Chains in Immature Thymocytes Ζ Competence of CD3-Independent TCR Surface Expression and Functional

Surface Expression and Functional Competence of CD3-Independent TCR ζ -Chains in Immature Thymocytes This information is current as Fabio Grassi, Eliane Barbier, Simona Porcellini, Harald von of October 2, 2021. Boehmer and Pierre-André Cazenave J Immunol 1999; 162:2589-2596; ; http://www.jimmunol.org/content/162/5/2589 Downloaded from References This article cites 77 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/162/5/2589.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 2, 2021 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Surface Expression and Functional Competence of CD3-Independent TCR z-Chains in Immature Thymocytes1 Fabio Grassi,2*† Eliane Barbier,† Simona Porcellini,* Harald von Boehmer,‡ and Pierre-AndreĆazenave† In recombinase-deficient (RAG-22/2) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that z-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-22/2 thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of z dimers with the CD3 complex in RAG-22/2 thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of z dimers with ZAP-70 in the RAG-22/2 thymocytes, suggest that these z-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-22/2 z-deficient thymocytes, ZAP-70 and p120cbl were only Downloaded from weakly phosphorylated. The Journal of Immunology, 1999, 162: 2589–2596. evelopment of ab thymocytes is characterized by the lacking both Lck and Fyn (8, 9) and mice lacking both ZAP-70 and transition of cells through an ordered sequence of dis- Syk (10) exhibit a phenotype that is strikingly similar to the one of D tinct phenotypes, which can be defined by the expression RAG2/2 mice (11), implying a critical role for these molecules in of the coreceptor molecules CD4 and CD8. Early in development, the proximal signal transduction by the pre-TCR. The role played http://www.jimmunol.org/ the transition of the most immature double-negative (DN)3 to the by CD3 and z-chains in pre-TCR assembly and signaling has been double-positive (DP) stage is largely dependent upon the produc- only partially elucidated. Studies with mutant mice have revealed tive rearrangement of TCRb genes and expression of TCR an absolute requirement for CD3e-chains (12), no essential re- b-chains in association with monomorphic pTa chains (1). Cell- quirement for CD3d-chains (13, 14), and a major role for z-chains autonomous signaling by the pre-TCR results in b selection, i.e., (15–18). z2/2 mice have a scarcely populated thymus and display b selection of TCR -expressing cells for survival; other conse- an anomalous progression from the DN to the DP stage, in that the quences include expansion, down-regulation of the IL-2R (CD25), CD4282252 stage that precedes the DP stage is absent (19). expression of both coreceptor molecules, and induction of a second Therefore, a specific role for z-chain in controlling proliferation a wave of RAG expression, allowing TCR rearrangements to and differentiation of DN cells has been hypothesized. by guest on October 2, 2021 occur (2, 3). In RAG-2-deficient mice, the absence of TCRb rearrangements ab Ligation of the TCR in mature T cells induces activation of leads to a developmental block at the DN CD251 stage (11). Src family kinases, such as Lck and Fyn, as well as phosphoryla- CD251 cells express low levels of CD3g, d, and e subunits at the tion of the immunoreceptor tyrosine-based activation motifs surface (20, 21). Efficient cellular proliferation, down-regulation of (ITAM) of CD3 and z subunits (4). The phosphorylation of the two CD25, and production of small cortical cells that are characteristic tyrosines present in one ITAM results in recruitment of the ty- of the physiologic transition driven by the pre-TCR can be induced rosine kinase ZAP-70 into the TCR/CD3/z complex through the in fetal thymus organ cultures by addition of anti-CD3e mAb (22) binding of tandem SH2 domains (5, 6). This association permits and, in vivo, by injection of mice with anti-CD3e Abs (20, 21). the enzymatic activity of ZAP-70, resulting in phosphorylation of Hence, CD3 appears functionally competent to promote these de- downstream targets (7). A