Notch1 and TGF β 1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

Notch and its ligands have been implicated in the regulation and differentiation of various CD4 + T-helper cells. Regulatory T cells (T reg ), which express the transcription factor Foxp3, suppress aberrant immune responses that are typically associated with autoimmunity or excessive inflammation. Previous studies have shown that transforming growth factor beta (TGF β 1) induces Foxp3 expression and a regulatory phenotype in peripheral T cells. Here, we show that pharmacologic inhibition of Notch signaling using γ -secretase inhibitor (GSI) treatment blocks i) TGF β 1-induced Foxp3 expression, ii) the upregulation of Foxp3-target genes, and iii) the ability to suppress naïve T cell proliferation. Additionally, the binding of Notch1, CSL, and Smad to conserved binding sites in the foxp3 promoter can be inhibited by treatment with GSI. Finally, in vivo administration of GSI results in reduced Foxp3 expression and development of symptoms consistent with autoimmune hepatitis, a disease previously found to result from dysregulation of TGF β signaling and regulatory T cells. Together, these findings indicate that the Notch and TGF β signaling pathways cooperatively regulate Foxp3 expression and regulatory T cell maintenance both in vitro and in vivo . required for the induction of Foxp3 expression and the regulatory T cell program in the periphery. Here, we demonstrate that in vitro GSI treatment in the presence of TGF β 1 blocks Notch-mediated upregulation of Foxp3, Foxp3-target genes, and the ability to suppress naïve T cell proliferation. Further, in vivo GSI treatment downregulated Foxp3 and resulted in a spontaneous lymphocyte infiltration of the liver. These data identify Foxp3 as a novel downstream target of Notch signaling, and its relevance is underscored by the fact that GSI therapy is entering the clinic, and presents a previously unrecognized side effect of long-term GSI treatment.