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Lymphocyte Early Activation Thrombospondin-1 Inhibits TCR

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Lymphocyte Early Activation Thrombospondin-1 Inhibits TCR Thrombospondin-1 Inhibits TCR-Mediated T Lymphocyte Early Activation Zhuqing Li, Liusheng He, Katherine E. Wilson and David D. Roberts This information is current as of September 28, 2021. J Immunol 2001; 166:2427-2436; ; doi: 10.4049/jimmunol.166.4.2427 http://www.jimmunol.org/content/166/4/2427 Downloaded from References This article cites 53 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/166/4/2427.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 September 28, 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Thrombospondin-1 Inhibits TCR-Mediated T Lymphocyte Early Activation Zhuqing Li, Liusheng He,1 Katherine E. Wilson,2 and David D. Roberts3 Biological activities of the matrix glycoprotein thrombospondin-1 (TSP1) are cell type specific and depend on the relative expression or activation of several TSP1 receptors. Although engaging individual TSP1 receptors in T lymphocytes can elicit costimulating signals, in this study we show that intact TSP1 inhibits TCR-mediated T cell activation, assessed globally using cDNA microarrays. TSP1 signaling suppressed expression of several genes induced in Jurkat T cells, including the T cell activation markers CD69, early growth response gene-1 (Egr-1), and phosphatase of activated cells (PAC-1). TCR-stimulated and CD47-costimulated IL-2 secretion and cell surface CD69 expression were also inhibited by TSP1. The specific inhibitory effect of TSP1 was verified in freshly isolated human PBMCs. TSP1 inhibited TCR-mediated but not protein kinase C-mediated T cell activation. Using CD69 expression as a marker, we demon- strated that the inhibitory activity of TSP1 depended on two TSP1 receptors, CD47 and integrin-associated protein heparan sulfate proteoglycans. Signals from these receptors inhibited TCR signaling downstream of ZAP70, but upstream of NF-AT. Therefore, the Downloaded from expression of TSP1 induced during wound repair and in tumor stroma may limit T cell activation at these sites. The Journal of Immunology, 2001, 166: 2427–2436. lymphocytes play key roles in host immune responses. types. TSP1 inhibits angiogenesis and tumor growth (reviewed in Activation of T cells enhances host immune responses to Refs. 16 and 17), activates latent TGF-␤ (18, 19), and is necessary T both foreign and self Ags. Because of its critical role in for maintenance of pulmonary homeostasis (20). The diverse bio- http://www.jimmunol.org/ host immune responses, T cell activation is tightly regulated by logical effects of TSP1 have been partially attributed to the mul- secreted cytokines and chemokines as well as by cell-cell contact tiple functional domains of the protein that engage corresponding and cell-matrix signaling. The activation of T cells is primarily receptors on the surface of the targeted cells. Differential expres- mediated by the TCR, but several important regulatory receptors sion or activation of cell surface receptors for TSP1, including have also been identified that can either costimulate or inhibit TCR integrins, CD36, CD47, low density lipoprotein receptor-related signals (1–3). Interest in the negative regulation of T cell activation protein, proteoglycans, and sulfatides, may dictate the specific re- has been stimulated by the need to develop improved therapies to sponses of each cell type to TSP1 (16). treat autoimmune diseases, AIDS, and cancer (4–6). Several studies have suggested that TSP1 can regulate T cell Interactions between T cells and extracellular matrix proteins play function. TSP1 mediates activation-dependent T cell adhesion by guest on September 28, 2021 ␣ ␤ ␣ ␤ pivotal roles in several T cell functions, including T cell homing, through binding to 4 1 and 5 1 integrins (21). TSP1 also mod- recruitment to inflammatory sites (7–9), and regulation of T cell ac- ulates intracellular signaling cascades in anti-CD3-activated T ␤ tivation (10–12). Several extracellular matrix proteins, including fi- cells, mediated by 1 integrins, CD47, and proteoglycans (22). bronectin, collagen, vitronectin, and laminin, have been shown to in- Peptides from TSP1 that bind to two of these TSP1 receptors syn- fluence T cell functions by inducing T cell adhesion, motility, ergize with TCR activation to activate Ras and mitogen-activated trafficking, and T cell coactivation (12–14). However, recent data us- protein (MAP) kinase signaling pathways (22). Functionally, TSP1 ing tenascin suggest that some matrix proteins can inhibit TCR-me- has been shown to selectively inhibit IL-12 production in mono- diated T cell activation (15). Thus, matrix proteins may have both cytes via