Essential Role of E3 Ligase Activity in Cbl-b−Regulated T Cell Functions Magdalena Paolino, Christine B. F. Thien, Thomas Gruber, Reinhard Hinterleitner, Gottfried Baier, Wallace Y. Langdon This information is current as and Josef M. Penninger of September 29, 2021. J Immunol 2011; 186:2138-2147; Prepublished online 19 January 2011; doi: 10.4049/jimmunol.1003390

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Supplementary http://www.jimmunol.org/content/suppl/2011/01/19/jimmunol.100339 Material 0.DC1 http://www.jimmunol.org/ References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/186/4/2138.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Essential Role of E3 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 Downloaded from 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.

xtensive research carried out in the past few years has interaction domains that allow them to interact with many different

placed E3 ubiquitin ligases among the critical molecules proteins in multiple signaling pathways (9, 10). Cbl proteins also http://www.jimmunol.org/ E involved in the regulation of T cell responses (1–3). contain a RING finger domain responsible for the E3 ligase acti- Mechanistically, E3 ligases participate in the final step of protein vity (11, 12). Functionally, this catalytic domain is responsible for ubiquitylation by selecting the target protein and catalyzing its binding the E2 conjugating enzyme and mediating the transfer of covalent binding to the 76-aa polypeptide ubiquitin (4). Once ubiquitin between the E2 and the target substrate (13, 14). In the thought to be merely a proteolytic recycling system, it is now absence of Cbl-b, T cells are hyperproliferative and able to be fully known that tagging a protein with ubiquitin is a multifunctional activated even without CD28 costimulation (6, 7). As a conse- process that can affect protein stability, intracellular trafficking, quence, Cbl-b total body knockout mice develop spontaneous au- or functional interactions (5). Hence, protein ubiquitylation by E3 toimmunity and are highly susceptible to developing experimental ligases is an essential dynamic mechanism by which T cell sig- autoimmunity (6, 7, 15, 16). Moreover, the lack of Cbl-b prevents by guest on September 29, 2021 naling pathways can be modified to regulate T cell responses. T cell tolerance induction in vivo (15). Importantly, our group and Cbl-b was the first E3 ubiquitin ligase to be directly implicated others have recently demonstrated that Cbl-b–deficient mice are in T cell activation and tolerance in vivo (6, 7). Cbl-b belongs to able to reject multiple types of tumors spontaneously (17, 18). the highly conserved Casitas B-cell Lymphoma (Cbl) family of pro- Besides Cbl-b, other E3 ubiquitin ligases have been identified teins, which in mammals consists of three homologues: c-Cbl, Cbl- as critical regulators of T cell activation and tolerance; namely, the b, and Cbl-3 (8). Structurally, Cbl proteins contain protein–protein HECT ( 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 *Institute of Molecular Biotechnology of the Austrian Academy of Science, A-1030 GRAIL deficient T cells hyperrespond to TCR stimulation (19, Vienna, Austria; †School of Pathology and Laboratory Medicine, University of West- ern Australia, Crawley, Perth, Western Australia 6009, Australia; and ‡Department of 20). Additionally, Itch and GRAIL expression is upregulated in Medical Genetics, Clinical and Molecular Pharmacology, Medical University of T cells under anergizing stimuli (21, 22), and ablation of either of Innsbruck, A-6020 Innsbruck, Austria these E3 ligases renders T cells resistant to anergy induction, both Received for publication October 14, 2010. Accepted for publication December 7, in vitro and in vivo (20, 23). Like Cbl-b–deficient mice, Itch and 2010. GRAIL knockout mice develop spontaneous autoimmune respon- This work was supported by grants from the European Community’s Sixth Frame- work Programme (EuroThymaide: LSHB-CT-2003-503410; EuroRA [Functional Ge- ses (19, 20). Recent studies using Cbl-b and Itch double-deficient nomic Approaches Targeting Arthritis]: MRTN-CT-2004-005693; Innovative Mouse mice suggest that these proteins cooperate in the control of T cell Models for Functional Genomics in Immunology: MRTN-CT-2004-005632), the responses and autoimmunity (24). Fonds zur Foerderung der Wissenschaftlichen Forschung (SFB F23), the Austrian National Bank, the Austrian Federal Ministry of Science and Research, and the Genetic knockout studies have firmly placed E3 ligases among Institute of Molecular Biotechnology of the Austrian Academy of Sciences (to the crucial T cell gatekeepers, regulating the balance between 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- immunostimulation and immunotolerance. Although some ubiq- ment No. 233407. uitylation substrates have been postulated (12, 15, 19, 25–29), it Address correspondence and reprint requests to Josef M. Penninger, IMBA, Institute still remains unknown if the E3 ligase activity of these E3 ligases, of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, and in particular Cbl-b, is responsible for its in vivo T cell func- A-1030 Vienna, Austria. E-mail address: [email protected] tions or if instead its multivalent adapter function is essential. To The online version of this article contains supplemental material. address the physiological relevance of Cbl-b catalytic activity, Abbreviations used in this article: ANA, anti-nuclear Ag; Cbl, Casitas B-cell lym- we analyzed mice carrying a loss-of-function mutation in Cbl-b phoma. RING finger domain for T cell proliferation, susceptibility to Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 autoimmunity, in vivo T cell tolerance responses, and tumor