similar sequence of events appears to velopmental steps in the absence of a fully assembled pre-TCR and take place also in signal transduction by the pre-TCR, since mice in fact in the absence of the pre-TCR a-chain (23). We found that z-chains, despite the lack of detectable physical *Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita`di Milano at association with the surface CD3 complex, are functionally cou- Department of Biological and Technological Research, San Raffaele Scientific Institute pled to the signaling cascade initiated by anti-CD3 treatment of † (HSR), Milan, Italy; Unite´d’Immunochimie Analytique, De´partement d’Immunologie, 2/2 Institut Pasteur, Unite´de Recherche Associeé, Centre National de la Recherche Sci- RAG-2 thymocytes since they become associated with phos- entifique D1961, and Universite´Pierre et Marie Curie, Paris, France; and ‡Institut phorylated ZAP-70. This complex can be immunoprecipitated Necker, Institut National de la Santeét Recherche Medicale, U373, Paris, France from the membrane fraction of anti-CD3-treated RAG-22/2 thy- Received for publication August 31, 1998. Accepted for publication November mocytes, suggesting its recruitment to the cell membrane. More- 16, 1998. over, the z-chain dimers that are not linked to CD3 can be detected The costs of publication of this article were defrayed in part by the payment of page vav charges. This article must therefore be hereby marked advertisement in accordance in pre-TCR-expressing cells. The comparison of ZAP-70, p95 , with 18 U.S.C. Section 1734 solely to indicate this fact. and p120cbl phosphorylation upon anti-CD3 treatment shows the 2/2 1 This work was supported by grants from the Ligue Nationale Contre le Cancer and same pattern in RAG-2 thymocytes and pre-TCR-expressing Association pour la Recherche sur le Cancer. cells, and differs from activated RAG-22/2z2/2 thymocytes in 2 Address correspondence and reprint requests to Dr. Fabio Grassi, DIBIT-HSR via which ZAP-70 and p120cbl phosphorylation is barely detectable. Olgettina 58 I-20132 Milan, Italy. E-mail address: [email protected] Therefore, an important role of the z-chain/ZAP-70 complex in 3 Abbreviations used in this paper: DN, double-negative; DP, double-positive; ECL, enhanced chemiluminescence; IEF, isoelectric focusing; ITAM, immunoreceptor ty- mediating the transition from the DN to the DP thymocyte stage is rosine-based activation motif. postulated. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2590 PHOSPHORYLATION OF CD3-INDEPENDENT z-CHAINS IN IMMATURE THYMOCYTES Materials and Methods Deglycosylation Mice, cell lines, and Abs 125I-labeled anti-CD3e immunoprecipitates were boiled twice: 2 min in 1% Young adult (4–6-wk-old) C57BL/6, RAG-22/2, RAG-12/2, and CB17 SDS and 2 min in deglycosylation buffer (20 mM sodium phosphate, pH 7.2, 10 mM NaN3, 50 mM EDTA, 0.5% Nonidet P-40). One sample was scid/scid (SCID) mice were obtained from the animal colony of the Insitut 2 Pasteur (Paris, France). z2/1 mice were kindly provided by Dr. B. Malissen stored at 20°C, another one was incubated at 37°C for 16 h, and a third (Centre d’Immunologie INSERM-CNRS, Marseille, France). SCB.29 (24) one was incubated at 37°C for 16 h in the presence of 2 mU neuraminidase, and M14T (25) thymocyte cell lines were used. The mouse mAbs em- 2.5 mU O-glycosidase, and 0.4 U N-glycosidase F (Boehringer Mannheim, ployed were anti-CD3e 145-2C11 (26), anti-z G3 (27), and anti- Mannheim, Germany). The deglycosylation products were analyzed by phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY). The fol- SDS-PAGE. lowing rabbit antisera were used: anti-calnexin C-terminal peptide (StressGen, Victoria, B.C.), anti-TCRz (kindly provided by Dr. L. Samel- son, National Institute of Health, Bethesda, MD), anti-ZAP-70 and anti- Cell fractionation vav cbl p95 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-p120 2 2 After two washes in ice-cold PBS, C57BL/6 and RAG-2 / thymocytes (kindly provided by Dr. A. Veillette, University of Toronto, Canada). were resuspended at 50 3 106/300 ml in hypotonic buffer (20 mM Tris- HCl, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, and protease inhibitors) and incubated 10 min on ice.

Load more