
S
Transplant Research Center, Chiara Cucchi de Alessandri e Gilberto Crespi, Mario Negri Institute for Pharmacological Research, Bergamo, Italy.
Cassis
Paola
P
Cassis
Linda
L
Tomasoni
Susanna
S
Benigni
Ariela
A
Pezzotta
Anna
A
Cavinato
Regiane A
RA
Cugini
Daniela
D
Azzollini
Nadia
N
Mister
Marilena
M
Longaretti
Lorena
L
Thomson
Angus W
AW
Remuzzi
Giuseppe
G
Noris
Marina
M
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D002454:Cell Differentiation / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000378:metabolism*; D006085:Graft Survival; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D007108:Immune Tolerance; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D016030:Kidney Transplantation / Q000276:immunology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010641:Phenotype; D051381:Rats; D012995:Solubility; D014184:Transplantation, Homologous / Q000276:immunology
D053645:Interleukin-2 Receptor alpha Subunit; D052247:Nitric Oxide Synthase Type II; D051550:I-kappa B Kinase; C518254:Ikbkb protein, rat