DENDRITIC CELLS (DCS) GENETICALLY ENGINEERED TO BLOCK NF-KB PATHWAY INDUCE THE FORMATION OF POTENT TYPE-1-LIKE T REGULATORY CELLS (TREG)

O248* It has been documented that NF-kB is central to DC immunostimulatory function as NF-kB decoys or dnIKK2 (dominant negative IkB kinase 2) overexpression blocked DC-induced T cell allostimulation. However the underlying mechanisms are still unknown. In this study we used immature DCs, obtained through transfection of BN rat bone marrow derived DCs with adenoviral vector encoding for dnIKK2 (dnIKK2-DCs), to test the hypothesis that immature DCs induced T cell-hyporesponse through the induction of Treg and to investigate the phenotype of induced Treg. DCs transfected with null adenovirus were used as control mature DCs (Ad0-DCs). FACS analysis showed that both dnIKK2-DCs and Ad0-DCs expressed CD11c, while MHC II and B7-2 molecule expression was very low in dnIKK2-DCs as compared to Ad0-DCs. DnIKK2-DCs failed to induce a proliferative response of allogeneic LW rat T cells during primary (1ry) MLR (480±162 cpm with dnIKK2-DCs vs 46,980±7,740 cpm with Ad0-DCs, n=5; DC/T cell ratio: 1/100). After 1ry MLR with dnIKK2-DCs, CD4+ T cells acquired a CD4+CD25-/dim phenotype and expressed high IL-10 and TGF-β, intermediate IFN-γ very low IL-2 while IL-4 was undetectable (by real time RT-PCR). In co-culture experiments, dnIKK2-DC-generated-CD4+ T cells potently suppressed a naïve MLR (106 LW T cells+106 irradiated BN splenocytes) up to 1/1000 ratio with naïve syngeneic T cells (185±43 cpm vs 21,500±5,545 of naïve co-culture with Ad0-DC-generated-CD4+ T cells, n=3). Addition of IL-2 to a co-culture with dnIKK2-DC-generated-CD4+ T cells failed to reverse the hyporesponsiveness, indicating that dnIKK2-DC-generated-CD4+ T cells did not induce a state of anergy. To evaluate the role of immunomodulatory cytokines, co-culture experiments were repeated in the presence of either anti-IL-10 or anti-TGF-β blocking antibodies. Only the combination of the two antibodies partially reverted the suppressive effect of dnIKK2-DC-generated CD4+ T cells on a naive allogeneic MLR (1,105±514 cpm, n=3). To investigate whether cell-to-cell contact was dispensable to the regulatory activity of dnIKK2-DC-generated-CD4+ T cells, co-cultures were repeated using a transwell system. The degree of inhibition was superimposable to that obtained in direct co-culture of dnIKK2-DC-generated-CD4+ T cells with a naïve MLR. To confirm the role of soluble factors, conditioned medium, harvested at the end of co-culture carried out in the presence of dnIKK2-DC-generated-CD4+ T cells was tested on a naïve MLR. Inhibition of a naïve MLR was observed till the dilution of 1/106 (naïve MLR: 15,633 cpm; naïve MLR+conditioned medium: 293 cpm), while medium collected at the end of co-culture with Ad0-DC-generated-CD4+ T cells had no effect (14,277 cpm). The above results indicate that dnIKK2-DCs generate Tr1-like cells which inhibit naïve T cell response through the release of soluble factors. This finding may have a relevant impact to the control of T cell reactivity in autoimmunity and transplantation.