The Wiskott Aldrich Syndrome protein (WASp) reduces T cell proliferation and regulatory T cell induction via increased sensitivity to TGFβ

Abstract

The Wiskott-Aldrich Syndrome is an x-linked immunodeficiency resulting from defects in the Wiskott Aldrich Syndrome protein (WASp). Activation of WASp initiates actin polymerization through its interaction with ARP2/3, a complex that has been linked to ulcerative colitis in a genome wide association study. WASp-deficiency in humans and mice results in development of a spontaneous colitis. Colitis in WASp-deficient mice is associated with a reduction in the number and function of CD4+CD25+Foxp3+ regulatory T cells (Tregs). In the following study, we demonstrate that WASp-deficiency alters de novo induction of Tregs and explore a role for altered responsiveness to TGFβ in the observed defects. Naive (CD4+CD25-) CFSE-labelled T cells from ovalbumin-specific TCRtransgenic wild type (WT) or WASp-deficient mice were cultured with WT irradiated T-depleted splenocytes, ova peptide (0-10 mcg/mL) and TGFβ. Cells were analyzed at 72 hours for expression of FoxP3 and dilution of CFSE. Cytokine bead array was performed on assay supernatants to determine the concentration of IL-2 and IFNγ. Sensitivity to TGFβ was assessed by incubating CD4+ WT or WASp-deficient T cells with TGFβ for 0-60 minutes. Whole cell lysates were fractionated by SDS-PAGE and western blotting was performed for phospho-SMAD2, total SMAD2 and CD3ε as a loading control. TGFβ receptor 1 and 2 expression was determined by western blotting of whole cell lysates prior to TGFβ exposure. Antigen-specific stimulation of naïve WASp-deficient T cells in the presence of TGFβ resulted in reduced proliferative responses at doses of antigen ranging from 0.01-10 mcg/mL ova peptide. Ten-fold increased antigen doses were required to achieve similar proliferation in WASp-deficient naïve T cells compared to WT. This correlated with an altered threshold for the maximal induction of Tregs from WASp-deficient T cells such that the peak induction of Tregs in the WT occurred at an antigen dose of 0.1 mcg/mL versus 1 mcg/mL in WASp-deficient T cells. The culture supernatants from WASp-deficient T cells demonstrated significantly reduced concentrations of IL-2 and IFNγ compared to WT. Interestingly, WASp-deficient T cells demonstrated increased phosphorylation of the TGFβ receptor signalling protein, SMAD2, at early time points following exposure (5 minutes) compared to WT suggesting increased TGFβ sensitivity in the absence of WASp. Expression of TGFβ receptor was not significantly altered in WASp-deficient T cells. In the presence of TGFβ, WASp-deficient T cells have reduced proliferative and cytokine responses compared to WT correlating with altered threshold for the maximal induction of Tregs. Sensitivity to TGFβ is increased in WASp-deficient T cells compared to WT. These data suggest that the ability of TGFβ to down-regulate the T cell proliferative and cytokine response may, under certain circumstances, outweigh its ability to induce Tregs. In addition, altered TGFβ signalling through this novel mechanism may contribute, at least in part, to the reduced number of Tregs and development of colitis in the absence of WASp. Further understanding of the complex pathways that lead to induction of Tregs may allow the development of novel therapies for inflammatory bowel disease.