www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003390 The Journal of Immunology 2139 rejection. Our results demonstrate an essential role of the E3 ligase First-Strand Beads (GE Healthcare), and a high-fidelity PCR reaction was activity for Cbl-b–mediated T cell functions. performed using primers flanking the RING finger domain of Cbl-b (for- ward primer, 59-CCGGATTATGTGAACCTACACCTC-39; reverse primer, 59-CGATCATCGTCATCGTCCAAGTCAAG-39). The resulted PCR frag- Materials and Methods ments were cloned into TOPO vectors for nucleotide sequencing. Mice In vitro proliferation Cbl-b E3 ligase-defective (C373AKI/KI) knock-in mice were generated by For in vitro proliferation studies, 2.5 3 105 CD3+ T cells were purified homologous recombination as previously described (30). In brief, a tar- from spleen and lymph nodes of young (8–12 wk old) mice using mouse geting vector was constructed to introduce a cysteine (TGC) to alanine T cell enrichment columns (R&D Systems) and cultured in duplicates or (GCG) substitution at amino acid position 373 encoded within the RING triplicates in 96 U-bottom wells precoated with serial concentrations of finger domain (at exon 8) of Cbl-b. The pGKNeo resistance cassette was anti-CD3ε Abs (clone 145-2C11; Pharmingen). Then, 1 mg/ml anti-CD28 removed by crossing C373A mice to b-actin–Cre transgenic mice. Cbl- Abs (clone 37.51; Pharmingen) was added soluble to the media. For TCR- b knockout mice were generated in our laboratory and have been pre- independent T cell stimulation, 100 ng/ml PMA and 80 ng/ml of the 2 2 viously described (6). Rag2 / and P14+ TCR transgenic mice were calcium ionophore ionomycin were added to the media. Cells were stim- obtained from our in-house breeding stock. For in vivo T cell tolerance ulated for 56 h in 200 ml RPMI 1640 medium containing 10% FCS, 50 U/ experiments, P14+ TCR mice were bred onto the C373A and Cbl-b de- ml penicillin–streptomycin, and 2 mM glutamine. Proliferation was mea- 2 2 ficient backgrounds to generate P14+Cbl-b / , P14+C373AKI/KI, and the sured by scintillation counting after a pulse with 1 mCi [3H]thymidine per respective control mice. All experimental procedures were performed on well for the last 12 h of incubation. For cytokine release analysis, culture mice back-crossed for at least six generations onto the C57BL/6J strain. supernatants were harvested after 48 h stimulation and cytokines con- Except for autoimmunity experiments and if not otherwise stated, only 6- centrations determined by Bioplex Technology (Bio-Rad). to 12-wk-old littermate mice were used in all experiments. All animal Downloaded from experiments were carried out according to an ethical animal license pro- In vivo tolerance tocol complying with the current Austrian law. In vivo CD8+ T cell tolerance was induced according to a previously de- scribed protocol (15). Briefly, the C373A Cbl-b knock-in mutation was Western blot and in vitro ubiquitylation assays back-crossed onto the P14+ TCR transgenic background, and littermate For Cbl-b and c-Cbl protein expression analysis, proteins were extracted mice were intravenously injected with high (5 mg) or low (0.5 mg) doses of from thymocytes and total lymph node cells using a protein lysis buffer the high-affinity peptide p33 on day 0, day 3, and day 6. Non-TCR- transgenic C373AKI/KI and P14+Cbl-b2/2 were used as controls. Mice (0.5% Nonidet P-40, 150 mM NaCl, 50 mM HEPES pH 7.8, 1 mM EDTA http://www.jimmunol.org/ pH 8) containing protease inhibitors (Thermo Scientific). After clearance were observed for the following 6–12 h, and those mice found dead or of the homogenates by centrifugation, 35 mg tissue lysate was resolved by moribund mice (laborious breathing, restricted movement, and spike veins 4–12% SDS-PAGE, electro-transferred to polyvinylidene difluoride in the ears) were dissected and examined for signs of toxic T cell acti- membranes, and probed with anti–Cbl-b (Santa Cruz Biotechnology) and vation, namely inflammation, edema formation, hemorrhages, and/or vas- anti–c-Cbl (Pharmingen) Abs according to standard procedures. For cular dilation. Blood was collected, and serum was prepared for the de- in vitro ubiquitylation assays, N-terminal regions (aa 29–483) containing tection of IL-2 and IFN-g levels using Bioplex systems (Bio-Rad). the RING finger domain of wild-type, C373A,orW400A Cbl-b were fused to GST using pGEX5x-1. Expression of the GST–fusion proteins in Detection of spontaneous autoimmune phenotypes Escherichia coli BL21 was induced with 0.3 mM isopropyl-b-D-thio- For detection of tissue infiltrates, the submandibular salivary gland, kidney, galactopyranosid at 20˚C overnight. The cells were lysed, and lysates were pancreas, lung, and liver were harvested from 6- to 8-mo-old mice and fixed incubated overnight with glutathione Sepharose 4B (GE Healthcare) at overnight at 4˚C in 4% formalin. Tissues were then embedded in paraffin by guest on September 29, 2021 4˚C. Sepharose beads were then washed five times with lysis buffer, and and sectioned (3.5 mm) at three depth levels (interval 200–300 mm). Tissue proteins bound to the beads were eluted with elution buffer (50 mM Tris- sections were stained with H&E for detection of mononuclear cell in- HCl, pH 8, 20 mM reduced glutathione). For Cbl-b auto-ubiquitylation filtration and with anti-CD3ε Abs (clone SP7; Neo Markers) for detection assays, the reaction mixture (50 ml) containing ubiquitylation buffer (Enzo of T cells. Two independent investigators scored the presence of lym- Life Sciences), 1 mM DTT, 20 U/ml inorganic pyrophosphatase, 5 mM phocyte infiltrates in a blind fashion. For the detection of serum auto- Mg-ATP, 2.5 mM biotinylated ubiquitin, 100 nM E1, 2.5 mM E2, and 100 antibodies, indirect immunofluorescence on Rag22/2 tissue sections was nM of the