SARA and Hgs attenuate susceptibility to TGF-beta1-mediated T cell supression

Kunzmann S, Wohlfahrt JG, Itoh S, Asao H, Komada M, Akdis CA, Blaser K and Schmidt-Weber CB
Source: FASEB J
Publication Date: (2003)
Issue: 17: 194-202
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Nucleofectorâ„¢ I/II/2b
Resting primary human CD4+ T lymphocytes were nucleofected with GFP as control or SARA or Hgs and the protein expression was analyzed by fluorescence microscopy in a time course. Both activated and resting CD4+ cells were nucleofected with a TGF-beta sensitive luciferase reporter gene prior to TGF-beta stimulation. Measurement of the luciferase activity revealed a twofold higher susceptibility of activated T cells to TGF-beta. Overexpression of Hgs or SARA together with a TGF-beta sensitive luciferase reporter gene inhibited luciferase activity in activated T cells 2-3 fold.
Transforming growth factor-beta1 (TGF-beta1) is a pluripotent cytokine that controls peripheral T cell tolerance mainly in mucosal immunity. It is secreted by regulatory T cells (Tr /Th3) but also by other immununologically active cells. Smad anchor for receptor activation (SARA) and hepatic growth factor-regulated tyrosine kinase substrate (Hgs) are involved in TGF-beta1 signaling. Both molecules are known to present Smad2 and Smad3 to the TGF-beta receptor complex. The role of SARA and Hgs in TGF-beta1 susceptibility of human CD4+ T cells is unclear. We demonstrate here that TGF-beta1 up-regulates SARA mRNA expression in CD4+ T cells similar to that of Smad7. However, the increase in SARA expression was lower (6.1+/-0.3-fold vs. 25+/-4.1-fold) compared with Smad7 and delayed, with a maximum at 12 h compared with 2 h. Th1 and Th2 cell subsets expressed the same levels of SARA and Hgs. Compared with resting cells, significantly lower levels of the two molecules were found in antigen/allergen- or anti-CD3/CD28-stimulated cells. Down-regulation of SARA and Hgs mRNA in preactivated CD4+ T cells was accompanied by a twofold increase in a TGF-beta1 responsive reporter gene assay. Overexpression of SARA and Hgs in T cells yielded a dose-dependent decrease in cotransfected reporter gene expression, indicating an inhibitory function of both molecules. Thus, SARA and Hgs are regulators of TGF-beta1 susceptibility in T cells and integrate regulatory signals into the influence of TGF-beta1-mediated suppression of human T cells.