GRAIL and Otubain-1 are Related to T Cell Hyporesponsiveness during Trypanosoma cruzi Infection

Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of "gene related to anergy in lymphocytes" (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection. Balb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation. We hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during Trypanosoma cruzi infection.