corresponding GST–Cbl-b proteins was incubated at 37˚C for performed as described (31). Briefly, 5-mm cryostat sections from several 1 h. The reaction was quenched by adding 50 mlof23 nonreducing gel organs of Rag2 knockout mice were incubated with 1/40 dilutions of sera loading buffer and heating at 95˚C for 5 min. Finally, the samples were obtained from 6- to 8-mo-old mice, followed by detection with Alexa immunoblotted with streptavidin–HRP for detecting ubiquitylated Cbl-b and Fluor-555 labeled anti-mouse IgG secondary Abs (Invitrogen). DAPI was for GST as a loading control. used for nuclear counterstaining. Slides were examined in a blinded fashion, and representative photographs were taken using a fluorescence Flow cytometry microscope (Axioplan2; Zeiss). Serum IgG1 levels and anti-nuclear Ag (ANA) and anti-dsDNA autoantibodies were detected by ELISA according Multiparameter flow cytometry analyses were performed on single-cell to the manufacturer’s protocol (Bethyl Lab for IgG1; a Diagnostic for anti- suspensions by immunostaining for 20 min at 4˚C in FACS buffer (PBS- nuclear and ds-DNA autoantibodies). Serum glucose was measured using 2% FCS). For staining T cells, Abs directly labeled with FITC, PE, PE- a glucose meter (One Touch Ultra Easy; LifeScan), and serum insulin Cy5, PE-Cy7, allophycocyanin, or allophycocyanin-Cy7 reactive against levels were detected by ELISA according to the manufacturer’s protocol ε b CD3 , CD4, CD8, CD25, TCR , CD62L, CD69, CD44, and CD45 were (Mercodia AB). used (all from Pharmingen). Regulatory T cells were detected using a mouse regulatory T cell staining (eBioscience). For labeling of thy- TC1 tumor rejection mocyte precursors, thymocytes were stained with FITC-CD25, PECy5- CD44, and PE-labeled Abs to CD4, CD8, TCRb, TCRgd, CD19, B220, TC1 cells are primary C57BL/6 mouse lung fibroblasts co-transformed with CD11b, CD11c, Gr-1, and NK1.1. PE-negative cells were analyzed for an activated c-H-ras and the HPV-16 E6 and E7 oncoproteins expression of CD25 and CD44. For labeling of splenic B cells, anti-B220– (32). Culture conditions and TC1 inoculation protocols have been de- allophycocyanin, anti-IgM–PE-Cy5, anti-IgD–FITC, anti-CD21–FITC, scribed elsewhere (18). In brief, 2.5 3 105 TC1 tumor cells were s.c. and anti-CD23–PE Abs were used (all from Pharmingen). Flow cytometry injected into the shaved left flank of young (8–12 wk old) mice (day 0), data were acquired using a six-color flow cytometer (FACSCanto; BD) and tumor growth was monitored three times per week with a caliper. equipped with the FACSDiva software (BD). Data analysis was performed Because TC1 cells are derived from female mice, only female animals using FlowJo software (Tree Star). were used for the described experiments. Mice were euthanized when tumor volume reached 1 cm3 in accordance with animal license protocols. + Nucleotide sequence analysis For CD8 cell depletion studies, mice were i.p. injected with 50 mg anti- CD8+ depletion Abs (clone 24.3) at day 24 and day 22 before TC1 in- To confirm the correct cysteine to alanine point mutation at Cbl-b aa po- oculation. Efficient and specific depletion of CD8+ cells was confirmed by sition 373, splenic and lymph node CD3ε+ T cells from C373A+/+ and FACS analysis at day 21 (see Supplemental Fig. 6). CD8+ depletion was C373AKI/KI mice were isolated, and total RNA was extracted using RNeasy repeated weekly throughout the whole experimental observation period. Mini Kit (Qiagen). Total RNA was reversed transcribed to cDNA using For immunohistochemistry analysis, TC1 tumors were harvested at day 14 2140 THE ROLE OF Cbl-b E3 LIGASE ACTIVITY IN VIVO

FIGURE 1. Cbl-b expression and T cell populations in Cbl-b E3 ligase-defective mice. A, In vitro ubiquitylation assay detecting auto- ubiquitylation of 75-kDa GST-tagged wild-type Cbl-b and RING finger mutants with a cysteine to alanine (C373A) or tryptophan to alanine (W400A) substitution. Both C373A and W400A point mutations abrogate Cbl-b E3 ligase ac- tivity (upper panel, anti-ubiquitin blot). As a loading control, an anti-GST blot is shown (bottom panel). B, Western blot for Cbl-b and Downloaded from c-Cbl protein expression in the thymus and lymph nodes of C373A+/+, C373A+/KI, and C373AKI/KI mice. b-Actin expression is shown as a loading control. Data are representative of a minimum of four different animals. C–E, T cell populations in naive 6- to 12-wk-old +/+ +/KI KI/KI 2/2

C373A , C373A , C373A ,andCbl-b http://www.jimmunol.org/ mice. Representative FACS blots for thymic (C) and splenic (D)CD4+/CD8+ T populations as well as splenic CD4+ CD25+ Foxp3+ T regula- tory cells (E) are shown. Numbers indicate per- centage of cells within a quadrant. Data are representative for a total of n =5(C373A+/+), n =6(C373A+/KI and C373AKI/KI), and n =2 (Cbl-b2/2)mice. by guest on September 29, 2021

after tumor inoculation, fixed overnight at 4˚C in 4% formalin, and em- Statistical analyses bedded in paraffin. Several 3.5-mm-thick sections (at two different tumor 6 depths, interval 200–300 mm) were stained for each mouse with Abs Unless otherwise stated, data are shown as mean values SEM. Illus- against cleaved (activated) caspase 3 (Asp175; ), Ki67 (Ncl- trations and statistical analyses were generated using GraphPad Prism 4 Ki67p; Novacastra), and CD3ε (SP7; Neo Markers) using the automated (GraphPad Software). Data were analyzed using the two-tailed Student Ventana Discovery System (Ventana). Slides were scanned on a Mirax t test for single comparisons or ANOVA for multiple analyses. Ordinal and Scanner (Zeiss), and Definiens Tissue Map software (Definiens AG) was not normally distributed data were analyzed using the Mann–Whitney U used for the automated determination of positively stained cells. All cells test. For analyzing the presence of autoantibodies, we set a cutoff at p # in the section (6–12 sections per mouse) were counted. 