Research Article

Influence of sirolimus-induced TGF-β secretion on mouse Treg cell proliferation

Abstract

We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-β (TGF-β) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-β mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-β signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-β neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-β secretion increased 2.5X over control levels (P 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P vs 40.2%, P in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-β secretion in vivo.

We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-β (TGF-β) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-β mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-β signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-β neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-β secretion increased 2.5X over control levels (P 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P vs 40.2%, P in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-β secretion in vivo.