Signaling at the SLP-76-Vav-Itk Signalosome Exchange Factor Vav1
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Mitogenic CD28 Signals Require the Exchange Factor Vav1 to Enhance TCR Signaling at the SLP-76-Vav-Itk Signalosome This information is current as Kevin M. Dennehy, Fernando Elias, Shin-Young Na, of September 25, 2021. Klaus-Dieter Fischer, Thomas Hünig and Fred Lühder J Immunol 2007; 178:1363-1371; ; doi: 10.4049/jimmunol.178.3.1363 http://www.jimmunol.org/content/178/3/1363 Downloaded from References This article cites 66 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/178/3/1363.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Mitogenic CD28 Signals Require the Exchange Factor Vav1 to Enhance TCR Signaling at the SLP-76-Vav-Itk Signalosome1 Kevin M. Dennehy,2,3* Fernando Elias,3* Shin-Young Na,* Klaus-Dieter Fischer,† Thomas Hu¨nig,4* and Fred Lu¨hder5* Almost all physiological T cell responses require costimulation—engagement of the clonotypic TCR with MHC/Ag and CD28 by its ligands CD80/86. Whether CD28 provides signals that are qualitatively unique or quantitatively amplify TCR signaling is poorly understood. In this study, we use superagonistic CD28 Abs, which induce T cell proliferation without TCR coligation, to determine how CD28 contributes to mitogenic responses. We show that mitogenic CD28 signals require but do not activate the proximal TCR components TCR and Zap-70 kinase. In cell lines lacking proximal TCR signaling, an early defect in the CD28 pathway is in phosphorylation of the adaptor molecule SLP-76, which we show is essential for recruitment of the exchange factor ؉ Vav leading to Ca2 flux and IL-2 production. Point mutations in CD28 that result in diminished Vav phosphorylation also result Downloaded from in defective Ca2؉ flux, IL-2 production, and Tec-kinase phosphorylation. Using Vav1-deficient mice, we further demonstrate the importance of Vav1 for efficient proliferation, IL-2 production, and Ca2؉ flux. Our results indicate that CD28 signals feed into the TCR signaling pathway at the level of the SLP-76 signalosome. The Journal of Immunology, 2007, 178: 1363–1371. lmost all physiological T cell responses require costimu- such Abs thus provides an exception to the costimulatory require- lation (1). Ag specificity is provided by recognition of ment of T cell responses, and a unique tool to define CD28-specific http://www.jimmunol.org/ A cognate peptide in the context of appropriate MHC by events leading to mitogenic responses. the clonotypic TCR, whereas the interaction of CD28 with its nat- Mitogenic CD28 signaling can also be induced after stimulation ural ligands CD80/86 provides the most important costimulatory with the recombinant natural ligand CD80 (15). However, such stimulus for activation of naive T cells (2–4). Although the signals activation only occurs if the physiologically monovalent CD28 transduced by CD28 have been extensively studied, the mecha- extracellular region is replaced with a bivalent receptor such as the nisms of TCR and CD28 signal integration are poorly understood. extracellular domains of CTLA-4. Ligation of such bivalent CD28 In particular, it is not clear whether CD28 contributes qualitatively chimeras leads to an artificially stable complex in a receptor lattice unique signals, separate and independent from TCR signaling (5– structure resulting in mitogenic responses. Studies of binding stoi- 8), or signals that quantitatively amplify TCR signaling (3, 9–11), chiometry, as well as crystallographic evidence suggests that su- by guest on September 25, 2021 or both (12). peragonistic CD28 Abs similarly induce the formation of a recep- We have previously generated two functionally distinct classes tor lattice structure (15, 16). Taken together, these results suggest of CD28 Abs (13, 14). Conventional CD28 Abs recognize an that CD28 superagonists induce a physiologically relevant, al- epitope adjacent to the CD80/86 binding site, and like natural li- though dysregulated signaling pathway at the extremity of CD28 gands induce proliferative responses only when combined with costimulatory signaling, in the same way that ligation with CD3 TCR stimulation. By contrast, superagonistic CD28 Abs recognize Abs or high-avidity lymphocytic choriomeningitis virus Ags in- a membrane proximal epitope and fully activate primary peripheral duce particularly strong, and therefore costimulatory independent T cells in the absence of TCR ligation (13, 14). Stimulation with signaling at the extremity of the TCR pathway (1, 17). In this study, we have used superagonistic Abs to dissect