Organ transplantation remains the only solution available for the treatment of numerous end-stage diseases. The administration of immunosuppressive drugs following solid organ transplantation is essential in order to control the activation of alloreactive T cells and so prevent rejection of the graft. Although calcineurin inhibitors (CNI), anti-proliferative agents and steroids all have proven efficacy, immunosuppression after heart transplantation may still be improved, with respect to increased efficacy (reduced incidence of acute rejection and chronic allograft vasculopathy (CAV) and reduced toxicity (less renal failure and malignancy). The proliferation signal inhibitor (PSI) Rapamycin (RAPA), and its derivative everolimus, belong to a new class of potent therapeutic agents that promise such an improvement. Research has shown that delayed treatment with RAPA effectively slows the progression of both allograft rejection 1 and pre-existing CAV in both rat 2 and non-human primate grafts 34. Recently, ex vivo studies have shown that, in both humans and mice, RAPA selectively blocks the proliferation of effector T cells (T_eff) while at the same time, expanding the CD4⁺CD25⁺FoxP3⁺ Treg population 5.

Tregs control various immune responses and play an important role in inhibiting transplant rejection 6. It has been reported that graft survival rates are positively correlated with the number of circulating Tregs78, and that the adoptive transfer of Tregs (polyclonally expanded in vivo using low-dose RAPA) promotes tolerance to allogeneic pancreatic islet grafts 9.

In a retrospective study, Segundo et al8 found that RAPA (but not CNI) induces an increase in the number of circulating Tregs in stable renal transplant recipients. Therefore, we analyzed both the number and the cytokine expression levels in Foxp3+ Tregs in the blood, spleen and lymph nodes of rat transplant recipients after a single post-operative dose of RAPA. Our data show that Foxp3+ Tregs inhibit T_eff cell activity in the peripheral lymphoid tissues by secreting cytokines such as TGF-β1. We also suggest that the number of circulating Tregs may not be a suitable predictor of transplant rejection.

Recently, it has been shown that RAPA selectively blocks the proliferation of effector T cells while at the same time expanding the population of both human and mouse CD4⁺CD25⁺FoxP3⁺ Tregs ex vivo 1. In patients transplanted with heart 14, lung 15, liver 7 or kidney 8 grafts, graft survival rates showed a positive correlation with the number of circulating Tregs. Therefore, in this study, we monitored the number of circulating CD3⁺CD25⁺Foxp3⁺ Tregs in the peripheral blood of rats transplanted with cardiac allografts. We show that RAPA did not affect the number of circulating Tregs when compared with normal controls, which is in agreement with the study of Segundo et al8, but contrary to others. This conflict may be due to the fact that that several of these studies compared patients receiving CNI-based immunosuppression with those receiving RAPA. Our results (which include a healthy control group for comparison) suggest that RAPA has no deleterious effect on Treg numbers. However, the number of Tregs in patients treated with CNI appears to be reduced compared with healthy controls 16. Therefore, Treg cell numbers in peripheral blood may not be a suitable predictor for transplant rejection.

It is known that Foxp3⁺ nTregs are not only found in recipient lymphoid tissue after transplantation 17, but also at the graft site18. We hypothesized that lymphoid-tissue Tregs might be effective at blocking the initiation of an aggressive response against the graft, whereas at the graft site they may inhibit the activity of aggressive cells that have escaped regulation and migrated into the graft tissue19. Therefore, we examined the Foxp3 expression in both the allograft and lymphoid tissues using IHC and real-time PCR. We found no Foxp3 expression in the allograft hearts at the selected time point, but the expression of Foxp3 mRNA and protein increased significantly Day 1 post-transplant in the Con group.

We then examined the number of Tregs/CD4+ T cells in the peripheral blood, spleen, lymph nodes and thymus of healthy rats using flow cytometry. We found the greatest number of Tregs in the lymph nodes (3.66 ± 0.84%) followed by spleen (2.24% ± 1.28%), indicating that the lymph nodes are the major site of Tregs activity.

When a single dose of RAPA was given on Day 3 post transplantation, Foxp3 expression was increased. This suggests that lymphoid tissue might be the site where Tregs suppress activated effector T cells. While, when RAPA was given on Day 1 post transplantation, Fxop3 expression was decreased. This was conformity with Wang et al. 19921, Poston et al. 19992, Ikonen et al. 20003 and Dambrin et al.20034, which delayed treatment with RAPA effectively slows the progression of both allograft rejection. Furthermore, RAPA, in clinical treatment, is not traditionally used after transplantation at once.

The efficacy of RAPA in preventing acute rejection is known to be related to trough drug levels2021. Therefore, we assessed the pharmacodynamics of RAPA using HPLC. We found RAPA was below its effective concentration (5-15 ng/ml) 5 days after treatment (Fig 2). Therefore, the second peak in Foxp3+ Treg cell numbers seen in the 3D group at Day 7 may reflect the normal alloresponse in the Lewis rat recipients. In addition, the last treatment time point should be in 5 days.

There are three general modes of suppression (cell contact-dependent suppression, the production of inhibitory cytokines or the consumption of limiting growth factors) that may explain the inhibitory actions of Tregs on activated T cells 22. It has been proposed that the production of inhibitory cytokines1423 such as IL-10 and TGF-β1 play an important role in this suppression. However, measurements of cytokine concentrations in cell culture supernatants are typically very low, and picomolar concentrations are unlikely to trigger cellular responses. Therefore, biologically active cytokine concentrations may only be achieved in the microenvironments surrounding the secreting cells. Therefore, we examined the IL-10 and TGF-β1 mRNA expression by Tregs both in vivo and in vitro. When RAPA was given on Day 1 post transplantation, Foxp3 expression was decreased while IL-10 expression was increased. However, on Day 3 post transplantation both Foxp3 and TGF-β1 expression increased, but not that of IL-10 (Fig 6). These results support the suggestion that there is a role for cell-surface or secreted TGF-β1 in CD4⁺CD25⁺ Treg-mediated suppression 2425262728.

Interestingly, our data show the time of RAPA administration has differing effects upon Foxp3 expression and cytokine secretion by Tregs. As far as we are aware, this is the first time that this has been reported. We propose that RAPA administration on Day 1 post-transplant enhances iTreg proliferation, which then suppresses Teff cells by secreting IL-10, while RAPA administration on Day 3 post-transplant increases nTreg proliferation that in turn, inhibits Teff cells by secreting TGF-β1. The mechanisms responsible for these effects need further investigation.

In conclusion, our data indicate that Foxp3+Tregs inhibit Teff cell activity within the peripheral lymphatic tissues by secreting cytokines such as TGF-β1. We also show that the number of circulating Tregs may not be a suitable predictor of transplant rejection.

Juan Shan and Yuchuan Huang carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Jie Zhang carried out the immunoassays. Chuntao Zhang participated in the sequence alignment. Youping Li and Yuchuan Huang participated in the design of the study and performed the statistical analysis. Li Feng and Shengfu Li conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.