Immuological Mechanism Of CD4~+CD25~+ Regulatory T Cells Acting On B Lymphocytes In Vitro

In 1969, Nishizaka found that there are one kind of suppressor T cells in nascent mousse removed thymus. These T cells can regulated activity T cells and was studied to express CD25(αchain of IL-2 receptor). So these T cells are called CD4⁺CD25⁺ T cell. Some studies have show that only 5-10 percent of CD4⁺ T cell in peripheral blood is CD4⁺CD25⁺ regulatory T cell. Besides those, because they have immunosuppressive activity, these T cells are also called regulatory T cells. CD4⁺CD25⁺ Tregs are a naturally occurring population of T lymphocytes with a key role in suppressing the response of self-reactive T cells that escape negative selection in the thymus. In addition to regulating responses against self antigens, it is also well established that Tregs can exert potent suppressive effects against most other types of T cell-mediated immune responses .Although CD4⁺CD25⁺ Tregs have been confirmed to suppress B cells, the Immuological mechanism of regulatory T cells acting on effect B lymphocytes is not clear. Besides this, the study was designed to using different quantities donor antigenic specificity CD4⁺CD25⁺Treg cells to induce allograft transplantation tolerance of kidney transplantation in rats and to study the dose-effect relationship of CD4⁺CD25⁺Treg cells tentatively in vivo.Part 1: MACS classifacation and the purity identification of regulatory T CellObjective: To establish a stable and high effciency method for ex vivo enrichment of CD4⁺CD25⁺ regulatory T cells in rats.Methods: CD4⁺CD25⁺ regulatory T cells were isolated from the rat splenocytes in two steps by magic cell sorting (MACS) system. The first step was negative selection of CD4⁺ T cells by cocktail antibodies and anti-IgG magic microbeads, the second step was positive selection of CD25⁺ T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of enriched cells were measured by flow cytometry and Trypan blue dyeing. The method of real-time fluorescence quantitative PCR(RT-PCR) wasused to analyse the expression level of Foxp3 mRNA in enriched cells and spleen cells, respectively.Results: CD4⁺CD25⁺ Regulatory T Cell( Treg) can be successfully classified by the MACS and the average classifying purity could reach 79.2%±2.8% and the activity of classified cell could be higher than 90 %. The expression level of Foxp3 mRNA in enriched cells is significant higher than in spleen cellsConclusion: We established an effective procedure for enrichment of CD4⁺CD25⁺ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.Part 2: Immunological mechanism of regulatory T cells acting on effector T cellsObjective: To study the role of Treg number on immune response intensity in model of MLR. Besides this, to detect the inhibitory co-stimulating cytologic T-lymphocyteassociatedantigen 4 ( CTLA4) and cytokines secreted by regulatory T(Treg) cells, and to explore the immunological mechanism of Treg cells acting on effector B cells in co-cultured system(CCS) and separate-cultured system(SCS).Methods: We adopted Nyloon wool purification column to enrich B cells of Wistar rats. The purity and viability of enriched cells were measured by flow cytometry and Trypan blue dyeing. CD4⁺CD25⁺ regulatory T cells of Wistar rats were classified by the MACS and were loaded with antigen of SD rats. Then, characterizations of allogeneic antigen-specific CD4⁺CD25⁺ regulatory T cells were tested by mixed lymphocyte reaction (MLR) consisting of SD and BN splenocytes responder and MMC-treated ICR splenocytes as stimulator.Treg cells and B cells were added to co-cultured system(CCS)and Trans Well Millicell-PCF separate-cultured system (SCS).The inhibitory effects of Treg cells on the MLR were detected after adding Anti-TGF-β₁ or/and Anti-CTLA4 to the reacting systems. After Treg cells were co-cultured with B cells for 5 days, concentrations of IgG and IgA of the reacting systems in supernatants were determined by Beckmancoulter IMMAGE 800. CD4⁺CD25⁺ regulatory T cells inhibited the proliferative responses of Th cells by MLR.Result: B Cell can be successfully classified by the Nyloon wool purification column and the average classifying purity could reach 80.4%±4.2% and the activity of classified cell could be 92.5%±4.4%. In the MLR, allogeneic antigen-specific T cells displayed little proliferation with allogeneic-antigen existence. Our result showed that the secretion level of IgG and IgA in SCS were not obviously reduced, compared with the secretion level in positive control(P>0.05). But the secretion level of IgG and IgA in CCS were obviously lower than in the positive control(P<0.01).When Anti-TGF-β₁ or/and Anti-CTLA4 were added into CCS, the secretion level of IgG and IgA were higher than in CCS(P<0.05)and lower in the positive control. The SR in Treg cells and Th cells co-cultured systems is significant lower than in positive control group.Conclusion: The suppression of CD4⁺CD25⁺ Treg is dose-dependent. Donor-specific antigen-induced CD4⁺CD25⁺ T cells were allogeneic antigen-specific.These data show that cell-to-cell contact mechanism plays primal role in the regulation of B cells by CD4⁺CD25⁺ regulatory T cells. TGF-β₁ and CTLA-4 have partial effect. Besides this, Treg can suppress B cell induced HMI by Th cells.