Investigation Of Regulatory Roles Of γδ T Cells

Regulatory T cells (Tregs), a subpopulation of suppressor T cells, play important roles in immune pathology and graft tolerance through inhibiting T cell proliferation and cytokine secretion, thereby inhibiting the body's immune response to prevent the sustained inflammation attacking healthy tissues and maintain immune balance. Deficiency or dysfunction of Tregs can lead to many human autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and myasthenia gravis. In addition, Tregs are also involved in aplastic anemia, graft-versus-host disease, and transplant rejection pathological process. Tregs can be divided into CD4+Tregs and CD8+Tregs, NKT Tregs and double negative Tregs, among which the CD4+CD25+Tregs are currently the most widely studied Tregs. CD4+CD25+Tregs also express other immunomodulatory markers, such as cytotoxic T lymphocyte-associated antigen-4(CTLA-4), glucocorticoid induced tumor necrosis factor receptor (GITR) on the cell surface, and forkhad box P3(Foxp3) in the nucleus.Foxp3is an essential molecule to maintain autoimmune balance. Moreover, in vivo and in vitro experiments have shown that Foxp3expression can be induced in CD4+T cells to confer immunosuppressive function of Tregs.γδ T cells, a small subset of T cells, possess a distinct T cell receptor (TCR) composed of two glycoprotein chains called γ-and δ-TCR chains on their surface. γδ T cells are mainly distributed in the mucous membrane and subcutaneous tissue of the skin, intestine, lung and reproductive organs. In human peripheral blood, γδ was approximately expressed in0.5%to10%of CD3+T cells. According to the composition of δ chain, γδ T cells are divided into two subsets:V81and Vδ2T cells. V81T cells mainly locate in the intestinal epithelial tissue as an important component of intestinal epithelial lymphocytes (iIELs), while V81in the peripheral blood constitute only20%of γδ T cells. Vδ2T cells were mainly seen in peripheral blood, accounting for70%of γδ T cells. Vδ2T cells have the ability of significant cytotoxicity and memory responses to repeated microbial infections. Presently, little is known about the biological characteristics and functions of V81T cells in the peripheral blood because of its less amount and difficulties to ex vivo expansion. ilELs locate within and between the columnar epithelial cells as loosely infiltrating lymphocytes. iIELs play critical roles in mucosal immunoreaction and therefore become a hot spot of mucosal immune system research. It has been demonstrated that the phenotype and function of iIELs are diversified. According to the differences of T cell receptor (TCR) on the surface, iIELs can be classified into αβ T cells and γδ T cells. αβ T cells possess significant cytotoxic activity, contributing to the mucosal immune response. The function of y8T cells has not been completely clarified. However, it has been reported that y8T cells play multiple roles in immunological defense and cytotoxic activity. Moreover, recent studies showed the potential regulatory ability of γδ iIELs in immune response and some subsets might assume this function. Therefore, this article will focus on the regulatory function of γδ T cells.In this study, we analyzed the characteristics of Vδ1T cells deriving from human peripheral blood mononuclear cells (PBMCs) and γδ T cells deriving from mouse iIELs. In the first part of this thesis, we observed the expression of intracellularFoxp3in anti-TCR Vδ1monoclonal antibody (mAb)-stimulated Vδ1T cells from human PBMCs in the absence or presence of exogenous transforming growth factor beta-1(TGF-β1). The results showed that Foxp3was stably expressed in Vδ1T cells stimulated with anti-human TCR V81mAb alone. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the level of TGF-β1was significantly increased in the supernatant of anti-human TCR Vδ1mAb stimulated PBMCs in comparison with the medium. Subsequently, we analyzed the Tregs-specific surface markers and cytokines using FASC method. The results showed that V81T cells could express surface molecules, such as GITR, CTLA-4and CD25, and intracellular cytokines, such as interleukin-10(IL-10) and TGF-β1. Finally, we evaluated the suppressive potential of Foxp3+Vδ1T cells on the proliferation of autologous CD4naive T cells using CFSE. The results showed that CD25highVδ1T cells could inhibit the proliferation of CD4naive T cells. Taken together, the above findings suggest that Foxp3+Vδ1T cells are regulatory T cells. This will attribute to the studies of incidence and treatment of certain intestinal inflammatory diseases and the immune tolerance in transplantation.In the second part of this thesis, we firstly prepared iIELs from the small intestines of the C57BL/6J mice. These iIELs were cocultured with TGF-β1and anti-γδ TCR antibodies and examined the expression of Foxp3using FACS. The results showed that TGF-β1and anti-γδ TCR antibody could co-induce the generation of Foxp3+γδ T cells. Subsequently, we evaluated the effect of Foxp3+γδ T cells on the proliferation of CD4+T cells using CFSE-based T-cell proliferation assay. The results showed that Foxp3+γδ T cells could significantly inhibit the proliferation of CD4+T cells. These results suggest that Foxp3+γδ T cells can be prepared from iIELs in vitro through co-induction of TGF-βland anti-γδ TCR antibody. And Foxp3+γδ T cells also have potential immunoregulatory functions. Taken together, our findings will contribute to the understanding the potential role of TGF-βlin the generation of immunoregulatory γδ T cells during the process of mucosal immune response. Furthermore, our study also provides some clues for pathogenesis study and therapy of intestinal autoimmune diseases.