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

Objective:To explore the effect on allogeneic T lymphocyte proliferation given by highly-expressing indoleamine2,3-dioxygenase (IDO) mouse myeloid dendritic cells (DCs) transfected by IDO gene combined with tryptophan metabolites,which may offer a new method to inhibit transplantation rejection and induce immune tolerance successfully.Methods:(1) Obtain the IDO gene from mouse epididymis by the method of RT-PCR and construct a eukaryotie expression vector containing mouse IDO gene.(2) 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.(3) The recombinant pEGFP-N1-IDO was transfected into mature DCs by liposome. The green fluorescence protein expression in mature DCs was observed under inverted fluorescence microscope and the mRNA and protein expressions of IDO were respectively detected by RT-PCR and immunocytochemical technique.(4) Separate allogeneic mouse spleen CD4+T lymphocyte by immunomagnetic microbeads, and inspect the purity of CD4+T cells by flow cytometry.(5) Take DCs transfected by IDO gene as stimulating cells and take CD4+T cells as responding cells in mixing lymphocyte culture. Meanwhile add the tryptophan metabolites into medium and investigate CD4+T lymphocyte proliferation by the method of MTT. Datas were analysised by useing data analysis software package SPSS17.0.Result:(1) Construct a eukaryotie expression vector containing mouse IDO gene,which was transfected into mature DCs and express IDO successfully.(2) Average5-6×106DCs per mouse were acquired in vitro,which have the typical structure. Flow cytometry identification showed high expression of CD80(97.7%)、 CD86(83.6%)、 MHC-Ⅱ (94.6%)、CD11c (87.9%).(3)The immunomagnetic microbeads could separate more than90%purity of CD4+T cells.(4)The groups of IDO+DC and IDO-DC combined with tryptophan metabolites (KYN,3-HK,3-HAA) could inhibit CD4+T cells proliferation obviously, while compared with the both, the group IDO+DC combined with tryptophan metabolites had inhibit the CD4+T cells proliferation more obviously (P<0.001). Among them, the inhibition is the most obvious in3-HAA group. The inhibition of IDO-expressing DCs with tryptophan metabolites on CD4+T cell proliferation is superimposed.Conclusion:Highly-expressing IDO mouse myeloid DCs combined with tryptophan metabolites inhibit CD4+T cell proliferation more obviously compared with the effects of single factor.