0.05 based on a Z-statistic for the serum concentration measured in each individual mouse related to the mean and SD of the wild-type population Tumor-infiltrating lymphocytes (X 2 Xwt/SDwt). Kaplan–Meier survival curves were analyzed using a log- rank test. A p value ,0.05 was considered to indicate statistical signifi- Analysis of tumor-infiltrating lymphocytes was performed in tumors isolated cance (*p , 0.05, **p , 0.01, ***p , 0.001). on day 14 after inoculation, a time point when all experimental mice have similar tumor volume. For immunofluorescence staining of CD8+ intra- tumoral cells, 5-mm tumor cryosections were stained with anti-CD8 Abs (clone KT15; Chemicon) and detected with Alexa Fluor-555 labeled anti-rat Results IgG secondary Abs (Invitrogen). DAPI was used for nuclear counterstaining. Inactivation of Cbl-b E3 ligase activity does not alter T cell Sections were examined and photographed in a fluorescence microscope populations (Axioplan2; Zeiss). For staining quantifications, slides were scanned on a Mirax Scanner (Zeiss), and positively stained cells at five different image To dissect genetically the enzymatic function of Cbl-b in vivo, we fields were quantified per mouse using the Metamorph Imaging Software analyzed mice that carry a RING finger inactivating point muta- (Molecular Devices). For flow cytometry analysis, tumor single-cell sus- tion (C373AKI/KI) (30). These mice are viable, fertile, and grossly pensions were stained with anti-CD45, anti-CD3ε, anti-CD4, and anti-CD8 Abs (all Pharmingen). FACS data were acquired using a six-color flow normal. The C373A mutation substitutes the N-terminal consensus cytometer (FACSCanto; BD) equipped with the FACSDiva software (BD). cysteine residue in the RING finger domain of Cbl-b for an ala- Data analysis was performed using the FlowJo software (Tree Star). nine. Nucleotide sequence analysis on total RNA extracted from The Journal of Immunology 2141

T cells of C373AKI/KI mice confirmed a single cysteine/alanine sufficient C373A+/+ and C373A+/KI control mice or Cbl-b2/2 mice substitution at the right 373 position within the Cbl-b RING (Supplemental Fig. 2C). In addition, splenic B cell populations finger domain (Supplemental Fig. 1). To verify the functional were unaffected by the C373A mutation (Supplemental Fig. 3). inactivation of Cbl-b by this particular point mutation, we per- Thus, genetic inactivation of the Cbl-b RING finger domain has no formed in vitro ubiquitylation assays using wild-type Cbl-b, overt effect on the development and subpopulations of T and C373A mutant Cbl-b, and Cbl-b mutants containing a tryptophan B cells. to alanine substitution at position 400 (W400A), a second residue KI/KI required for E3 ligase activity (13, 25). Whereas Cbl-b auto- C373A T cells hyperrespond to TCR stimulation ubiquitylation was clearly detected using wild-type Cbl-b pro- It has previously been shown that Cbl-b–deficient T cells hyper- tein, the capacity to auto-ubiquitylate was virtually abolished in proliferate and can be activated even without CD28 costimulation both C373A and W400A Cbl-b mutant proteins (Fig. 1A). We next (6, 7). To determine whether negative regulation of T cell activa- examined whether this loss-of-function mutation affects Cbl-b tion is dependent on the E3 ligase activity of Cbl-b, purified pe- protein expression. Western blot analysis on whole thymus and ripheral CD3+ T cells from C373A+/+, C373A+/KI, C373AKI/KI, and lymph node lysates revealed similar Cbl-b protein levels between Cbl-b2/2 mice were stimulated in vitro with different concentra- C373A+/+, C373A+/KI, and C373AKI/KI mice (Fig. 1B). tions of anti-CD3ε Abs alone or together with anti-CD28 Abs. A Genetic ablation of Cbl-b neither alters T cell populations nor significant, stronger proliferation of T cells from C373AKI/KI mice TCR expression in naive mice (6, 7). Nonetheless, as an important was observed under these stimulatory conditions compared with control for our experiments and because an equivalent loss-of- that of C373A+/+ (not shown) or C373A+/KI T cells (Fig. 2A). Im- function mutation in the c-Cbl RING finger domain led to addi- portantly, peripheral T cells lacking Cbl-b enzymatic function were Downloaded from tional and more severe phenotypic changes than those observed in able to proliferate at suboptimal concentrations of anti-CD3ε Abs whole-body c-Cbl knockout mice (33), we assessed whether the and even in the absence of CD28 costimulation (Fig. 2A). In all Cbl-b C373A mutation might affect thymocyte development or stimulatory conditions tested, the hyperproliferative responses of peripheral T cell subpopulations. Flow cytometry analysis on total C373AKI/KI T cells were similar to that observed in T cells lacking thymus showed that C373AKI/KI mice exhibit normal thymocyte Cbl-b (Fig. 2A). TCR/CD28 independent T cell stimulation with

development (Fig. 1C, Supplemental Fig. 2A). Moreover, similar PMA and the calcium ionophore ionomycin was comparable be- http://www.jimmunol.org/ to total Cbl-b mutant mice, C373AKI/KI animals exhibited normal tween all genotypes (Fig. 2A). mature CD4+ and CD8+ peripheral T cell populations (Fig. 1D). TCR stimulation of C373AKI/KI mutant T cells not only resulted Of note, TCR and CD3 surface expression levels on developing in hyperproliferation but also in a marked increase in the secre- thymocytes as well as gated on CD4+ and CD8+ splenocytes tion of IL-2, IL-17, and IFN-g (Fig. 2B). Strikingly, stimulation of (Supplemental Fig. 2B) and lymph node cells (not shown) were C373AKI/KI T cells with anti-CD3ε Abs alone triggered equal also comparable between C373A+/+, C373A+/KI, C373AKI/KI, and cytokine levels as the stimulation of control C373A+/KI T cells Cbl-b2/2 mice. In addition, these animals contain comparable with anti-CD3ε plus anti-CD28 costimulation. As in the case of numbers of regulatory CD4+CD25+Foxp3+ T cells (Fig. 1E). proliferation, the elevated levels of IL-2, IL-17, and IFN-g pro- Naive/memory T cell populations as defined by the CD25, CD69, duced by C373AKI/KI T cells were comparable with those gener- by guest on September 29, 2021 CD62L, and CD44 surface markers were also normally repre- ated by Cbl-b–deficient T cells (Fig. 2B). Taken together, these sented in C373AKI/KI mice compared with those in E3 ligase- in vitro data show that inactivation of Cbl-b RING finger domain

