The effects of adenoviral overexpression of Indoleamine 2.3- dioxygenase in bone-marrow derived rat dendritic cells on T cell proliferation and phenotype

Background: Targeting the interaction between antigen presenting cells and T cells represents an appealing tool for acquiring graft-specific tolerance. In this study, we investigated the feasibility of enhancing the expression of Indoleamine 2.3-dioxygenase (IDO) in rat dendritic cells (DC), as a strategy to obtain tolerogenic dendritic cells. The phenotype of these adenoviral-transduced dendritic cells overexpressing IDO was analyzed. Moreover, the effects of transduced DC on the proliferation and the phenotype of the T cells were investigated in vitro. Methods: DC were obtained from bone marrow of Brown Norway rats. Nine days after harvesting, DC were transduced with an RGD modified adenovirus carrying the gene for human IDO (RGD-AdTIDO) or the gene for luciferase (RGD-AdTL). Untransduced DC served as control. At day 11, DC were used as stimulators in a mixed leukocyte reaction (MLR) with naïve Lewis rat lymphocytes as responders. Phenotype of DC and T-cells was assessed by FACS and real-time PCR. Results: Both in RGD-AdTIDO and RGD-AdTL transduced cells CD86 membrane expression was decreased in DC compared with the untransduced cells. However, in the MLR only the DC overexpressing IDO suppressed the proliferation of the naïve T cells, which was associated with an up-regulation of foxp3, IL-10, IL-13, IL-2 and IL-17 gene expression. Conclusion: Although both RGD-AdTL and RGD-AdTIDO transduced DC showed an immature phenotype, only the IDO-overexpressing DC inhibited the proliferation of the naïve T cells in vitro. Moreover, these T cells exhibited a mixture of tolerogenic and proinflammatory cytokine profile. Whether the antior the pro-inflammatory effects of IDO transduced DC will predominate in vivo remains to be established. Rijksuniversiteit Groningen IDO in renal transplantation 81 IDO overexpressing DC suppress T cells Introduction Dendritic cells (DC) are highly-specialized antigen presenting cells able to modulate the T-cell reactivity to foreign antigens. While DC are key regulators of the immune outcome, they are capable of both promoting and suppressing T-cell responses, depending on their functional state of maturation. Mature DC possess a strong immunostimulatory capacity, which is reflected by high levels of cell surface class II major histocompatibility complex (MHC II), integrin CD11c and co-stimulatory molecules such as CD80 (B7-1), CD86 (B7-2) and CD40. Consequently, fully mature DC induce a potent, antigen-specific response characterized by the induction of T helper (Th) type 1 CD4 T cells, cytotoxic CD8+ T cells and the activation of natural killer cells. In contrast, immature DC, which were previously regarded as mere sentinels for antigens, diminish Tcell reactivity and induce tolerance. Immature DC, characterized by a low expression of both MHC II antigens, CD11c integrin and co-stimulatory molecules, promote peripheral tolerance by several mechanisms including T-cell anergy, immune deviation (i.e. shifting from a Th1-dominated response toward a Th2 response and inducing infectious tolerance), T-cell apoptosis and stimulation of regulatory T cell (Treg) formation. Specifically, immature DC can differentiate naïve CD4+ T cells into different Treg classes, including CD4+CD25+foxp3 Treg and type 1 T regulatory cells (Tr1), which inhibit proliferation of Th1 cells. Thus, mature DC induce immunity, while immature DC promote tolerance to the presented antigen. Because of the central role of DC in modulating T cell reactivity, modification of their function by, for instance, genetic manipulation with tolerogenic molecules, is an attractive approach to treat immune-mediated diseases, including transplant allograft rejection. Indoleamine 2.3-dioxygenase (IDO) is an enzyme that catabolizes the essential aminoacid tryptophan into kynurenines and plays a crucial role in the foeto-maternal tolerance. Tryptophan starvation, accumulation of kynurenines and induction of Treg are considered as the mechanisms underlying the tolerogenic effects of IDO. Overexpression of IDO in solid organ transplantation was previously found to attenuate acute rejection of the lung, heart, islets and skin allograft. We have previously shown that adenovirusmediated gene therapy with IDO attenuates the features of both acute rejection and CTD (Chapter 3, this thesis) in animal models of kidney transplantation. During the last decade, the importance of IDO expression in DC has been recognized. It has been observed that induction of IDO by DC is necessary for the protection from graft versus host disease (GVHD), as INFγ -/mice succumbed to GVHD very rapidly due to the failure of IDO expression in DC. Moreover, IDO is able to steer the phenotype of DC towards