| Organ transplantation has had a considerable effect in extending and improving the quality of life of patients with endstage organ failure. As most transplants involve genetically non-identical donor–recipient combinations (allografts) , it has been necessary to apply potent combinations of drugs such as cyclosporine A (CsA) that suppress the host immune response, to prevent rejection. These drugs have many side effects, and need to be given life-long. Long-term immuno- suppression increases the risk of life-threatening infections and cancer in transplant recipients, and patients receiving immunosuppressive drugs still experience chronic graft rejection. An alternative to immunosuppression would be to reprogram the host's immune system to selectively ignore, or'tolerate', the transplant without compromising protection against pathogens.Medawar, Brent and Billingham1 demonstrated the feasibility of transplantation tolerance induction in newborn mice in 1953, yet full induction of graft tolerance has not been translated into a therapeutic procedure for humans.The immune response leading to graft rejection requires the participation of thymus-processed lymphocytes (T cells), which are the main target of tolerance induction protocols2. T cells have clonally distributed antigen-receptors designed to recognize processed antigen fragments that are presented on antigen-presenting cells (APCs) in the context of major histocompatibility complex (MHC) molecules. The immune response to allograft is that normally the immune system processes all foreign antigens through direct presentation and indirect presentation.T cells and APCs interact through a collection of surface proteins. Both the engagement of the T-cell receptor with the MHC–antigen complex and a second signal (co-stimulation), such as CD40 and CD40L,CD28 and CD80/CD86, are needed for T-cell activation. Interruption of this signaling pathway with blockers of costimulation, such as CD40 or CD28 antagonists, results in the suppression of the immune response, and antigen-specific tolerance.In a series of studies on tolerance induction by combinations of non-depleting CD4 and CD8 antibodies. Another strategy to prevent transplant rejection is to partially ablate the host hemopoietic system and to replace it with that of the donor through bone marrow (BM) transplantation.Indoleamine 2,3 dioxygenase (IDO)-expressing APCs negatively regulate T-cell response and play a critical role in the induction of transplantation tolerance. Immunosuppression mediated via IDO-APCs may therefore be augmented by promoting the development of regulatory T cells (Tregs). In this study, we overexpressed IDO in donor BMDCs using an adenovirus expression vector containing IDO cDNA (Ad-IDO) to investigate the IDO regulatory mechanism(s) in a mouse model of acute allograft rejection.1. Production of adenoviral-IDO particlesTo obtain adenoviral-IDO plasmid adenoviral particles were propagated, plaque-titered in HEK293T cells and purified by double centrifugation on CsCl gradients. The plasmid was proved to contain the full-length cDNA for the mouse IDO gene by PCR. IDO expression was determined by Western blots using a rabbit anti-murine IDO polyclonal antibody. IDO functional activity was measured in vitro by determining its ability to convert trytophan to kynurenine by HPLC. Adenoviral-GFP plasmid was generated in the laboratory and used as a negative control.2. IDO mediate survival prolongation of cardiac allograftAdenovirus-mediated gene transfer of IDO prolongs cardiac allograft survival. The mean survival time + SD of cardiac allografts injected with 1010 PFU of the adenovirus Ad-GFP (8.1+1.0, n= 8) was indistinguishable from that of control untreated hearts (8.3+0.7, n= 8). Cardiac allografts injected with 1010 PFU of AdIDO showed significant prolongation (MST+SD=16.9+1.5, n= 8).Graft-infiltrating cells from animals bearing AdIDO-treated grafts Inhibited of the MLR responses. Histological analysis of allografts on day 7 post-transplantation revealed much higher inflammatory infiltrations in either allografts pre-treated with AdGFP or untreated as compared to that of allografts pre-treated with AdIDO. Quantification of mRNA levels for cytokines and IDO expressed within transplanted hearts 5 days after transplantation showed that hearts treated with AdIDO contained significantly reduced transcript levels for IL-2, IFN-γand IL-17, whereas Foxp3 levels were increased. Heart allograft pre-treated with AdIDO had a significant higher proportion of Tregs as compared to those recipients pre-treated with AdGFP or untreated.3. Steady state of dendritic cells with ectopic IDO expression mediate skin allograft tolerance by induction of regulatory T cellsWe first checked the maturation status of BMDCs generated in our culture system.After 10 days of culture, > 90% of the bone marrow progenitors were positive for CD11c, a characteristic maker for DCs. BMDCs after IDO transduction showed a very similar phenotype as that of untransduced DCs, while LPS treated BMDCs showed a more mature status. High levels of ectopic IDO were detected in BMDCs transduced with Ad-IDO. Ad-IDO can effectively mediate ectopic IDO expression in DCs both in vitro and in vivo, demonstrating the feasibility of AdIDO-transduced BMDCs for our proposed studies in animal models. we next sought to establish the role of IDO in DCs for the induction of skin allograft tolerance. The longest mean graft survival time (MST 36.5±3.4 days) was observed in the recipient mice received IDO-transduced donor-specific BMDCs. IDO also increases the capability of DCs to suppress antigen-specific T cell response. The expression for IL-2, IFN-γand IL-17 in splenic T cells originated from recipients pre-treated with IDO-transduced BMDCs was 2-fold and 4-fold lower than that of those recipients pre-treated with GFP-transduced BMDCs or untransduced BMDCs. IDO could augment the capacity of DCs for the induction of Tregs in vitro. We therefore, performed an adoptive Treg transfer study. CD4+CD25+ Tregs or CD4+CD25- T cells were islolated from C57BL/6 recipient mice pre-treated with IDO-transduced donor-specific BMDCs after day 15 of skin allograft transplantation. It was found that the MST (29.3±1.2 days) in those recipient mice transferred with CD4+CD25+ Tregs was almost 3fold higher than that of recipient mice transferred with CD4+CD25- T cells (9.7±1.2days, P < 0.01). To further demonstrate whether those Tregs induced a donor-specific tolerance, we performed secondary cardiac allograft transplantations in those skin allograft recipient mice pre-treated with IDO-transduced BMDCs. For this purpose, the C57BL/6 recipients after 15 days of their first skin allograft transplantation were then transplanted with either BALB/c- or C3H-derived cardiac allografts. BALB/c-derived cardiac allografts survived two times longer than that of CH3-derived cardiac allografts (MST 15.7±1.0 days vs. 8±1 days, P <0.01). In conclusion, our studies show that IDO gene transfer can prolong murine skin and cardiac allograft survival. Our findings suggest that IDO may be a good target for immunotherapy particularly at the CD4+CD25+ Tregs T cell subset. |
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