FIGURE 2. Inactivation of Cbl-b enzymatic ac- tivity results in T cell hyperactivation. A, In vitro proliferation of CD3+ T cells from C373A+/KI, C373AKI/KI, and Cbl-b2/2 mice. Peripheral CD3+ T cells were isolated from 8- to 12-wk-old mice and stimulated (2.5 3 105 cells/well) with the indicated concentrations of plate-bound anti-CD3ε and soluble anti-CD28 Abs, PMA (100 ng/ml), and ionomycin (ION; 80 ng/ml). Proliferation was measured after a pulse with 1 mCi [3H]thymidine for the last 12 h of a 56-h incubation period. All data are mean values 6 SEM of three different mice per genotype. One ex- periment representative of a minimum of three is shown. *p , 0.05, **p , 0.01 (comparing the C373A+/KI versus C373AKI/KI or Cbl-b2/2 groups). B, Quantification of IL-2, IL-17, and IFN-g in the cul- ture supernatants of peripheral CD3+ T cells from C373A+/KI, C373AKI/KI, and Cbl-b2/2 mice stimu- lated for 48 h with plate-bound anti-CD3ε (1 mg/ml) or anti-CD3ε (1 mg/ml) plus soluble anti-CD28 (1 mg/ml). All data are mean values of n = 6 to 9 mice per genotype 6 SEM. Data are representative of three independent experiments. Cytokines were de- termined by BioPlex. *p , 0.05, **p , 0.01 (com- paring the C373A+/KI versus C373AKI/KI or Cbl-b2/2 groups). 2142 THE ROLE OF Cbl-b E3 LIGASE ACTIVITY IN VIVO is functionally equivalent to total Cbl-b ablation, rendering T cells showed no overt phenotype in response to repetitive p33 injec- hyperresponsive to TCR stimulation, even in the absence of CD28 tions (Supplemental Fig. 4), P14+C373AKI/KI mice died upon Ag costimulation. Thus, Cbl-b E3 ligase activity is essentially re- rechallenge (Fig. 3B). Approximately 50% of the P14+C373AKI/KI quired in the negative regulation of TCR/CD28-induced T cell mice died after the second injection of p33 at day 3, and with the proliferation and cytokine production. exception of only one mouse injected with a low dose of p33, no P14+C373AKI/KI mouse survived a third Ag encounter on day 6. In Cbl-b E3 ligase activity is critically required for inducing all cases, lethality occurred within 6–12 h after the last p33 ad- T cell tolerance in vivo ministration. Moribund C373AKI/KI mice displayed restricted To investigate whether Cbl-b functions as an E3 ligase in the in- movements, severely impaired breathing, and vascular dilation duction of T cell tolerance in vivo, we decided to challenge Cbl-b (Fig. 3C). Dissection of diseased P14+C373AKI/KI mice further E3 ligase mutant mice in a mouse model for Ag-specific CD8+ revealed signs of severe edema formation, capillary dilation, and T cell tolerance. For this purpose, we crossed the C373A mutation hemorrhagic areas in several organs (Fig. 3C). After p33 rechal- onto a P14+ TCR transgenic background, where the P14+ TCR lenge, serum levels of IL-2 and IFN-g were up to several hundred- specifically recognizes with high affinity the cognate Ag p33 (de- fold elevated in P14+C373AKI/KI mice compared with those in rived from the lymphocytic choriomeningitis virus) presented in P14+C373A+/KI mice (Fig. 3D). Challenge of P14+Cbl-b knockout the MHC class I complex (34). Non-TCR-transgenic C373AKI/KI as mice either at low or high doses of p33 resulted in 100% lethally well as P14+Cbl-b2/2 mice were included as controls. (Fig. 3B). The inflammatory features as well as the massive in- For T cell anergy induction, mice received i.v. injections of either crease in serum IL-2 and IFN-g were comparable among P14+ 2 2 high (5 mg) or low (0.5 mg) doses of p33 peptides at day 0, day 3, C373AKI/KI and P14+Cbl-b / mice (data not shown). These Downloaded from and day 6 (Fig. 3A). Whereas all P14+C373A+/KI survived and results show that the functional inactivation of the Cbl-b RING http://www.jimmunol.org/

FIGURE 3. Cbl-b E3 ligase activity is required for in vivo T cell tolerance. A, Schematic of the protocol used to induce peptide-specific CD8+ T cell tolerance in vivo. For the induction of CD8+ T cell tolerance, P14+C373A+/KI, P14+C373AKI/KI, and P14+Cbl-b2/2 mice were injected (i.v.) on day 0, day 3, and day 6 by guest on September 29, 2021 either with high (5 mg) and low (0.5 mg) doses of p33. Non-TCR-transgenic C373AKI/KI mice were used as controls. After p33 rechallenge, mice were observed for signs of generalized inflammation or eventual death. B, Survival table. Number of mice injected per genotype and total number and percentage of surviving mice for each dose of p33 are shown. Lethality was scored as mice found dead and/or morbid mice sacri- ficed according to ethical guidelines. Of note, in all instances lethality occurred within 6–12 h after the last p33 injection. C, Representative photographs of sev- eral organs depicting inflammation in P14+C373AKI/KI mice after rechallenge with p33 peptide. Severe ede- mas, capillary dilation, and hemorrhages (arrows) were observed in various organs: ear veins (a), feet (b), thy- mus (c), mesenteric lymph nodes (d), Peyer’s patches (e), and lung (f). Whereas the same inflammatory phe- notype was observed in P14+Cbl-b2/2 mice, P14+ C737A+/KI and non-TCR-transgenic C373AKI/KI litter- mate mice showed no signs of inflammatory edema or vascular dilatation (Supplemental Fig. 4, and data not shown). D,IL-2andIFN-g serum levels in P14+ C373A+/KI (n = 13 to 15) and P14+C373AKI/KI (n =8to 10) mice measured 6–8 h after p33 rechallenge on days 3 and 6. Cytokine concentrations were measured by BioPlex. **p , 0.01, ***p , 0.001. The Journal of Immunology 2143

