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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
Foxp3 regulates human natural CD4+CD25+ regulatory T-cell-mediated suppression of xenogeneic response
Lei Sun1, Shounan Yi, Philip J O'Connell
1Center for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Westmead, NSW, Australia.
Backgrounds:
Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Foxp3 expressing human naturally occurring CD4+CD25+ regulatory T cells (nTregs) have been shown to suppress pathological and physiological immune responses, including the CD4+ T-cell-mediated anti-pig xenogeneic response in vitro. Although Foxp3 is required for nTreg development and their function, the precise role of Foxp3 in regulating Treg suppressive function in xenoimmune response remains to be identified.
Methods:
In vitro expanded human nTregs were transfected with fluorescein isothiocyanate -conjugated Foxp3 small interfering RNA (siRNA) by Lipofectamine 2000. Transfected nTregs were sorted by fluorescence-activated cell sorting, and then analyzed for Foxp3 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25- T cells were stimulated with xenogeneic pig peripheral blood mononuclear cell in the presence or absence of nTregs in a coculture or transwell system for evaluation of nTreg suppressive activity. The production of effector cytokines by xenoreactive CD4+CD25- T cells as well as suppressive cytokine by nTregs in their cocultures was examined by ELISA.
Results:
The siRNA-mediated Foxp3 knockdown resulted in impaired nTreg anergic state, downregulated expression of nTreg function associated molecules, and reduced production of suppressive cytokines by nTregs, which together leading to impaired nTreg-mediated suppression of CD4+CD25- T-cell proliferation and their effector cytokine production in response to xenogeneic stimulation.
Conclusions:
This study demonstrates that Foxp3 expression is required for human nTregs to maintain their suppressive function in the xenoimmune response.
