Study On The Effect Of CCL17 Secreted By Dendritic Cells On The Function Of Regulatory T Lymphocytes

Objective:To investigate the effect of dendritic cells expressing chemokine CCL17 on the proliferation of Treg cells and the possible molecular mechanisms,,and further explore the function of CD8~+T on Treg cells affected by CCL17~+DC.This study may provide new ideas and methods for the application of CCL17~+DC in tumor immunotherapy.Method:First,take the BABL/C male mice as a model(1)Isolation of spleen lymphocytes and induction of Treg cells in vitro,isolation of bone marrow cells and differentiation into BMDC cells in vitro;detection of Treg cells and BMDC cell surface marker molecule expression by flow cytometry,detection of CCL17 in BMDC by fluorescence quantitative PCR Expression,while observing the morphological features of BMDC cells.(2)siRNA CCL17 was transfected into Treg cells by liposome Lipofection2000 by lipofection.The transfection efficiency was detected by FAM control group,and the expression of CCL17 was detected by fluorescent quantitative PCR.(3)Treg cells were stained by CFSE staining,and Treg cells were co-cultured with exogenous recombinant protein CCL17,BMDC expressing CCL17,and BMDC cells transfected with siRNA CCL17,and the expression of Treg cells were detected by flow cytometry and cell counting.(4)Western blot was to detect the expression of STAT5 in Treg cells after co-culture with CCL17~+BMDC cells.(5)A simple tumor microenvironment was constructed in vitro,and four cells were selected,which were CD8~+T cells,melanoma cells,CCL17~+BMDC cells,and Treg cells.The recombinant protein CCL17,siRNA CCL17 BMDC,CCL17~+BMDC and Treg cells were co-culturede for several days,then co-cultured with CD8~+T cells and melanoma cells.The effect of CD8~+T cells on the killing activity of melanoma cells was examined by a microplate reader.Second,take human dendritic cells as a model(1)Using the pcDNA3.1(-)vector,the human Foxp3 gene fragment was successfully constructed into the vector by genetic engineering,and the non-mutated recombinant plasmid pcDNA3.1-Foxp3 was successfully cloned by sequencing.(2)It is known that Jurkat cells are peripheral blood CD4~+T lymphomas,and the constructed recombinant plasmid pcDNA3.1-Foxp3 is electroporated into Jurkat cells by electroporation,and the expression of cell surface related molecules are detected by real-time PCR.(3)Isolation of healthy human PBMC,acquisition of monocytes and induction into mature DC cells in vitro,detection of CCL17 expression by PCR.(4)Exogenous recombinant protein CCL17,the DC cells treated with neutralizing antibodies CCL17 and CCL17+DC were co-cultured with Jurkat-Treg-like cells respectively.And the proliferation of Treg-like cells was detected by flow cytometry and fluorescence counting.Result:In the mouse model,mature BMDC cells induced in vitro expressed high expression of chemokine CCL17,and siRNA CCL17 was transfected into BMDC cells by lipofection.The results showed that the mRNA expression of CCL17 in the transfected group was significantly lower than that in non-transformed cells.Recombinant proteins CCL17,CCL17~+BMDC and siRNA CCL17 BMDC were co-cultured with Treg-CFSE cells respectively,and the expression of CFSE~+CD25~+double positive cells in Treg was detected by flow cytometry.The results showed that the expression of CFSE~+CD25~+double positive cells was significantly lower in CCL17~+BMDC co-cultured groups than that in siRNA CCL17 transfection group.Next,the growth of Treg cells was observed by fluorescence counting.With the co-cultivation time prolonged.The results showed that the proliferation rate of Treg cells was significantly lower than that of siRNA CCL17 transfection group.Then,the molecular mechanism of CCL17 affecting Treg cells was explored,and the expression of protein p-STAT5 was detected by Western Blot.The results showed that the expression of p-STAT5 was decreased in Treg cells treated with CCL17~+BMDC compared with the siRNA CCL17 transfected group.A simple tumor microenvironment was constructed in vitro we divided into five groups:CD8~+T cells,B16F10;Treg,CD8~+T cells,B16F10;rmCCL17,Treg,CD8~+T cells,B16F10;CCL17~+BMDC,Treg,CD8~+T cells,B16F10;siRNA CCL17 BMDC,Treg,CD8~+T cells,B16F10.The results shows that compared with siRNA CCL17BMDC group,the cytotoxicity of CD8~+T cells in the recombinant protein CCL17,CCL17~+BMDC group is increased.Next,using human dendritic cells as a model,the recombinant plasmid pcDNA3.1-Foxp3 was successfully constructed by genetic engineering and electroporated into Jurkat cells to construct Jurkat-Treg like cells.The surface molecules of Treg cells were detected by fluorescent quantitative PCR.Surface molecules include Foxp3,GZMB,CTLA-4,and PRF1 were found to have an increased expression.The constructed Jurkat-Treg-like cells were co-cultured with the recombinant protein CCL17?DC cells which were added to the neutralizing antibody CCL17 and CCL17+DC cells,respectively.And expression of CD4CD25 was detected by flow cytometry.Compared with the DC group of neutralizing antibody CCL17,the expression of surface molecule CD4CD25 positive cells were significantly decreased in CCL17~+DC group.While counting under fluorescence,it was found that with the time of co-culture prolonged.Compared to the DC to which the neutralizing antibody CCL17.,the proliferation of Jurkat-Treg like cells were inhibited in the CCL17~+DC group.Conclusion:In a mouse model,BMDC cells expressing the chemokine CCL17 are able to inhibit the proliferation of Treg cells,and studies may influence the proliferation of Treg cells through the STAT5 signaling pathway,and Treg cells treated with CCL17~+BMDC also has an impact on the function of CD8~+T cells.Furthermore,CCL17~+DC in a human cell model also have the effect of inhibiting the proliferation of Jurkat-Treg like cells..In addition,in the human cell model,CCL17+DC cells also have the effect of inhibiting the proliferation of Jurkat-Treg-like cells.In summary,dendritic cells expressing the chemokine CCL17 may have the function of interfering with the proliferation of Treg cells.