finger domain leads to a massive activation of P14+ T cells, a se- C373A+/+ and C373A+/KI littermate mice, lymphocytic infiltrates vere cytokine storm, and death due to a T cell “shock.” Thus, Cbl- were also observed, albeit at significantly reduced incidence and b E3 ligase activity is essential for the induction of immunotol- severity (Fig. 4A, Supplemental Fig. 5A,5B, and not shown). erance of CD8+ P14+ T cells in vivo. In addition to cellular infiltrates in multiple organs, immuno- fluorescence staining revealed the presence of autoantibodies Cbl-b E3 ligase-defective mutants develop systemic against several tissue Ags in the serum of C373AKI/KI mice (Fig. autoimmune responses 4C). We could also detect a higher prevalence for anti-nuclear The observations that Cbl-b RING finger mutant mice present (ANA) (Fig. 4D) and anti-dsDNA autoantibodies (Supplemental hyperproliferative T cells that cannot be tolerized in vivo promp- Fig. 5C)inC373AKI/KI E3 ligase-defective mice compared with ted us to test whether, similar to Cbl-b knockout mice (6), Cbl-b that for C373A+/+ and C373A+/KI littermates. Additionally, E3 ligase-defective mutants develop autoimmunity. To this end, C373AKI/KI mice present augmented serum titers of total IgG1 +/+ +/KI KI/KI 2/2 C373A , C373A , C373A , and Cbl-b mice were ana- (Supplemental Fig. 5D). In accordance with previously published lyzed for the presence of spontaneous pathological tissue infiltrates data (6), 6- to 8-mo-old Cbl-b2/2 mice exhibited highly elevated and autoantibodies. Exhaustive histological analyses revealed serum levels of autoantibodies (Fig. 4D, Supplemental Fig. 5C). a high incidence of mononuclear inflammatory tissue infiltrates in These data show that mice carrying a loss-of-function mutation in KI/KI several organs of C373A as well as age-matched Cbl-b–de- Cbl-b RING finger domain develop a spontaneous autoimmune ficient mice (Fig. 4A, Supplemental Fig. 5A). Lung, submandi- phenotype. bular salivary glands, and kidney were the most affected organs, Cbl-b E3 ligase-defective mice can spontaneously reject TC1 followed by liver and pancreas (Fig. 4A, Supplemental Fig. 5B, Downloaded from and data not shown). For most tissues, cellular infiltrates were tumors mostly located adjacent to tissue vasculature. In the particular case Recently, our group and others have demonstrated that genetic of pancreas, virtually all infiltrates were located within islets of ablation of Cbl-b renders mice able to reject several different types Langerhans. However, the presence of cellular infiltrates in of tumors (17, 18). Based on these observations, we set out to C373AKI/KI and Cbl-b2/2 mice did not result in pancreatic mal- investigate whether inactivation of Cbl-b E3 ligase function is

function, as determined by physiological levels of glucose and sufficient to mediate tumor rejection in vivo. For this purpose, we http://www.jimmunol.org/ insulin in serum (Supplemental Fig. 5E,5F). Staining with anti- challenged C373A ligase-defective mice with TC1 tumor cells CD3ε Abs revealed significant numbers of T cells among the in- expressing the human papilloma virus oncoproteins (32). We used filtrating cells (Fig. 4B). In tissue cross-sections of age-matched this tumor because we have previously shown that Cbl-b2/2 mice by guest on September 29, 2021 FIGURE 4. Cbl-b E3 ligase-defective mice develop spontaneous autoimmunity. A, H&E staining on lung, submandibular salivary gland, and kidney from 6- to 8-mo-old C373A+/KI, C373AKI/KI, and Cbl-b2/2 mice. Cellular infiltrates are marked with an asterisk or arrows. Representative images for a total of n =10 (C373A+/KI, C373AKI/KI) and n =5(Cbl-b2/2) mice are shown. Original magnification 320. B, Anti-CD3ε immunostaining showing T cells among the cellular infiltrates in lung, pancreas, and submandibular salivary gland of C373AKI/KI and Cbl-b2/2 mice. Representative images are shown. Original magnification 320. Scale bars, 200 mm. C, Detection of autoantibodies (red) by indirect immunofluorescence staining on tissue sec- tions of Rag22/2 mice using serum obtained from 6- to 8-mo-old C373A+/KI and C373AKI/KI littermates. Autoantibodies were visualised using Alexa Fluor-555 labeled anti-mouse IgG secondary Abs. Representative images are shown. Nuclei were counterstained using DAPI (blue). Original magnifications 363 and 340 (for liver). D, Quantification of serum autoantibodies reactive against nuclear Ags (ANA) in the serum of 6- to 8-mo-old C373A+/+ (n = 6), C373A+/KI (n = 10), C373AKI/KI (n = 11), and Cbl-b2/2 (n = 5) mice, de- termined by ELISA. OD values for individual mice are shown. Red dots represent significantly increased ANA autoantibodies serum concentration based on a Z-scored cutoff of 1.65 (dashed line, p # 0.05) related to the serum concentration measured in wild-type mice. 2144 THE ROLE OF Cbl-b E3 LIGASE ACTIVITY IN VIVO can spontaneously reject TC1 cells (18). Subcutaneous injections C373AKI/KI mice were able to efficiently and spontaneously of 2.5 3 105 TC1 cells in the flanks of C373A+/+ and C373A+/KI reject TC1 tumor (Fig. 5A). It should be noted that we injected mice lead to a fast and progressive growth of palpable tumors. For a tumor cell number (2.5 3 105) that is 10 times higher than the both cohorts, tumors reached 1 cm3 (the end point of the experi- dose that is lethal for wild-type mice (35). Tumor rejection by ment) within the first 3–4 wk after inoculation (Fig. 5A). Cbl-b ligase-defective mutant mice started between 2 and 3 wk Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Spontaneous rejection of TC1 tumors in Cbl-b E3 ligase mutant mice. A, Kinetics of TC1 tumor cell growth in C373A+/+ (n = 9), C373A+/KI (n = 13), C373AKI/KI (n = 14), and Cbl-b2/2 (n = 9) mice. TC1 cells (2.5 3 105) were injected into the left flanks of 8- to 12-wk-old littermate mice, and tumor volume was measured over time (days). Data are pooled from four different experiments. B, Photographs showing tumor rejection by C373AKI/KI mice at day 26 post-TC1 inoculation. Note that the mutant mouse carried a tumor of similar size as the control mouse on day 14 post-TC1 inoculation. C, Kaplan–Meier survival curves of C373A+/+ (n = 9), C373A+/KI (n = 13), C373AKI/KI (n = 14), and Cbl-b2/2 (n = 9) mice. ***p , 0.001 (C373A+/+ versus C373AKI/KI or Cbl-b2/2 and C373A+/KI versus C373AKI/KI or Cbl-b2/2). D, Immunohistochemistry for proliferation (Ki67), apoptosis (cleaved caspase 3), and T cell (CD3ε) markers in tumors from C373A+/+, C373A+/KI, C373AKI/KI, and Cbl-b2/2 mice at day 14 posttumor inoculation. For immunostaining, see Materials and Methods. Left panels, Representative images of individual mice for each immunostaining at low (320) and high (3200, insets) magnifi- cation. Right panels, Percentages of positive cells for each immunostaining and genotype. Data are shown as mean 6 SEM. Staining quantification was performed using Definiens Tissue Map software. For each mouse (n = 4 to 7 mice per group), sections were quantified at two different intervals (2 or 3 sections per interval). All the cells in 4–6 sections per mouse were counted. *p , 0.05, **p , 0.01, ***p , 0.001 (comparing the C373A+/+ or C373A+/KI versus C373AKI/KI or Cbl-b2/2 cohorts). The Journal of Immunology 2145 after tumor inoculation, when tumor mass was clearly visible, and average, whereas half of the C373AKI/KI and Cbl-b2/2 mice could this rejection progressed extremely fast for ∼10 d (Fig. 5A,5B). reject TC1 cells for up to 100 d, the median survival time for The kinetics of tumor rejection in C373AKI/KI mice were very C373A+/+ and C373A+/KI mice was ∼30 d. Remarkably, whereas similar to that observed in Cbl-b–deficient mice (Fig. 5A). On not a single C373A+/+ or C373A+/KI mouse was able to survive Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. CD8+ T cells are required for tumor rejection in Cbl-b E3 ligase-defective mice. A, Flow cytometry analysis of lymphocytes infiltrating TC1 tumors isolated from C373A+/+, C373A+/KI, C373AKI/KI, and Cbl-b2/2 mice at day 14 post-tumor inoculation. Representative FACS blots of CD4+ and CD8+ intratumoral T cells and their percentages per group are shown (top panels). Bottom panel, Quantification of intratumoral CD4+ and CD8+ T cells (mean 6 SEM). n = 4 to 7 animals per group. *p , 0.05 versus C373AKI/KI or Cbl-b2/2 (t test). B, Representative images of individual mice showing tumor-infiltrating CD8+ cells at day 14 after TC1 inoculation (top panel). Bottom panel, Quantification. Five optical fields were counted for each mouse (n = 4 to 7 mice per group); that is, data from 20–35 optical fields were counted per genotype. Tumor infiltrating CD8+ cells were detected with anti-CD8 Abs followed by Alexa Fluor-555 labeled anti-rat IgG secondary Abs. Data are shown as mean numbers of intratumoral CD8+ T cells/mm2 6 SEM. ***p , 0.001 versus C373AKI/KI. C, Kinetics of TC1 tumor cell growth in untreated (left panels, control) or CD8+ cell-depleted (right panels) C373A+/KI and C373AKI/KI mice. Data are pooled from two different experiments. D, Kaplan–Meier survival curves for untreated C373A+/KI (n = 7) and C373AKI/KI (n =8) mice and C373A+/KI (n = 6) and C373AKI/KI (n = 9) CD8+ cell-depleted mice. **p , 0.01 (C373A+/KI control versus C373A+/KI CD8+ depleted mice), ***p , 0.01 (C373AKI/KI untreated versus C373AKI/KI CD8+ depleted mice). 2146 THE ROLE OF Cbl-b E3 LIGASE ACTIVITY IN VIVO beyond 48 d, ∼30% of C373AKI/KI and Cbl-b2/2 animals remained whether the in vivo functions of any of these key E3 ligases, and in tumor-free their whole lives, in some cases for up to 2 y (Fig. 5C). particular Cbl-b, are indeed dependent on the E3 ligase catalytic To understand better the mechanism involved in tumor rejection activity or whether these molecules function in vivo as multivalent by Cbl-b E3 ligase inactivation, we performed histological anal- adapters (9, 10, 36). The generation of point mutant Cbl-b E3 yses on tumors isolated at day 14 post-TC1 inoculation. Of note, ligase-defective mice provided us with the unique possibility to this time point immediately preceded tumor rejection, and thus ultimately dissect the relative contribution of Cbl-b enzymatic and tumor volume was still comparable between all experimental adapter functions in vivo. mice groups. Staining for apoptosis markers revealed markedly Our data show that a loss-of-function mutation in the Cbl-b increased cell death in the tumors from C373AKI/KI and Cbl-b2/2 RING finger domain renders peripheral T cells hyperproliferative mice compared with that in E3 ligase-sufficient control mice (Fig. and able to be fully activated in the absence of CD28 costimula- 5D). We also observed slightly reduced proliferation of TC1 cells tion. Hence, a functional RING finger catalytic domain is required as detected by Ki67 immunostaining. Moreover, whereas tumors for Cbl-b to repress T cell activation. More strikingly, by crossing from C373A+/+ or C373A+/KI mice contained few CD3+ cells, the C373A point mutation into a mouse model of T cell tolerance, tumors isolated from C373AKI/KI and Cbl-b2/2 mice exhibited we could show that Cbl-b controls T cell anergy to specific Ags a marked increase in tumor-infiltrating CD3ε+ T cells (Fig. 5D). and