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Updated: Jun 12, 2026
In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol
Published on: December 30, 2016
CD4+Foxp3+ regulatory T cells converted by rapamycin from peripheral CD4+CD25(-) naive T cells display more potent
Jian-Fei Chen1, Jie Gao, Dong Zhang
1Department of Hepatobiliary Surgery, Peking University People's Hospital, Peking University Center for Transplantation, Beijing 100044, China.
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.
Conclusion:
Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.
Insights
Rapamycin (RAPA) converts T-effector (Teff) cells into T-regulator (Treg) cells. This novel mechanism of RAPA demonstrates its potential for inducing immune tolerance, offering new therapeutic strategies in transplantation.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Rapamycin (RAPA) is an immunosuppressant that inhibits T-effector (Teff) cell activation.
- RAPA is known to expand the T-regulator (Treg) cell population.
- The mechanism by which RAPA converts Teff to Treg cells remains unexamined.
Purpose of the Study:
- To investigate the mechanism of Rapamycin-induced conversion of Teff cells to Treg cells.
- To determine if RAPA can induce the differentiation of naive T cells into functional Treg cells.
- To explore RAPA's potential role in inducing immune tolerance.
Main Methods:
- Cultured peripheral CD4(+)CD25(-) naive T cells with RAPA and B cells as antigen-presenting cells (APCs) in vitro.
- Analyzed T cell conversion to CD4(+)Foxp3(+) Treg cells using flow cytometry and quantitative real-time PCR.
- Assessed the suppressive function of converted Treg cells on Teff cell proliferation.
Main Results:
- Rapamycin (RAPA) effectively converts peripheral CD4(+)CD25(-) naive T cells into CD4(+)Foxp3(+) Treg cells when B cells act as APCs.
- The RAPA-induced Treg cells demonstrate potent suppression of Teff cell proliferation.
- These converted Treg cells maintain antigenic specificity.
Conclusions:
- Rapamycin (RAPA) directly induces the conversion of Teff cells into Treg cells.
- This study uncovers a new mechanism for RAPA-mediated tolerance induction.
- Findings suggest RAPA as a potential therapeutic agent for enhancing immune tolerance.
