Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X

The T cell hyperproliferation and autoimmune phenotypes that manifest in mice lacking E3 ubiquitin ligases such as Cbl, ITCH, or GRAIL highlight the importance of ubiquitination for the maintenance of peripheral T cell tolerance. Less is known, however, about the deubiquitinating enzymes that regulate T cell proliferation and effector function. Here, we define a cell intrinsic role for the deubiquitinase Usp9X during proximal TCR signaling. Usp9X-deficient T cells were hypoproliferative, yet mice with T cell–specific Usp9x deletion had elevated numbers of antigen-experienced T cells and expanded PD-1 and OX40-expressing populations consistent with immune hyperactivity. Aged Usp9x KO mice developed lupus-like autoimmunity and lymphoproliferative disease, indicating that ubiquitin ligases and deubiquitinases maintain the delicate balance between effective immunity and self-tolerance.Ignorance of self-antigens is one example of a cell intrinsic mechanism for inducing tolerance in peripheral T cell populations and results from either a failure to encounter self-antigens or more pertinently, because the avidity or amount of self-antigen is not sufficient to trigger a response. Naive CD4⁺ T cells function at the apex of the adaptive immune response, where, during an initial encounter with antigen, their principal function is to discriminate between low and high signal strength. If a signal is high, as is typically the case for encounters with foreign antigens, then the antigen-specific T cell will undergo clonal expansion; however, if the signal strength is low, an immune response fails to occur. Insights into the regulation of the earliest signaling events downstream of TCR engagement will inform our understanding of how signal strength is modulated during this critical decision making process.The enzymatic conjugation of the 76-aa protein ubiquitin to lysine residues, either singly or as polymeric chains, impacts cell signaling by modifying the stability, localization, or interactions of a protein (Komander and Rape, 2012). Ubiquitin is attached by the concerted actions of E1, E2, and E3 enzymes and it is removed by ubiquitin hydrolases (also called deubiquitinating enzymes). Therefore, the balance between these activities will determine the magnitude and duration of signaling. The E3 ubiquitin ligases Cbl, ITCH, and GRAIL are established regulators of peripheral T cell tolerance due to their provision of negative signals that shift the balance toward lower TCR signal strength. For example, in activated peripheral T cells, Cbl-b binds and ubiquitinates the TCR-proximal kinase ZAP70, resulting in its proteasomal-mediated degradation (Lupher et al., 1996; Rao et al., 2000). The fact that deubiquitinating enzymes counteract these ligases to shift the balance toward higher signal strength has received little attention.Ubiquitin-specific protease 9X (Usp9X) is a mammalian orthologue of the Drosophila developmental gene fat facets (Wood et al., 1997) that exhibits specificity for diverse ubiquitin linkages, not only removing degradative K48-linked ubiquitin chains (Nagai et al., 2009; Schwickart et al., 2010) but also nondegradative monoubiquitin from SMAD4 (Dupont et al., 2009) and atypical Lys29/33 polyubiquitin chains from NUAK1 or MARK4 (Al-Hakim et al., 2008). Here, we show that Usp9X is a positive regulator of proximal TCR signaling in peripheral T cells and also contributes to T cell tolerance established during intrathymic development.