determines the survival/lethal outcome after repetitive en- Thus, inactivation of the Cbl-b RING finger catalytic domain counter with the same Ag in an E3 ligase-dependent manner. results in slightly reduced tumor proliferation, increased tumor Additionally, Cbl-b enzymatic function is essential in the control cell death, and higher numbers of intratumoral T cells. Most im- of autoimmune responses, as inactivation of Cbl-b catalytic ac- portantly, these data using TC1 tumor cells show that inactivation tivity phenocopies the spontaneous autoimmunity observed in Downloaded from of Cbl-b enzymatic function is sufficient to confer antitumor ac- Cbl-b knockout mice. Moreover, inactivation of Cbl-b E3 ligase tivity in vivo. activity in mice resulted in a spontaneous capacity to reject TC1 tumors. Although in our model CD8+ T cells are a major pop- CD8+ T cells control tumor rejection in Cbl-b E3 ligase mutant ulation contributing to tumor rejection, additional studies are mice needed to understand better the cellular mechanisms implicated in +

As greater numbers of CD3 T cells were present in the tumors of the remarkable tumor rejection capacity of Cbl-b E3 ligase- http://www.jimmunol.org/ KI/KI C373A animals, we speculated that, as in the case of Cbl-b– defective mice. Nonetheless, our study importantly suggests that deficient mice (17, 18), T cells might be relevant mediators of Cbl-b ligase inactivation could be a novel modality to confer tumor rejection in these mutant mice. Whereas flow cytometry antitumor activity in vivo. analysis of tumor tissue revealed similar numbers of intratumoral Cbl-b is ubiquitously expressed and participates in the modula- + + CD4 T cells in all animal cohorts, the percentages of CD8 tion of other cellular immune responses beyond T cells, for example KI/KI 2/2 T lymphocytes present in the tumors of C373A and Cbl-b in B cells (37, 38), mast cells (39), and macrophages (40). Recent mice were significantly elevated compared with those observed in studies have confirmed that Cbl-b functions as an E3 ligase in many KI/KI +/KI C373A and C373A mice (Fig. 6A). Further immunofluo- but interestingly not all cellular contexts. By analyzing the same rescence analyses on tumor cross-sections confirmed the increased Cbl-b C373A ligase-defective mice, very recent publications have by guest on September 29, 2021 + KI/KI numbers of CD8 cells within the tumors of C373A and Cbl- demonstrated that Cbl-b enzymatic function is required to induce 2/2 b mice (Fig. 6B). anergy in NKT cells (41) as well as for Foxp3 expression in + To examine whether CD8 T cells are indeed required for the TGF-b–induced regulatory T cells (42). In contrast, Cbl-b ligase spontaneous rejection of TC1 tumor cells in Cbl-b E3 ligase- activity has been reported to be differentially required for IgE + defective mice, we depleted mice of CD8 cells prior to tumor -mediated mast cell functions. Whereas loss of Cbl-b inoculation by i.p. injections of specific depleting Abs (Supple- enzymatic activity altered some signaling pathways downstream +/KI mental Fig. 6). As expected, C373A could not reject tumors, of the FcεRI receptor, Cbl-b negative regulation of the signaling + and absence of CD8 cells in these mice led to an even faster pathway leading to the production of inflammatory cytokines KI/KI tumor growth (Fig. 6C,6D). Importantly, whereas C373A was found to be largely independent of the RING finger domain untreated control mice exhibited efficient and spontaneous tumor (30). Thus, Cbl-b may biochemically function as both E3 ligase KI/KI + rejection, C373A mice depleted of CD8 T cells were unable and multivalent adapter protein depending on the cellular con- to reject TC1 cells, displaying instead a progressive and lethal text or even in different signaling pathways within each cell + tumor growth (Fig. 6C,6D). Thus, Cbl-b E3 ligase-deficient CD8 type. Our results establish the essential in vivo role of Cbl-b E3 T cells mediate spontaneous tumor rejection. ligase activity in the regulation of T cell tolerance, autoimmunity, and tumor immunosurveillance. A more complete understanding Discussion of the cellular and molecular mechanism involved in Cbl-b ubi- The characterization of Cbl-b knockout mice during the past de- quitylation-dependent immune regulation could offer a unique cade has established the essential role of Cbl-b in the regulation possibility for the development of future therapies to modulate of T cell activation, immunotolerance, and autoimmunity. The re- Cbl-b E3 ligase activity to control T cell immunity in vacci- markably evolutionary conservation of the RING finger domain of nation, cancer biology, or autoimmunity. Cbl proteins (8), together with the confirmation that this domain is directly responsible for protein ubiquitylation (13, 14), suggested Acknowledgments that Cbl-b physiological functions are mediated by the catalytic We thank Gabi Stengl, Mihaela Zeba, Karin Aumayr, Pawel Pasierbek, Har- E3 ligase activity. However, Cbl proteins contain also highly ald Scheuch, Maria Novatchkova, and Ricardo de Matos Simoes for tech- evolutionarily conserved protein-interacting domains (8), and nical support, Cornelis J.M. Melief for providing TC1 cells, and Stefanie Cbl-b binds to multiple key molecules of the TCR signalosome (6, Loeser and all other current and former members of the Penninger labora- 7) that, so far known, are not being ubiquitylated. Similarly, it has tory for helpful discussions. been shown that other E3 ubiquitin ligases, such as GRAIL and Itch, play critical roles in T cell activation and immunological Disclosures tolerance (19, 20, 22–24). However, it was entirely unknown J.M.P. holds shares in Apeiron Biologics. The Journal of Immunology 2147

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