CD4+Foxp3+ regulatory T cells converted by rapamycin from peripheral CD4+CD25(-) naive T cells display more potent regulatory ability in vitro.
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 1 x 10(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) (0.25 x 10(5)/well) in 96-well round-bottom plates for 6 days. Then 1 x 10(5) or 0.25 x 10(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.


CONCLUSIONS
Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.
Chen
Jian-Fei
JF
Department of Hepatobiliary Surgery, Peking University People's Hospital, Peking University Center for Transplantation, Beijing 100044, China.
Gao
Jie
J
Zhang
Dong
D
Wang
Zi-Han
ZH
Zhu
Ji-Ye
JY
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000938:Antigen-Presenting Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D016685:Mitomycin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D016685:Mitomycin; D020123:Sirolimus