Preoperative application of combination of portal venous injection of donor spleen cells and intraperitoneal injection of rapamycin prolongs the survival of cardiac allografts in mice.

OBJECTIVE: To investigate the effects of preoperative portal venous injection of donor spleen cells (PVIDSC) and intraperitoneal injection of rapamycin in the acute rejection of cardiac allograft in mice and the underlying mechanisms. METHODS: Homogenous female B6 mice and BALB/c mice were used as recipients and donors of heart transplantation. These mice were randomly divided into different groups and received PVIDSC alone, rapamycin alone, or PVIDSC and rapamycin combined therapy. In addition, the underlying mechanism was studied by measuring a number of cytokines. RESULTS: Preoperative combination of PVIDSC and intraperitoneal injection of rapamycin significantly prolonged the survival of heterotopic cardiac allograft in mice, but had no effects on the survival time of cardiac allografts in mice pre-sensitized by skin grafting. Preoperative combination of PVIDSC and intraperitoneal injection of rapamycin increased the expression of IL-10 and Foxp3 and reduced the expression of INF-. Short-term preoperative administration of rapamycin promotes the expression of CD4+CD25+Foxp3+ regulator T cells. However, preoperative using alone of rapamycin, or combination of PVIDSC and rapamycin had no effects on the inhibition of proliferation of memory T cells. CONCLUSIONS: Preoperative application of combination of PVIDSC and rapamycin significantly prolonged the survival time of cardiac allografts in mice but not in mice pre-sensitized by skin grafting. This may be explained by the fact that combination of PVIDSC and rapamycin inhibited the cellular immune response and induced the expression of IL-10 from Tr1 cells and CD4+CD25+FoxP3+ regulatory T cells.

The recall alloresponse following retransplantation is more intense compared with the T cell memory-transfer model.

The presence of alloreactive memory T cells in recipient is a critical handicap to achieving transplantation tolerance. To make a mouse model that can as closely as possible mimic the presensitized transplant patient is important for research on this subject. Thus, we developed a novel retransplant model and compared the alloresponse in this model with that in the memory T cells-transfer model (transfer control). Mean survival time of allograft was compared between 3 groups, including blank transplant control, memory transfer control and retransplant groups. Cellular rejection activity in allografts was evaluated via HE staining of cardiac graft section. Proliferation and differentiation of the alloreactive effector T cells were assayed by in vitro mixed lymphocyte reaction and flow cytometry, respectively. Real-time quantitive RT-PCR was used to assess gene expression of cytokines and surum IFN-gamma was measured via ELISA. It showed that the median survival time of allograft in retransplant recipients was significantly shortened compared to that of transfer control, and it was the same in rejection score of graft. Moreover, proliferation and differentiation of the alloreactive effector T cells were more intensive in retransplant recipients than that in transfer control, which was confirmed by in vitro mixed lymphocyte reaction and by flow cytometry of the splenocytes for detecting CD44highCD62L- memory/effector phenotype cells. Furthermore, activation of CD4+ memory T cells is reflected by high level of surum IFN-gamma and the intensive gene expression of IFN-gamma and IL-2 at cardiac allograft in retransplant recipients. Collectively, the recall alloresponse in retransplantation is more intensive than that in a memory-transfer setting, and this retransplant model is closer to the clinic situation than the memory-transfer model in rodents.

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4(+) memory T cells in mice.

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4(+) Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5days to 9days in recipient mice with CD4(+) Tm infusion (P<0.01), which however was not significantly longer than that (8days) in untreated recipient mice without CD4(+) Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4(+) Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4(+) Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44(high)CD62L(-) effector/memory as well as CD69(+)/CD25(+) activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4(+) Tm, for serum level of IFN-gamma and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4(+) Tm cells responding to the alloantigen, indicating that alloreactive CD4(+) Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4(+) Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.