Inhibitory Effects Of IDO Transfected Dendritic Cells And Tryptophan Catabolites In Transplantation Rejection

Objective:To discuss the effect on allogeneic T lymphocyte proliferation and apoptosis given by highly-expressing indoleamine2,3-dioxygenase (IDO) mouse myeloid dendritic cells (DCs) transfected by IDO gene combined with tryptophan catabolites in vivo and in vitro,which may offer a new method to inhibit transplantation rejection and induce immune tolerance successfully.Methods:(1) In vitro culture of the mice myeloid DCs was performed by selective medium containing essential cytokines for its growth. The expression of CD80, CD86, MHCII and CD11c antigen was assayed by flow cytometry.(2) DCs were infected by the recombinant IDO gene adenoviral vector. And the green fluorescence protein expression in mature DCs was observed under inverted fluorescence microscope and protein expressions of IDO were respectively detected by HPLC.(3) Separate allogeneic mouse spleen CD4+T lymphocyte by immunomagnetic microbeads, and inspect the purity of CD4+T cells by flow cytometry.(4) In vitro test, isolated CD4+T cells from the spleen of untreated recipient (BALB/c) mouse as responding cells, and then take either donor DC, DC+TC, IDO+DC or IDO+DC+TC as stimulating cells in one-way mixing lymphocyte culture. In vivo experiments, DC、TC、IDO+DC or IDO+DC+TC were injected into recipient mouse, and CD4+T cells were isolated as responding cells five days after injection and donor DCs were treated in mixing lymphocyte culture. In group IDO+DC+TC, CD4+T cells isolated from recipient mouse were mix cultured with donor DC and DC of tertiary origin from C3H/He mouse. Investigate CD4+T lymphocyte proliferation by the method of MTS,and its apoptosis was determined by Annexin-v and PI double staining method.Datas were analysised by useing data analysis software package SPSS18.0.Result:(1) Average5-6×106DCs per mouse were acquired in vitro,which have the typical structure. Flow cytometry identification showed high expression of CD80(97.5%±2.1)、CD86(92.0%±1.3)、MHC-Ⅱ (97.0%±1.6)、CDllc (89.1%±3.1).(2) When the multiplicity of infection was300, DCs which was infected by the recombinant IDO gene adenoviral vector were in good condition,and its infection efficiency was74.3%.To detect the expression of the IDO gene in DCs by HPLC, the kynurenine specific metabolic rate was61%.(3)The immunomagnetic microbeads could separate more than96%purity of CD4+T cells,which were used as responding cells in one-way mixing lymphocyte culture.(4) In vitro and vivo experiments, group of IDO+DC and TC could both inhibit CD4+T cells proliferation and induce apoptosis obviously(P<0.01), while compared to the first two group, group IDO+DC+TC had inhibit the CD4+T cells proliferation and induce apoptosis more obviously (P<0.01). In group IDO+DC+TC, contract to group C3H DC,group C57DC could inhibit CD4+T cells proliferation and induce apoptosis significantly (P<0.01). Conclusion: Highly-expressing IDO mouse myeloid DCs combined with tryptophan catabolites inhibit CD4+T cell proliferation more obviously compared with the effects of single factor, and might be able to induce antigen-specific immune suppression.