Study On The Regulatory T Cells

Regulatory T cells (Tregs) belong to a subpopulation of T cells that can functionally suppress an immune response by influencing the activity of other cell types. In recent years, regulatory T cells had become a hot spot in the immunological field and had evolved rapidly: the role of classical CD4+CD25+Foxp3+Treg had been thoroughly researched, naive peripheral CD4+CD25- T cells can be converted to CD4+CD25+Treg through costimulation with T cell receptors (TCRs) and transforming growth factor (TGF-β) which expressed the specific marker of Treg—Foxp3, and exerted potent immunosuppressive functions.γδT cells, despite small in numbers, have their own distinct characteristics such as direct antigen recognition without requirement for antigen processing and presentation as well as less TCR diversity, has played a critical role in immunodefense and immunosurveillance. In recent years, growing evidences suggest that y8 T cells also have immunoregulatory function. So we are interested in whether the murineγδT cells have a subpopulation of immunoregulatory function and whether it can be induced through costimulation with TCRs and TGF-β. We use mouse spleen T cells as the research system, the expression of Foxp3 in freshly isolated mouse y8T cells was detected by RT-PCR and flow cytometry. The results showed freshly isolated mouseγδT cells did not express Foxp3. Then we stimulated mouse spleen T cells with immobilized anti-TCRγδin the absence or presence of TGF-β(5ng/ml) for 5-8 days, and the the expression of Foxp3 as well as other immunoregulatory associated molecules were detected by Realtime PCR and/or flow cytometry. The results showed anti-TCRγδand TGF-βinduced y8T cells expressed large amount of Foxp3 and most of which coexpressed CD25, where uninducedγδT cells rarely expressed Foxp3. In addition, compared with induced TCRγδ+CD25-T cells, induced TCRy8+CD25+T cells not only expressed Foxp3, but also had increased TGF-βand GITR expression. Furthermore, we performed in vitro functional analysis which confirmed that the TGF-βinduced y8 T cells could mediate a potent immunosuppressive effect on anti-CD3 stimulated T cell activation and proliferation. In a word, our results suggest that mouse spleenγδT cells with both immunoregulatory phenotype and immunoregulatory function can be induced through TCRs and TGF-βcostimulation. y8T cells, with their distinct characteristics, are different fromαβT cells. In recent years, studies showed that activated y8T cells displayed potent cytotoxic activity against various solid and blood tumors. As a consequence,γδT cells are considered to be the promising candidate for adoptive cell therapy against tumor. Yet whetherγδT cells could distribute effectively among the tumor tissues is the prerequisite for their anti-tumor activity. During the last few years, our laboratory had established a method forγδT cells expansion in vitro using immobilized anti-TCRγδ, and the expanded y8T cell reagents displayed notable anti-tumor activity. In order to evaluate the distribution ofγδT cell reagents in vivo, especially in tumor tissues, we performed the following studies:first we analyzed the chemokine receptor expressions of the in vitro expanded y8T cell; then using colorectal carcinoma as the study objects, we analyzed the chemokine expressions in colorectal carcinoma cell lines and colorectal carcinoma tissue samples; last we investigated the chemotactic effects ofγδT cells towards colorectal carcinoma using chemotaxis experiments and chemotaxis blocking experiments.y8T cell reagents were expanded from peripheral blood mononuclear cells using immobilized anti-TCRγδin vitro, the chemokine receptor expressions ofγδT cells before and after the expansion were measured by immuno-fluorescent staining and flow cytometry. The results showed that CCR5 and CXCR3 were the main chemokine receptors expressed on the in vitro expanded Vδ1 T cells and V82 T cells. The chemokine expressions of 4 types of colorectal carcinoma cell lines and 10 cases of colorectal carcinoma tissue samples (each case included tumor tissues and para-tumor tissues) were detected by RT-PCR. The results showed that different types of colorectal carcinoma cell lines expressed different chemokines; and various chemokines including the ligands for CCR5 and CXCR3 were detected in the tumor tissues and para-tumor tissues of the colorectal carcinoma tissue samples. Therefore, we performed the chemotaxis experiments of y8T cells using two representative colorectal carcinoma cell lines (HT-29 and HR8348), and adding the blocking monoclonal antibodies for CCR5 and CXCR3 to investigate the contribution of CCR5 and CXCR3 to the chemotactic effects of theγδT cells. The results showed that in vitro expandedγδT cells possessed the ability to migrate toward tumor cells, and this effect could be partially blocked by anti-CCR5 and anti-CXCR3 monoclonal antibody. Our results showed that in vitro expandedγδT cells possessed the ability to distribute among colorectal carcinoma tissues which established a basis for their further cytotoxic effects in focal tumor tissues.