Aims: T helper (Th)-2 cells are the major players in allergic asthma; however, the mechanisms that control Th2-mediated inflammation are poorly understood. Our previous studies showed that E3 ubiquitin ligase, Grail, is associated with T cell tolerance. In addition to tolerant T cells, Grail mRNA is upregulated during normal T cell activation, suggesting that Grail function might not be restricted to T cell tolerance. In fact, we determined a significant expression of Grail in Th2 cells compared to other T helper subsets, suggesting the role of Grail in controlling Th2 programming.

Methods: The regulation of Grail expression in Th2 cells was assessed by quantitative Real-Time PCR, Chromatin Immunoprecipitation (ChIP) assay, luciferase reporter assay and retroviral transduction. Wild-type and Grail deficient T cells were utilized for T helper cell differentiation assay to evaluate the role of Grail in Th2 responses in vitro. For in vivo studies wild-type and Grail deficient mice were subjected to Ova immunization and asthma model. Immunoblot analysis and ubiquitination assay were performed to explore the mechanism whereby Grail controls Stat6 expression in Th2 cells.

 Results: In the current study, we found that Grail is selectively induced upon IL-4 stimulation in a time dependent manner and depends on Th2-specific factors Stat6 and Gata3 that bind to and transactivate the Grail promoter. Grail deficiency in T cells leads to enhanced Th2 development in vitro and in vivo; Grail deficient mice are more susceptible to allergic asthma. Mechanistically, the enhanced effector function of Grail-deficient Th2 cells is mediated by increased expression of Stat6 and IL-4 receptor α-chain. Grail interacts with Stat6 and targets it for ubiquitination and degradation.

Conclusions: Our results suggest an important link between the Th2 specific expression of Grail and its role in control of Th2 developmentand Th2-mediated pathogenesis and immunity through a negative feedback loop.