The Effect Of Extracellular Ubiquitin On Immunosuppression Of Treg Cells

Objective:CD4+CD25+ regulatory T (Treg) cells were proved to play important roles in immune homeostasis, especially immune tolerance, mainly by regulating the function of CD4+CD25-effector T (Teff) cells. As the reduced activity of Treg cells is closely related with autoimmune diseases, development of strategies for modulating the activity of Treg cells has become the focus of recent studies. In our preliminary findings, the concentration of ubiquitin increased significantly in the process of blood storage, and there was a certain correlation with save time. CXCR4,ubiquitin receptor, is highly expressed on surface of CD4+CD25+ Treg cells, and closely related to chemotaxis migration of Treg cells, the induction of immune tolerance and antitumor immunity. So whether does ubiquitin affect the function of Treg cells by combining CXCR4? In our research, we will discuss the combination of ubiquitin and Treg cells and the influence of Treg cells’immunosuppressive function upon ubiquitin treatment by detecting the combination of extracellular ubiquitin and Treg cells.Methods:We first determined the binding efficiency of Treg cells in human peripheral blood mononuclear cells (PBMCs), from 8 healthy volunteers, to different concentration of FITC-labeled ubiquitin at 37℃ for 1 h by flow cytometry, and then obtain the optimal concentration of ubiquitin. Using transwell assays, we evaluated the effect of ubiquitin(0,0.2,0.4,0.6 and0.8 μM) on chemotactic migration of primary Treg cells. By multi-parametric flow cytometry analysis, we determined the influence of extracellular ubiquitin on the regulatory function of Treg cells by the co-culture system of Treg cells and Teff cells(1:0,1:1,1:2 and 1:3), and measured the ubiquitin-induced apoptotic effect to Treg cells.Results:We observed that primary Treg cells and FITC-labeled extracellular ubiquitin show affinity for each other, and the mean combined rate of Treg cells in PBMCs and pure Treg cells are 1.26±0.11% and 17.93±1.81%,95% CI are 1.00%-1.52% and 13.64%-22.23%, and the optimal concentration of ubiquitin is 0.6 μM; In addition, we found that ubiquitin exhibited enhancing effect on chemotactic migration of Treg cells; The addition of ubiquitin to co-cultures of Treg and Teff cells enhanced the inhibitory effect of Treg cells on Teff cell proliferation; Moreover, ubiquitin could reduce apoptosis of Treg cells and the effect is more stronger when the concentration of ubiquitin is 0.6 μM.Conclusions:We proved that extracellular Ub could significantly affect the regulatory function of Treg cells by direct binding to them, and thus modulate immune response likely through the indirect control of Teff cells.