Effect Of Rapamycin On CD4～+ CD25～+ Regulatory T Cells In Allo-Transplantation Tolerance Model In Vitro

Objective:CD4~+CD25~+ Treg are one of the important types of regulatory T cells. Recent studies have demonstrated that CD4~+CD25~+ regulatory T cells (Treg) participated in a variety of ways in allo-transplantation immunological tolerance. However, the effect of immunosuppressant on CD4~+CD25~+ regulatory T cells is uncertainty so far. Rapamycin (Rapa) as a novel macrolide immunosuppressant, its unique role in the field of organ transplantation has been paying close attention. Experiments results show that Rapa selectively expands the murine naturally occurring CD4~+ CD25~+ Foxp3~+ in vitro and does not block activation-induced cell death and proliferation of CD4~+ T cells, but the exact mechanism was not clear. IL-2 regulates the progress of immune tolerance induced by the regulatory T cells; meanwhile IL-2 signal infects the function of regulatory T cells and maintains their competitiveness and adaptability.The previously study in our laboratory found that the expression of CD4~+ CD25~+ Foxp3~+ is higher in allo-transplantation tolerance model induced by CsA. But whether Rapa could expand the Treg in allo-transplantation tolerance model and maintain their inhibitory in vitro was not clear. The purpose of this paper was to study the effect of Rapa on CD4~+ CD25~+ Foxp3~+ in tolerance-induced period in murine allotransplantation model and its suitable dose and the effective pathway, and to study that whether CD4~+ T cells from tolerance model, treated with Rapa in vitro, could mediate the adoptive tolerance in allograft mice.Methods:The model of skin allo-transplantation was established. The recipient BALB/c mice were injected with allogeneic donor spleen cells from B6 mice on the day before grafting and with CsA or Rapa after transplantation. T cells and CD4~+ T cells were purified on the day 14 from the tolerant group and cultured with Rapa and/or IL-2 in different concentrations in vitro. Mixed lymphocyte reaction (MLR) wasused to analyze the specific recall reaction of T cells. Percentages of CD4~+CD25~+ regulatory T cells were examined by flow cytometry (FCM). The expression of Foxp3 mRNA was measured by RT-PCR, and the level of IL-10 in supernatant of T cells cultured in vitro was determined by ELISA. BALB/c-SCID mice underwent allo-transplantation were given adoptive transfer of CD4~+ T cells treated with Rapa and/or IL-2, then the survival conditions of the grafted skin were observed daily.Results:1.Compared with the control group (13.2±1.5 d), Rapa plus donor splenocyte injection can prolong allograft of BALB/c significantly(22.8±2.1d), (P <0.05); CsA plus donor splenocyte injection were also prolonged allograft of BALB/c significantly(22.0±1.9d),there is no significant difference between two groups, but the general condition of CsA group is poor, there is a significant loss of body weight.2.T cells from tolerant group were treated with Rapa in different time(24,48,72,96h), MLR shows the specific memory response of T cells from Rapa group is decreased on the 72h compared with control group, but the co-culture group's specific memory response of T cells is the lowest. There are same changes for the level of IL-10 in supernatant of T cells cultured in vitro. 3.Compared with the control group, The number of CD4~+CD25~+ regulatory T cells and the expression of Foxp3 mRNA for T cell in the tolerant group treated with low concentrations of Rapa(100 nM) for 72h in vitro were obviously increased. If combined with the group of low concentrations of IL-2 (50IU/ml), the effect of up-regulation CD4~+CD25~+T cells ratio will improved significantly.4.Compared with control group, CD4~+T cells from tolerant group were treated with Rapa in vitro can prolonged the graft survival time obviously after adoptive transfer immunity (P<0.05) .Conclusions:1. Application of Rapa or CsA plus donor splenocyte injection in vivo induced the transplantation tolerance, prolonged the allograft survival time significantly. The effect of Rapa is better than CsA.2. The suitable dose of Rapa is 100 nmol/L and best effect time is 72 hours in induce T reg from tolerant group in vitro.3. Rapa can increase the ratio of CD4~+CD25~+ regulatory T cells and the expression of Foxp3 mRNA from tolerant model in vitro, and the effect was enhanced by low-dose of IL-2 (50IU/ml).4. The function of Treg induced by Rapa is independent on the IL-10, but its function's activation is depends on the existence of IL-2.5. The inhibitory activity of Treg which come from tolerance model was enhanced by Rapa in vitro and adoptive immunity of those cells could prolong the graft survival time obviously.