The Effects Of Transcription Factor Foxp3on The Phenotype And Genes Associated With Immune Suppression In RAW264.7Cell

Recently, with the increasing level of medical treatment, transplant technology hasbecome an important means for the treatment of diseases. However, graft transplant rejectionhas become a key problem to hinder the success of transplantation technology. Therefore,preventing transplant rejection becomes the urgent task of contemporary medicine in thefield of transplantation.Transcription factor Foxp3(Forkhead box protein3) is specially expressed on theregulatory T cells (Tregs), and it is important for differentiation, development andmaintenance of suppression function in Tregs. It has been confirmed that Tregs suppress theactivation of effector cells and control immune response in tolerant recipients. In this regard,Tregs is considered to be a potential measure for the treatment of organ transplantation.Cytotoxic T-cell-associated antigen4(CTLA-4), a transmembrane receptor on T cells, ishigh sustainably expressed on Tregs,. The binding of CTLA-4with B7can induce anergy ofT cells and can be involved in the negative regulation of immune response. In addition,CTLA-4can also function in a cell-extrinsic manner by mediating transendocytosis of CD80and CD86resulting in depletion of these costimulatory molecules. Thus, CTLA-4isimportant regulation factor in the T lymphocyte response, which may function throughinducing the activity or development of Tregs. glucocorticoid-induced TNF receptorfamily-related gene (GITR) is also high sustainably expressed on Tregs, and it is one of thetumor necrosis factor receptor superfamily (TNFRSF18) members. GITR promotes Tregsproliferation in vivo and in vitro experiments. GITR ligand (GITRL) can promote theproliferation of Tregs through combining to GITR. Presently, GITRL has been proved to be auseful tool in induced immune tolerance.MDSC (myeloid-derived suppressor cell) is a heterogeneous cell population comprisedof macrophages, granulocytes, and dendritic cells at various stages of maturation. A largenumber of studies have confirmed that inflammatory environments induce the productionand the accumulation of MDSC, and MDSC can block the immune response of CD4andCD8T cells. Compared with Tregs cells, the role of MDSC has not yet been fully describedin transplant rejection. MDSC inhibited proliferation of effector T cells and induced a contact-dependent apoptosis in an iNOS-dependent manner. The iNOS inhibitor inducedgraft rejection, thus confirming the important role of iNOS in MDSC suppression. Similarly,functional MDSC generated in vitro from murine embryonic stem cells prevented the GVHDvia IL-10and iNOS, and also able to induce the development of CD4+CD25+Foxp3+Treg.Also, research has shown that bone marrow-derived MDSC can inhibit the occurrence ofGVHD through Arg1dependent pathway, thus confirming that MDSC can play an importantrole in transplant rejection.Based on the above background, we hypothesized that Foxp3trtansfected RAW264.7cells, a mouse macrophage cell line, may be endowed with immune suppress function asTregs and MDSC. This may provide new cell therapeutic measures for transplant rejection.The study contains the following three aspects:1. Establishment of stable transfectant expressing Foxp3in RAW264.7cell lineInitially, pIRES2-EGFP vector expressing the full-length murine Foxp3gene(pIRES2-EGFP/mFoxp3) was consructed. After transfection with pIRES2-EGFP/mFoxp3orpIRES2-EGFP and selection in media containing G418, RAW264.7cells stably expressingGFP-Foxp3protein or GFP protein was identified by immunofluorescence microscopy, flowcytometry and RT-PCR.2. The changes of surface molecules on RAW264.7cells overexpressing Foxp3geneIn order to evaluate the effects of Foxp3on the expression of CTLA-4and GITR, whichare associated with immunosuppression of Tregs, a semi-quantitative RT-PCR was adoptedto detect the expression of CTLA-4and GITR. The results showed that the mRNAexpression of CTLA-4and GITR in RAW264.7cells overexpressing Foxp3gene weresignificantly increased compared with control groups. These results indicate that Foxp3canpromte the expression of CTLA-4and GITR, which are associated with immunosuppressionof Tregs, and mediate the regulation of immune response. At the same time, we found thatthe mRNA expression of GITRL in RAW264.7cells overexpressing Foxp3gene weresignificantly increased compared with control groups. This result indicates that the bindingof GITR and GITRL can promote the proliferation of Tregs. We speculate that RAW264.7cells transfected with Foxp3gene may simulate the immunosuppressive function of Tregs tonegatively regulate the immune response.3. The changes in immunosuppressive gene of RAW264.7cells overexpressing Foxp3geneIn order to evaluate the effects of Foxp3on iNOS and Arg1, which were associated with immunosuppression of MDSC, a real-time quantitative RT-PCR was adopted to detectthe expression of iNOS and Arg1. The results showed that the mRNA expression of iNOSand Arg1in RAW264.7cells overexpressing Foxp3gene were significantly increasedcompared with control groups. These results indicate that RAW264.7cells overexpressingFoxp3gene may prevent graft-versus-host diseases in iNOS and Arg1dependent pathway.We speculate that RAW264.7cells transfected with Foxp3gene may simulate MDSC toprevent the GVHD.From the studies of above, the conclusions are:1. Mouse macrophage-like cell line (RAW264.7) stably expressing GFP-Foxp3proteinor GFP protein were successfully established.2. The mRNA expression of CTLA-4, GITR and GITRL in RAW264.7cellsoverexpressing Foxp3gene were significantly increased compared with control groups.3. The mRNA expression of iNOS and Arg1in RAW264.7cells overexpressing Foxp3gene were significantly increased compared with control groups.The significance of this study is to make macrophage cells have the ability of immunesuppression so that new therapeutic measures can be provided for the treatment of transplantrejection.