NF-κB Blockade Induces Apoptosis and Decreases Costimulatory Molecules and IL-12 Secretion of Dendritic Cells, Altering DC Maturation: Role in Induction of Tolerance after Transplantation.

In steady state, dendritic cells (DC) are in an immature status and maintain tolerance to self antigens. By contrast, if DC maturation is stimulated, as it occurs in the proinflamatory milieu induced by allogeneic transplantation, they trigger effector and memory T cells and, for this reason, they play an essential role in the development of graft versus host disease (GVHD). DC maturation and survival is dependent on NF-kB. Thus, we explored the ability of the proteosome inhibitor bortezomib (B), which prevents Nuclear Factor kB (NF-kB) activation, to induce DC apoptosis and block maturation as a new strategy to prevent GVHD. DC were obtained from PBMN after culture with GM-CSF and IL-4 and were stimulated to maturate and express costimulatory molecules (CD80, CD83, CD86, CD40L) adding TNF and LPS. Even after this stimulation, B at doses ranging from 10 to 50 nM was able to decrease costimulatory molecules as well as HLA-DR expression, as assessed by flow cytometry. In addition, at 50 nM, B increased DC apoptosis (2650 vs 1404 anex/7AAD positive events among DC cultured with or w/o B, respectively; p=0.05). Moreover, B significantly decreased the production of IL-12, while IL10 secretion was unaffected (1,4% vs 6,2% DC cultured with or w/o B secreting IL-12, respectively; p=0.01). Cytokines and proinflamatory molecules genes expression profiling was also analysed by microarrays in DC cultured with or without B. Finally, after culture with bortezomib, DC were cocultured with T lymphocytes and, interestingly, allogeneic mixed lymphocyte reaction was markedly inhibited also inducing a decrease in Th1 cytokine secretion among T cells.

In conclusion, bortezomib induces apoptosis and decreases DC maturation and cytokine secretion and favours tolerance to alloantigens. This new strategy based on DC therapy supports the use of proteosome inhibitors in the management of GVHD.