Twisted Gastrulation (Tsg), an Evolutionarily Conserved, Secreted, Morphogenetic Protein Enhances TGF-b Signaling in T Lymphocytes.
Authors: Tzachanis Dimitrios , Lafuente Esther M. , Li Lequn , Berezovskaya Alla , Freeman Gordon J. , Boussiotis Vassiliki A. 
Published: April 2020

Abstract
We have previously determined that quiescent T cells express Tob, a member of the novel APRO gene family, which functions to repress T cell activation. Tob is as a transcriptional regulator and may regulate mRNA transcription. To identify genes that are regulated by Tob, Jurkat T cells were transfected with Tob cDNA or empty vector. Differential gene expression was determined by suppression subtractive hybridization. Twisted Gastrulation (Tsg) was one of the genes suppressed by Tob. Tsg is an evolutionarily conserved secreted protein that interacts with Drosophila Decapentaplegic (Dpp) and its vertebrate orthologs BMP2/4, and also the extracellular Dpp/BMP inhibitors short gastrulation (sog) and chordin. As a result, Tsg affects the binding of Dpp/BMP2/4 to their receptors and subsequent downstream signaling. BMPs belong to a family of secreted signaling molecules the founder of which, TGF-b, is essential for immune homeostasis and maintenance of T cell quiescence. In the thymus, Tsg functions as a regulator of thymocyte development by inhibiting BMP2/4 signaling which arrests thymocyte differentiation. Here, we examined Tsg expression and function in mature CD4+ human T cells. Tsg mRNA was expressed at very low levels in unstimulated T cells and was highly upregulated after activation by TCR/CD3 and either CD28, IL-2 or PMA. To determine the function of Tsg, we generated recombinant human Tsg tagged with V5 epitope. Based on its effects on thymocytes, we hypothesized that Tsg may regulate BMP2/4-mediated effects on mature T cells. Culture of CD4+ T cells with various concentrations of BMP2 or BMP4 in the presence or absence of anti-CD3 mAb did not affect T cell proliferation or cytokine production. Addition of Tsg to cultures containing BMP2/4 did not alter T cell responses. Because our results showed that Tsg mRNA was induced after activation, we tested pre-activated, polyclonal CD4+ alloreactive T cell lines. Neither BMP2 nor BMP4 had any effect on proliferation of alloreactive T cell lines in the presence or absence of anti-CD3 mAb. Strikingly, Tsg induced a significant inhibitory effect on CD3-mediated proliferation and cytokine production, including IL-2, IL-4, IFN-g and IL-10. This effect was not altered by the presence of BMP2 or BMP4. In contrast, Tsg enhanced the inhibitory effect of TGF-b1 suggesting that Tsg regulates TGF-b and not BMP downstream signaling in pre-activated, mature CD4+ T cells. Consistent with this hypothesis, Tsg did not affect phosphorylation of the BMP-specific Smad1, but induced phosphorylation of the TGF-b-specific Smad2 and mediated DNA binding on Smad3/4 consensus sites. In vitro association assays using recombinant Tsg-V5 and TGF-b revealed a direct interaction of these proteins as determined by co-immunoprecipitation followed by western blot with V5-specific and TGF-b-specific antibodies. Moreover, soluble anti-TGF-b receptor reversed the inhibitory effect of Tsg on pre-activated T cells either in the presence or in the absence of TGF-b, providing functional evidence for the biological significance of the Tsg/TGF-b interaction. Our results show that Tsg is a potent agonist of TGF-b downstream signaling in human CD4+ T cells and suggest that enhancement of TGF-b mediating signaling by Tsg may represent a novel target for molecular intervention for induction of transplantation tolerance.