a CD47-dependent mechanism (23). However, CD47 is a stimulatory and inhibitory effects on T cell activation. costimulatory receptor on T cells (24). In addition, TSP1 null Thrombospondin-1 (TSP1)4 is an extracellular matrix glycop- transgenic mice seem to be more susceptible to pulmonary bacte- rotein that displays distinct biological activities on different cell rial infection, suggesting an impact of TSP1 on host immune re- sponses (20). A recent study using an autoreactive T cell clone isolated from rheumatoid arthritis synovium reported a stimulatory Laboratory of Pathology, Division of Clinical Science, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 activity of immobilized TSP1 (25). These observations demon- Received for publication August 1, 2000. Accepted for publication December 5, 2000. strate that TSP1 regulates T cell activation, but are consistent with The costs of publication of this article were defrayed in part by the payment of page both inhibitory and costimulatory activities. To clarify this issue, charges. This article must therefore be hereby marked advertisement in accordance we have investigated the global effects of TSP1 on T cell activation with 18 U.S.C. Section 1734 solely to indicate this fact. and identified two TSP1 receptors that mediate its activity. We 1 Current address: Autoimmunity Branch, National Institute of Arthritis and Muscu- show that intact TSP1 is a potent inhibitor of TCR-mediated T cell loskeletal and Skin Diseases, Bethesda, MD 20892-0975. activation. Although this inhibition is TCR specific, it is not due to 2 Current address: Molecular Medicine Unit, St. James’s University Hospital, Leeds, U.K. direct interference with upstream signaling events of the TCR sig- 3 Address correspondence and reprint requests to Dr. David D. Roberts, Building 10, nal transduction pathway. Room 2A33, 10 Center Drive, MSC 1500, National Institutes of Health, Bethesda, MD 20892-1500. E-mail address: [email protected] 4 Abbreviations used in this paper: TSP1, thrombospondin-1; CAT, chloramphenicol Materials and Methods acetyltransferase; CD27BP, CD27-binding protein; Egr, early growth response gene; Antibodies FAST, Fas-activated serine/threonine kinase; HSPG, heparan sulfate proteoglycan; LAT, linker for activation of T cells; MAP, mitogen-activated protein; PAC-1, phos- The following Abs were used: anti-human CD3 (clone HIT3a; BD PharM- phatase of activated cells; PP2A, protein phosphatase 2A; TIEG, TGF-␤-inducible ingen, San Diego, CA), human CD47-blocking Ab (clone B6H12.2; BD early gene. PharMingen), human CD47-stimulating Ab (clone CIKm 1; ICN, Costa Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2428 TSP1 INHIBITS T CELL ACTIVATION Mesa, CA), PE-conjugated anti-human CD69 (Coulter Immunotech, Mi- IL-2 ELISA ami, FL), anti-ZAP70 (a general gift from Dr. Larry Samelson’s laboratory, National Cancer Institute, Bethesda, MD), anti-phosphotyrosine Ab (RC Cells were cultured as described above and stimulated in a flat-bottom 20; BD Transduction Laboratories, Lexington, KY), and activated TGF- 96-well plate. Culture supernatants were used to measure secreted IL-2 ␤1-neutralizing Ab (Life Technologies, Rockville, MD). level using a commercially available ELISA kit (R&D Systems, Minne- apolis, MN). The ELISA was performed according to the manufacturer’s Cell cultures and stimulation instruction. Briefly, 100 ␮l of the culture supernatant was incubated with anti-IL-2 Ab precoated on the wells. After extensive washing, bound IL-2 The Jurkat T cell line (provided by Dr. Kevin Gardner, National Cancer was detected by an HRP-conjugated second Ab. The cytokine expression Institute) was maintained in RPMI 1640 medium supplemented with 10% level was quantified by regression analysis using IL-2 standard curve. FCS, 2 mM L-glutamine, and 1ϫ penicillin and streptomycin (unless spec- ified, all culture medium and medium supplements were purchased from Flow cytometry analysis Biofluids, Rockville, MD). Human PBMCs were prepared by gradient cen- trifugation. In brief, fresh human blood buffy coat (obtained from the Na- The Jurkat T cells or freshly isolated human PBMCs, treated as described tional Institutes of Health blood bank under National Institutes of Health above and in the text, were isolated and washed with PBS containing 1% ϫ 6 ␮ Multiple Project Assurance M-1000) was diluted 1/5 with sterile 1ϫ PBS. BSA. About 1 10 cells were mixed with 10 l of PE-conjugated anti- Human PBMCs were isolated by mixing Ficoll Plus (Pharmacia, Piscat- human CD69 Ab and incubated in the dark on ice for 90 min. Unbound Abs away, NJ) and the diluted buffy coat and centrifuged at 100 ϫ g at room were washed off, and the cells were fixed with 0.1% paraformaldehyde on temperature for 30 min.
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