Recombinant Adenovirus Mediated Expression Of Ido By Dcs Affects Alloreactive T Cells Differentiation

Objective: Previously, we reported that recombinant adenovirus mediated expression of IDO in donor-derived BMDCs could prolong skin allograft survival by negative regulation of T-cell response. Here we investigated the changes in T cell subsets in vitro and in vivo in a mice skin allograft model, in which they were treated with donor-derived BMDCs with/without overexpression of IDO.Methods: Adenoviral-IDO or adenovirus used for control was propagated in HEK293T cells, and crude viral lysate was obtained by freezing and thawing for lysing the cells to release the viruses. IDO expression was determined by RT-PCR and Western Blotting. Donor derived BMDCs from BALB/c mice were obtained by inducing culture of bone marrow cells with GM-CSF (10 ng/ml) plus IL-4 (1 ng/ml) for 7 days. The mRNA levels of IDO expression in BMDC transduced with Ad-IDO were detected by RT-PCR, and its protein levels were determined by Western Blotting using a rabbit anti-murine IDO polyclonal antibody. The proportion of T cell subsets (Th1 and Th17) in an MLC was analyzed using flow cytometry. Thereafter the BMDCs transduced with/without Ad-IDO were infused into C57BL/6 mice via caudal vein. Allogenic skin transplantation was performed 24 hrs after cells infusion, and the alloreatcive T-cell subsets from spleen and lymphocyte nodes in recipient mice were determined by flow cytometry.Results:1. Adenovirus vector containing IDO cDNA propagation and crude viral lysate preparation?First, the adenovirus production was packaged into HEK293T cells, and crude viral lysate were obtained by freezing (-80°C) and thawing (37°C water bath) for lysing these cells to release the viruses at 5 to 6 cycles. As a result, highly purified and enough titers of Ad-IDO viral vectors were prepared for use.2. Recombinant adenovirus mediated overexpression of IDO can inhibit alloreactive T cells differentiation to Th1 and Th17 cellsThe BMDCs exhibited high purity to be infected by adenovirus vector with high efficiency which was determined by FCM after Ad-GFP transduction. RT-PCR and Western blot demonstrated the expression of IDO in Ad-IDO infected BMDCs.In an MLC, after coculture of BMDCs with/without Ad transduction and allogeneic na?ve T cells, it was found that the differentiation of alloreactive T-cell subsets IFN-γ+Th1 and IL-17+T cell were both strongly suppressed by IDO-expressing BMDC in comparison with the control groups.In vivo, because the proportion of IL-17+ T cell from the recipient's spleen was actually always very tiny, the influences of infusion of Ad-IDO treated BMDC on alloreative Th17 differentiation thus was not detectable. Whereas it is obvious that BMDCs transduced with IDO inhibit na?ve T cell differentiation to IFN-γ+Th1 after skin allograft transplantation. Also we tested the IL-17+T and IFN-γ+Th1 level in the draining lymphocyte node, but it showed that these alloreative T-cell subsets were always at very low levels.Conclusions: Our findings indicated that recombinant adenovirus mediated overexpression of IDO on donor-derived BMDCs can strongly suppress the differentiation of na?ve T cells to alloreative IFN-γ+Th1 and IL-17+T cells in vitro; in vivo, although we did not verify its suppression on na?ve T cells to IL-17+T cells, its inhibition on na?ve T cells to IFN-γ+Th1 was apparent. This suggested that IDO may play pivotal roles in mediating alloimmune tolerance.