The Journal of Immunology Essential Role of E3 Ubiquitin Ligase Activity in Cbl-b–Regulated T Cell Functions Magdalena Paolino,* Christine B. F. Thien, † Thomas Gruber, ‡ Reinhard Hinterleitner, ‡ Gottfried Baier, ‡ Wallace Y. Langdon, † and Josef M. Penninger* E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tol- erance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or auto- immunity. The Journal of Immunology, 2011, 186: 2138–2147. E xtensive research carried out in the past few years has placed E3 ubiquitin ligases among the critical molecules involved in the regulation of T cell responses (1–3). Mechanistically, E3 ligases participate in the final step of protein ubiquitylation by selecting the target protein and catalyzing its covalent binding to the 76-aa polypeptide ubiquitin (4). Once thought to be merely a proteolytic recycling system, it is now known that tagging a protein with ubiquitin is a multifunctional process that can affect protein stability, intracellular trafficking, or functional interactions (5). Hence, protein ubiquitylation by E3 ligases is an essential dynamic mechanism by which T cell sig- naling pathways can be modified to regulate T cell responses. Cbl-b was the first E3 ubiquitin ligase to be directly implicated in T cell activation and tolerance in vivo (6, 7). Cbl-b belongs to the highly conserved Casitas B-cell Lymphoma (Cbl) family of pro- teins, which in mammals consists of three homologues: c-Cbl, Cbl- b, and Cbl-3 (8). Structurally, Cbl proteins contain protein–protein interaction domains that allow them to interact with many different proteins in multiple signaling pathways (9, 10). Cbl proteins also contain a RING finger domain responsible for the E3 ligase acti- vity (11, 12). Functionally, this catalytic domain is responsible for binding the E2 conjugating enzyme and mediating the transfer of ubiquitin between the E2 and the target substrate (13, 14). In the absence of Cbl-b, T cells are hyperproliferative and able to be fully activated even without CD28 costimulation (6, 7). As a conse- quence, Cbl-b total body knockout mice develop spontaneous au- toimmunity and are highly susceptible to developing experimental autoimmunity (6, 7, 15, 16). Moreover, the lack of Cbl-b prevents T cell tolerance induction in vivo (15). Importantly, our group and others have recently demonstrated that Cbl-b–deficient mice are able to reject multiple types of tumors spontaneously (17, 18). Besides Cbl-b, other E3 ubiquitin ligases have been identified as critical regulators of T cell activation and tolerance; namely, the HECT (homology to the E6 associated protein C terminus)-type E3 ligase Itch and the RING-type E3 ligase GRAIL (gene related to anergy in lymphocytes). As in the case of Cbl-b, Itch and GRAIL deficient T cells hyperrespond to TCR stimulation (19, 20). Additionally, Itch and GRAIL expression is upregulated in T cells under anergizing stimuli (21, 22), and ablation of either of these E3 ligases renders T cells resistant to anergy induction, both in vitro and in vivo (20, 23). Like Cbl-b–deficient mice, Itch and GRAIL knockout mice develop spontaneous autoimmune respon- ses (19, 20). Recent studies using Cbl-b and Itch double-deficient mice suggest that these proteins cooperate in the control of T cell responses and autoimmunity (24). Genetic knockout studies have firmly placed E3 ligases among the crucial T cell gatekeepers, regulating the balance between immunostimulation and immunotolerance. Although some ubiq- uitylation substrates have been postulated (12, 15, 19, 25–29), it still remains unknown if the E3 ligase activity of these E3 ligases, and in particular Cbl-b, is responsible for its in vivo T cell func- tions or if instead its multivalent adapter function is essential. To address the physiological relevance of Cbl-b catalytic activity, we analyzed mice carrying a loss-of-function mutation in Cbl-b RING finger domain for T cell proliferation, susceptibility to autoimmunity, in vivo T cell tolerance responses, and tumor *Institute of Molecular Biotechnology of the Austrian Academy of Science, A-1030 Vienna, Austria; † School of Pathology and Laboratory Medicine, University of West- ern Australia, Crawley, Perth, Western Australia 6009, Australia; and ‡ Department of Medical Genetics, Clinical and Molecular Pharmacology, Medical University of Innsbruck, A-6020 Innsbruck, Austria Received for publication October 14, 2010. Accepted for publication December 7, 2010. This work was supported by grants from the European Community’s Sixth Frame- work Programme (EuroThymaide: LSHB-CT-2003-503410; EuroRA [Functional Ge- nomic Approaches Targeting Arthritis]: MRTN-CT-2004-005693; Innovative Mouse Models for Functional Genomics in Immunology: MRTN-CT-2004-005632), the Fonds zur Foerderung der Wissenschaftlichen Forschung (SFB F23), the Austrian National Bank, the Austrian Federal Ministry of Science and Research, and the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (to J.M.P.). M.P. and J.M.P. are supported by the European Community’s Seventh Frame- work Programme (FP7/2007-2013) under European Research Council Grant Agree- ment No. 233407. Address correspondence and reprint requests to Josef M. Penninger, IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria. E-mail address: josef.penninger@imba.oeaw.ac.at The online version of this article contains supplemental material. Abbreviations used in this article: ANA, anti-nuclear Ag; Cbl, Casitas B-cell lym- phoma. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003390