Absence Of Grail Promotes CD8~+ T Cell Anti-Tumor Activity

T cell tolerance is a major obstacle to successful cancer immunotherapy;thus,it is of high priority to develop strategies to break immune tolerance.Here we report that expression of the E3 ubiquitin ligase Grail is significantly upregulated in CD8~+ T cells infiltrated into transplanted lymphoma tumors and Grail-deficiency confers long-term tumor control.Importantly,therapeutic transfer of Grail-deficient CD8~+ T cells was sufficient to repress established tumors.Mechanistically,loss of Grail enhanced anti-tumor reactivity and functionality of CD8~+ T cells.In addition,Grail deficient CD8~+ T cells exhibited increased IL-21 R expression and hyper-responsiveness to IL-21 signaling as Grail promotes IL-21 R ubiquitination and degradation.Moreover,CD8~+ T cells isolated from lymphoma patients expressed high levels of Grail and lower levels of IL-21 R compared with normal donors.Altogether,our data demonstrates that Grail is a crucial factor controlling CD8~+ T cell function and is a potential target to improve CTL activity.It is accepted that the adaptive immune system,especially cytotoxic CD8~+ T lymphocytes(CTLs),play a crucial role in controlling the development of neoplastic lesions [1].Effector CD8~+ T cells can efficiently destroy target cells by using death cell ligands such as TNF-related apoptosis-inducing ligand(TRAIL)or execution of the perforin/granzyme and IFN-? dependent machinery [2].While CD4~+ T cells also play an important role in anticancer immunity,their predominant role at the tumor site is to maintain function of tumor-specific CTLs by providing cytokines [3,4].Cancer immunotherapy aims to reactivate a patient's immune system to fight against tumors.However,T cell tolerance induced by the inhibitory tumor microenvironment is an obstacle in achieving successful cancer immunotherapy [5].Therefore,understanding the cellular and molecular mechanisms that underlie T cell tolerance in cancer would guide the development of novel effective therapies for cancer.Recent data have demonstrated that E3 ubiquitin ligases,including Cbl-b,Itch,and Grail are key regulators of T cell tolerance [6].Cbl-b and Itch have been reported to be involved in tumor development [7-10].Grail is a type I transmembrane protein localized to the endosomal compartment whose expression is associated with T cell anergy induction [11].Recently we generated and analyzed Grail-deficient mice and found that they were resistant to immune tolerance induction in vitro and in vivo12,13.Grail is required for properly downregulating TCR signaling in recently activated CD4~+ T cells;lack of Grail leads to hyperproliferation and excessive cytokine production 12.In addition,we found that lack of Grail abrogated suppressive function of regulatory T(Treg)cells [12].However,the role of Grail in CD8~+ T cells remains unclear.In the current study,we found significantly high level of Grail expression in mouse CD8~+ T lymphocytes infiltrated into lymphoma tumors and examined the role of Grail in transplanted EL-4 and EG-7 lymphoma models.We found that Grail deficiency provided the host with spontaneous protection against tumors,which was mediated mainly by CD8~+ T cells in Grail-deficient mice.In tumors,loss of Grail enhanced anti-tumor reactivity of CD8~+ T cells.Moreover,in mouse CD8~+ T cells,Grail controls IL-21 R expression and na?ve Grail-/-CD8~+ T cells exhibit hyper-responsiveness to IL-21 cytokine in vitro due to augmented IL-21 R levels,which was regulated through ubiquitination and degradation by Grail.Importantly,similar to the mouse model,we found high expression of Grail and diminished expression of IL-21 R in CD8~+ T cells from lymphoma patients compared to healthy donors.Altogether,our results indicate a suppressive function of Grail in CD8~+ T cells and suggest that Grail-ablated CD8~+ T cells could be an efficient tool for eliciting immune responses against tumors.