Natural And Induced Regulatory T Cell Inhibited The Proliferation Of Lymphocytes Through TIM-3-TIM-3L Pathway

Part I. TGF-β1 influenced the proliferation and CD25 expression of alloreactive T cells[Objective] To investigate the roles of TGF-β1 in the proliferation and CD25 expression of alloreactive T cells.[Methods] In MLC, purified C57BL/6 T cells labeled with CFDA-SE were added to the nude Balb/c splenocytes pretreated with mitomycin. Five groups were established: control group(TGF-β1 0ng/ml), low concentration group(TGF-β11.0ng/ml), middle concentration group(TGF-β1 5.0ng/ml), high concentration group(TGF-β1 10.0ng/ml) and IL-2 group(TGF-β1 5.0ng/ml +exogenous IL-2 100u/ml). After MLC completed, the proliferation of T cells was tested by Alamar Blue method, and the expression of CD3 and CD25 was detected by flow cytometry.[Results] TGF-β1 suppressed the proliferation of T cells activated by alloantigen(control group vs low concentration group, p<0.05) and the suppression could be enhanced as the TGF-β1 concentration increased (low concentration group vs middle concentration group, p<0.05). At the same time, TGF-β1 increased the ratio of CD25+ T cells (control group vs low concentration group, p<0.05) with dose-independent manner. In addition, the exogenous IL-2 could strengthen the proliferation of alloreactive T cells (IL-2 group vs middle concentration group, p<0.05), and decreased the ratio of CD25+ T cells(IL-2 group vs middle concentration group, p<0.05).[Conclusion] TGF-β1 suppressed the proliferation of alloreactive T cells, and increased the ratio of CD25+T cells. However, the biological effect of TGF-β1 can be reversed when adding the exogenous IL-2.Part II. TGF-β1 induced CD4+CD25+ regulatory T cells from CD4+CD25-T cells[Objective] To investigate the roles of TGF-β1 to induce the differentiation of CD4+CD25+ regulatory T cells and the mechanisms.[Methods] 1. The nude Balb/c splenocytes pretreated with mitomycin as the stimulator were added to C57BL/6 T cells with or without TGF-β1 for a 6-day culture. Four groups were established: control group(0ng/ml TGF-β1), low concentration group(0.1ng/ml), middle concentration group(5.0ng/ml) and high concentration group(10.0ng/ml). At the end-point of culture, the ratio of CD4+CD25+T cells and the expression of Foxp3 mRNA were measured with FACS and RT-PCR respectively. 2. CD4+CD25-T cells were isolated by MACS and stimulated with alloantigen and TGF-β1. After a 6-day culture, CD4+CD25+T cells were isolated and we measured the immunosuppressive activity of these cells.[Results] 5.0ng/ml and 10.0ng/ml TGF-β1 could increase the ratio of CD4+CD25+T cell and the expression of Foxp3 mRNA in T cells stimulated by alloantigen(p<0.05). TGF-β1 induced CD4+CD25-T cells to convert to CD4+CD25+T cells which could suppress the proliferation of lymphocytes.[Conclusion] TGF-β1 induces CD4+CD25+ regulatory T cells from CD4+CD25-T cells, which could express Foxp3 and suppress the proliferation of lymphocytes.Part III. Transfer of regulatory T cells induced by TGF-β1 prolonged the skin-graft survival in mice[Objective] TGF-β1 induced na?ve T cells to differentiate into regulatory T cells ex vivo. This work explored the probability of transferring the induced cells to prolong the allograft survival and researched the mechanisms involved.[Methods] According to the different culture conditions, three experimental groups were established: control group(T cells from C57BL/6 mice cultured with IL-2), MLR group(T cells from C57BL/6 mice activated by alloantigen) and TGF-βgroup(T cells from C57BL/6 mice activated by alloantigen and cultured with 5.0ng/ml TGF-β1). After the culture, the ratio of CD4+CD25+T and the Foxp3 expression were measured with FACS and RT-PCR respectively. On 9 day, the pathologic analysis was performed and the ratios of Th1, Th2 and Treg and the proliferation of lymphocytes were measured.[Results] The ratio of CD4+CD25+T in TGF-βgroup was higher than that in control group and MLR group (p<0.05), and CD4+CD25+T in TGF-βgroup expressed Foxp3. After transferring of the cells, the allograft survival time in TGF-βgroup was prolonged and its MST was (22.8±1.9)d, which was longer than the MLR group and control group (p<0.05), but MST of MLR group was (9.4±1.3)d, which was shorter than that of control group (p<0.05). In TGF-βgroup, the ratio of Th1 was lower than that of control group and MLR group (p<0.05), but the ratio of Th2 was similar with MLR group (p>0.05), lower than that of control group (p<0.05). And the ratio of CD4+CD25+T in TGF-βgroup was higher than control and MLR group obviously (p<0.05). In addition, the proliferation of lymphocytes in TGF-βgroup was impaired.[Conclusion] TGF-β1 could induce na?ve T cells to differentiate into regulatory T cells. After to transfer these cells, in mice skin transplant model, the ratio of CD4+CD25+Treg was increased, and the differentiation of Th1 and Th2 and the proliferation of lymphocytes were inhibited. Finally, the allograft survival time was prolonged successfully.Part IV. Regulatory T cells inhibit the proliferation of lymphocytes through TIM-3-TIM-3L pathway[Objective] To explore the role of TIM-3-TIM-3L pathway to modify the immunosuppressive effect of natural Treg (nTreg) and induced Treg (iTreg).[Methods] Firstly, we separate nTreg from C57BL/6 by MACS and induce na?ve T cells to differentiate into iTreg by TGF-β1. And then Foxp3 and TIM-3L expression in these Treg, and the ability of inhibiting the proliferation of lymphocytes were measured. The TIM-3-TIM-3L pathway was blocked with TIM-3.Ig and anti-TIM-3, which could combine with TIM-3L and TIM-3 expressed on the surface of nTreg and iTreg respectively. And then, the immunosuppressive ability of these Treg was measured by MLC.[Results] Both of nTreg and iTreg expressed Foxp3, and could inhibit proliferation of lymphocytes. TIM-3L(galectin-9) mRNA was measured in nTreg and iTreg with RT-PCR. The expression of galectin-9 in nTreg and iTreg was lower than that in splenocytes(p<0.05). Immunosuppressive effects of nTreg and iTreg were partially reduced with TIM-3.Ig interference. It was weaker than control group(p<0.05). But anti-TIM-3 did not affect nTreg and iTreg obviously(p>0.05).[Conclusion] TGF-β1 could induce iTreg population. nTreg and iTreg could express TIM-3L, and inhibited the proliferation of lymphocytes through TIM-3-TIM-3L pathway. Even while TIM-3-TIM-3L pathway was blocked, Treg could still inhibit the lymphocytes to proliferate partly. These results imply that TIM-3-TIM-3L pathway was not the only mechanism of Treg function.