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Background: Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells.

Methods: Peripheral CD4 CD25~. naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4 CD25- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4 CD25 CD127~. subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4 CD25 T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1x10 5 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4 CD25-. T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25x10 5/well) in 96-well round-bottom plates for 6 days. Then 1x10 5 or 0.25x10 5 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4 CD25-. T cells using flow cytometry. Data were analyzed using CellQuest software.

Results: We found that RAPA can convert peripheral CD4 CD25~. naive T Cells to CD4 Foxp3 Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity.

Conclusion: Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.

(C) 2010 Chinese Medical Association