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Tolerogenic dendritic cells (DCs) have the potential to prolong graft survival after transplantation. Tolerogenic DCs are in general characterized by a low expression of co-stimulatory molecule and a high IL-10:IL-12 production ratio. Based on promising results with earlier used alternatively activated DCs, we aimed to generate in culture potentially tolerogenic DC by simultaneously blocking GSK3 by lithium chloride (LiCl) and stimulating TLR2 by PAM 3 CysSerLys 4 .    Materials and Methods  Bone marrow-derived LiClPAM 3  DCs were generated by the addition of LiCl 24 hours before harvesting, and one hour later PAM 3 CysSerLys 4 . The phenotype of the DCs was assessed by determining the expression of co-stimulatory molecules in flow cytometry and cytokine production in ELISA, whereas their functional properties were tested in a mixed lymphocyte reaction. A fully MHC mismatched heterotopic heart transplant preceded by infusion of donor-derived LiClPAM 3  DC was performed to assess the tolerogenic potential of LiClPAM 3  DCs  in vivo .    Results  LiClPAM 3  DCs displayed a tolerogenic phenotype accompanied with a low expression of co-stimulatory molecules and a high IL-10:IL-12 production ratio. However, in mixed lymphocyte reaction, LiClPAM 3  DCs appeared superior in T cell stimulation, and induced Th1 and Th17 differentiation. Moreover, mice pretreated with LiClPAM 3  DC displayed a reduced graft survival. Analysis of LiClPAM 3  DC culture supernatant revealed high levels of CXCL-1, which was also found in supernatants of co-cultures of LiClPAM 3  DC and T cells. Nevertheless, we could not show a role for CXCL-1 in T cell proliferation or activation  in vitro .    Discussion  LiClPAM 3  DCs display  in vitro  a tolerogenic phenotype with a high IL-10:IL-12 ratio, but appeared to be highly immunogenic, since allograft rejection was accelerated. As yet unidentified LiClPAM 3  DC-derived factors, may explain the immunogenic character of LiClPAM 3  DCs  in vivo .

Allostimulatory Effects of Dendritic Cells with Characteristic Features of a Regulatory Phenotype