Regulatory T cells are a subpopulation of T cells that have the ability to suppress immune reactions and maintain tolerance. They were believed to play a key role in transplant tolerance induction. The most important representative of regulatory T cells is the naturally occurring regulatory T cells (Tregs), with a CD4 + CD25 + Foxp3 + phenotype. They were found to mediate donor-specific Figure 2 . DCs from recipients treated with Flt3L/Rapa display a tolerogenic phenotype. Spleen cells isolated from mice treated with Flt3L/ Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were stained with PE-cy7-labeled anti-mouse CD11c mAb, Per-cy5.5-labeled anti-mouse B220 mAb, APC-cy7-labeled anti-mouse CD8a mAb, FITC-labeled anti-mouse CCR9 mAb, APC-labeled anti-mouse CD80 mAb and APC-labeled anti-mouse CD40 mAb. CD11c + and CD11c + B220 + gated DCs were analyzed by flow cytometry for expression of the various DC markers. (A) The bar graph was a summary of percentages of pDC and CD8a + DC in the recipients. (B) CD11c + -gated DCs were analyzed for expression of costimulatory markers CD80 and CD40 to assess their maturation state. (C) The percentage of CCR9 + cell of gated pDC subsets was assessed by FACS analysis. The results are representative of three independently performed experiments (* P,0.05). doi:10.1371/journal.pone.0046230.g002 Figure 3 . Flt3L combined with Rapa promotes the production of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + T cells. Spleen cells isolated from mice treated with Flt3L/Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were analyzed to determine the proportion of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + T cells. (A, B) The expression of Foxp3 by CD4 + CD25 + and CD8 + CD25 + T cells was analyzed by flow cytometry after intracellular staining. The bar graph was a summary of percentages of CD4 + CD25 + Foxp3 + T cells and CD8 + CD25 + Foxp3 + T cells in the recipients. The data shown are representative of three independent experiments that yielded comparable results (* P,0.05). doi:10.1371/journal.pone.0046230.g003 unresponsiveness in some stable renal [32, 33, 34, 35, 36] and liver [37] transplant patients. In addition, adoptive transfers of Tregs could prevent allograft rejection and prolong graft survival in the mouse models [38] . Previously, addition of rapamycin to human and murine T cell cultures preferentially expanded regulatory T cells [39, 40] . Our data indicated that infusion of Flt3L with Rapa and Rapa alone enhances the production of CD4 + CD25 + Foxp3 + Tregs. In addition, we found a significant increase of splenic CD8 + CD25 + Foxp3 + T cell populations in the recipient mice treated with Flt3L/Rapa, while CD8 + CD25 + Foxp3 + T cell have been found being able to inhibit antigen-specific CD4 + and CD8 + T cell responses by cell contact inhibition, secretion of cytokines and other ways [41] .