CD28 signals at the extremity of costimulatory signaling, and thus in the absence of *Institute of Virology and Immunobiology, University of Wu¨rzburg, Wu¨rzburg, signaling events induced by TCR ligation. Germany; and †Department of Physiological Chemistry, University of Ulm, Ulm, Germany We have previously shown that stimulation of primary T cells with CD28 superagonists induces phosphorylation of the exchange Received for publication September 15, 2005. Accepted for publication November 15, 2006. factor Vav and adaptor SLP-76, activation of phospholipase C ␥ 6 2ϩ The costs of publication of this article were defrayed in part by the payment of page (PLC ) leading to delayed but sustained Ca flux, and activation charges. This article must therefore be hereby marked advertisement in accordance of the PKC-NF-B pathway (18). Surprisingly, these events oc- with 18 U.S.C. Section 1734 solely to indicate this fact. cur in the absence of inducible phosphorylation of the proximal 1 This work was supported by grants from Bundesministerium fu¨r Bildung und For- TCR signaling components TCR, Zap-70 kinase, and linker for schung, project 01 KX 9820 E; Bayerisches Staatsministerium fu¨r Wissenschaft, For- schung und Bildung (Bayerischer Habilitationsfo¨rderpreis 2000) (to F.L.); Deutsche activation of T cells (LAT), suggesting that CD28 can contribute Forschungsgemeinschaft Graduate College 520 Immunomodulation (to F.E.); and autonomous and qualitatively unique mitogenic signals (13, 18, Deutsche Forschungsgemeinschaft Grant Hu 295/7-1, 7-2 (to K.M.D.). 19). Using a genetic approach, we show in this study that this is not 2 Current address: Institute of Infectious Disease and Molecular Medicine, University the case. Mitogenic CD28 signaling is not autonomous but depen- of Cape Town 7925, South Africa. dent on constitutive proximal TCR signaling. We demonstrate that 3 K.M.D. and F.E. authors contributed equally to this work. 4 Address correspondence and reprint requests to Dr. Thomas Hu¨nig, Institute of Virology and Immunobiology, University of Wu¨rzburg, Versbacher Straße 7, 6 Abbreviations used in this paper: PLC, phospholipase C; LAT, linker for activation D-97078 Wu¨rzburg, Germany. E-mail address: [email protected] of T cell; WT, wild type; PKB, protein kinase B. 5 Current address: Institute for Multiple Sclerosis Research, University of Go¨ttingen and Gemeinnu¨tzige Hertie-Stiftung, D-37073 Go¨ttingen, Germany. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 1364 MITOGENIC CD28 SIGNALS AMPLIFY TCR SIGNALING constitutive TCR signals are required for CD28-mediated phos- phorylation of the adaptor SLP-76, and that CD28 signals feed into the TCR signaling pathway at the level of the SLP-76 signalosome, with Vav1 as an essential component of this complex. Materials and Methods Abs, cell lines, and constructs Generation of Abs to rat ␣TCR (R73), conventional (JJ319), and super- agonistic (JJ316) Abs to rat CD28 have been described previously (14, 20). Superagonistic (5.11A1) and conventional (7.3B6) Abs to human CD28 (13) was provided by TeGenero or purchased (CD28.2) (BD Biosciences). Mouse anti-human TCR V8 (16G8), anti-human CD3 (UCHT1), anti-human Itk (2F12), and anti-phosphotyrosine (4G10) were obtained from BD Bio- sciences. Mouse anti-human Zap-70 (3.3.1) (18) was provided by Dr. A. D. Beyers (University of Stellenbosch, Matieland, South Africa), and polyclonal anti-CD3 Ab was obtained from DakoCytomation. Rabbit anti-Vav (C-14), mouse anti-TCR (6B10.2), and anti-SLP-76 (F-7) were obtained from Santa Cruz Biotechnology, and rabbit anti-phospho-Akt(S473), rabbit anti-phospho- PLC␥1(Y783), and rabbit anti-PLC␥1 were from Cell Signaling Technology. Sheep anti-mouse IgG was from Roche, and rat anti-mouse IgG, goat anti- mouse IgG-peroxidase, and goat anti-rabbit IgG-peroxidase were from Downloaded from Dianova. Jurkat P116 and J14 cell lines (21) were provided by Drs. R. T. Abraham (The Burnham Institute, La Jolla, CA) and B. Schraven (Institute of Im- munology, Magdeburg, Germany), Jurkat E6.1 and TCR-deficient JRT3- T3.5 were provided by Dr. W. Kolanus (University of Munich, Munich, Germany), and TCR-deficient 3A9 cells were obtained from Dr. D. Vignali (St. Jude Children’s Research Hospital, Memphis, TN). The TCRϪ T cell hybridoma cell line 58, which expresses low levels of endogenous CD28, http://www.jimmunol.org/ was described previously (13, 22). TCR-negative 58 cells expressing chi- FIGURE 1. Mitogenic signals through CD28 require but do not activate meric CD25-TCR (23) were obtained from Dr. E. Vivier (Centre proximal TCR signaling components.