DUAL BLOCKADE OF STAT4 AND NF-κB PATHWAYS ENHANCES DENDRITIC CELL TOLEROGENICITY

Abstract

P889 Aims: Administration of immature dendritic cells (DC) inhibits T cell responses and prolongs allograft survival. The tolerogenic effect is however, limited due to late activation of DC by interaction with recipient allogeneic T cells. Decoy oligodeoxyribomucleotides (ODN) specific for NF-κB prevent DC maturation in response to cytokines, but do not block maturation in response to allo-T cell stimulation. Methods and Results: We investigated immune responses of DC engineered with NF-κB ODN and sirolimus both in vivo and in vitro, since sirolimus has been shown to block Stat4 phosphorylation, a pivotal molecule mediating ignaling DC activation. B10 (H-2b) mouse bone marrow-derived DC propagated in GM-CSH and IL-4 were transduced with NF-κB ODN (10 μM). Sirolimus (20 ng/ml) was added on day 3 and extensively washed out at the end of culture. DC phenotype was evaluated by FACS. The allostimulatory function was determined by MLR and CTL assays in vitro. Cytokine profiles were analyzed by ELISA and RNase protection assay. For in vivo evaluation, DC were injected into C3H (H-2k) recipients 7 days prior to a B10 cardiac transplantation. Grafts were inspected for cytokine expression by RNase protection assay, and apoptosis of graft infiltrating lymphocytes (GIC) by TUNEL staining. Both DNA binding capacity of NF-κB (assayed by gel shift) and IL-12-stimulated Stat4 phosphorylation (determine by Western blotting) were completely blocked in sirolimus/ODN DC. Expression of CD40, CD80 and CD86 (not MHC class I, II and CD11c) was markedly inhibited by ODN alone. This inhibition appeared resistant to LPS activation, but not resistant to allo-T cell stimulation. Importantly, the inhibition of costimulatory molecule expression on sirolimus/ODN DC maintained despite allo-T cell stimulation, indicating the stable immature status. The proliferative responses of C3H spleen T cells were significantly inhibited by stimulation with B10 ODN DC (cpm 37316 ± 2149), and more profound inhibition was achieved by stimulation with sirolimus/ODN DC (14588 ± 1561, both p<0.05, compared with 73761 ± 4265 of control DC). Donor-specific CTL activity (4h 51Cr release) was also suppressed in ODN group, and further suppressed by sirolimus/ODN DC. The supernatant levels of IL-2 and IFN-γ were correlated with this suppressive pattern in MLR and CTL assays. Injection of C3H mice with sirolimus/ODN DC significantly prolonged B10 cardiac allograft survival, associated with increased apoptosis of GIC. Conclusion: DC engineered to inhibit both Stat4 and NF-κB pathways enhance their tolerogenicity.