Inhibition Of T Cell Proliferation By Combined Low-Tryptophan Medium And Tryptophan Metabolites In Vitro

Objective:The subject application from mouse myeloid dendritic cells (Dendritic Cell, DC) to stimulate allogeneic T lymphocytes in mice, application of low-tryptophan medium and tryptophan metabolites in inhibition of T lymphocyte proliferation, and to reveal the mechanism of the inhibitory T lymphocyte proliferation in vitro.Methods: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. Adding tryptophan metabolites in the low-tryptophan medium, and observe the different lines of T lymphocyte proliferation in mixing lymphocyte culture. Using Annexin-V and PI double staining method determines T cell apoptosis. Datas were analysised by useing data analysis software package SPSS 15.0.Result:Average5-6×106 DCs per mouse were acquired in vitro. Dendritic cells have the typical structure. Flow cytometry identification showed high expression of CD80 (94.56%),CD86 (88.42),MHC-Ⅱ(86.12%) and CD11c (96.79%).The immunomagnetic microbeads could separate more than 90% purity of CD4+T cells. Mixed lymphocyte culture showed that low-tryptophan group compared with normal tryptophan group, stimulation index (SI) decreased significantly (P<0.001).When adding tryptophan metabolites, the KYN group,3-HK group,3-HAA group SI values are decreasing along with its concentration rised. Compared with the control group, the change has significant difference (P<0.001). Among them, the decrease of SI is the most obvious in 3-HAA group.The inhibition of low-tryptophan medium with tryptophan metabolites on CD4+Tcell proliferation is superimposed.When Annexin-V and PI double staining by measuring the apoptosis of CD4+Tcells, compared with the control group, the apoptosis rate of low-tryptophan goup was significantly higher. And when adding tryptophan metabolites, the rate of apoptosis is further increased (P <0.001).Conclusion:In vitro culture of the mice myeloid DCs by selective medium is a reliable method to acquire sufficient DCs. The immunomagnetic microbeads could separate highly purity of CD4+T cells. Mixed lymphocyte culture showed that both low-tryptophan and ryptophan metabolites can inhibit the proliferation of CD4+T cell.And the effect is superimposed. The mechanism of causing the inhibition of allogeneic CD4+T cell proliferation is "tryptophan hunger" and "toxic effects of tryptophan metabolites",it cause cell growth arrest or apoptosis. This provides the experimental basis for further study of immune tolerance after organ transplantation.