Background: Regulatory T cell therapy is generally used for bone marrow transplantation and autoimmune diseases but is less frequently applied for transplantation of the heart. In this work, we adopted a drug regimen developed by Battaglia et al. to induce CD4+Foxp3+Tregs and T regulatory type 1 cells in vivo to maintain long-term tolerance after heart transplantation.

Methods: Hearts from BALB/c mice were transplanted into C57BL/6 mice. We then administered (hu)IL-10 and rapamycin to generate CD4+Foxp3+Tregs and Tr1 cells (CD4+IL-10+IL-4-) in vivo. Levels of rejection were analyzed using hematoxylin and eosin (H&E) staining and ELISA, and cell subtypes were identified using flow cytometry.

Results: Combined treatment with (hu)IL-10 and rapamycin induced tolerance to the heart allografts in all recipient mice, and levels of IL-2 and INF-γ were also clearly decreased in the rapa+IL-10 group. Compared to the rapa group, the subset of Th1 was decreased in the combined group. The proportion of CD4+Foxp3+Tregs induced by this treatment continued to be high during the administration period and recovered to normal levels on the 100th day after transplantation, yet the proportion of Tr1 cells remained high after drug withdrawal. Mixed lymphocyte reactions and adoptive transfer experiment showed that the recipient’s T cells were tolerant (showed no response) to the donor’s antigen presenting cells (APCs) compared to the third-party donor’s APCs, and it was the induced regulatory T cells that inhibited this proliferation.

Conclusion: The combination of (hu)IL-10 and rapamycin was able to induce CD4+Foxp3+Tregs and Tr1 cells in vivo . The two subsets showed different regulatory effects and antigen specificity in the mouse model of heart transplantation and were able to maintain a low immune response environment to facilitate long-term graft survival.