Alternative Splicing Generates Putative Soluble CD83 Proteins That Inhibit T Cell Proliferation This information is current as Diana Dudziak, Falk Nimmerjahn, Georg W. Bornkamm and of September 23, 2021. Gerhard Laux J Immunol 2005; 174:6672-6676; ; doi: 10.4049/jimmunol.174.11.6672 http://www.jimmunol.org/content/174/11/6672 Downloaded from References This article cites 36 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/174/11/6672.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 23, 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Alternative Splicing Generates Putative Soluble CD83 Proteins That Inhibit T Cell Proliferation1 Diana Dudziak,2* Falk Nimmerjahn,3† Georg W. Bornkamm,* and Gerhard Laux* CD83 is expressed on mature dendritic cells and activated lymphocytes and has been implicated to play an important role during T cell development in the thymus. In contrast, not much is known about the function of CD83 in the periphery. Soluble forms of CD83 have been detected in the serum, but neither the function nor the mechanism of how these soluble forms of CD83 are generated are fully understood. In this study, we report the identification of four different transcripts of CD83 in unstimulated PBMCs. Sequence analysis demonstrated that the longest form codes for transmembrane CD83 (CD83-TM), whereas the smaller transcripts are splice variants of full-length CD83, coding for putative soluble CD83 proteins. Stimulation of PBMCs with PHA, TNF-␣, or LPS leads to the up-regulation of the full-length CD83 transcript and to a strong down-regulation of two of the three smaller transcripts. The smallest CD83 splice product can be translated efficiently into protein, and recombinant soluble CD83 shows a strong inhibitory effect on T cell proliferation in MLRs. Our results suggest that the constitutive production of soluble Downloaded from forms of CD83 under steady-state conditions may have an important function in regulating immune homeostasis. The Journal of Immunology, 2005, 174: 6672–6676. he surface glycoprotein CD83 has a molecular mass of are rejected after injection into C3H/HeN mice, whereas wild-type 40–45 kDa and belongs to the Ig superfamily (1, 2). It melanoma cells showed tumor growth (16, 17). consists of an extracellular V-type Ig-like domain at the N The addition of a human CD83-Ig fusion protein or a human http://www.jimmunol.org/ T 4 terminus, one transmembrane (TM) domain, and a short intracel- soluble CD83 protein lacking the TM and cytosolic domain lular cytoplasmic domain of 39 aa (2). CD83 is described as a cell strongly inhibited MLR or DC-mediated allogeneic T cell prolif- surface marker of mature dendritic cells including Langerhans eration, demonstrating that soluble CD83 could have an inhibitory cells in the skin and interdigitating reticulum cells in the T cell function (18). Moreover, soluble CD83 was able to prevent exper- zones of the lymph nodes (2–5). In immature monocyte-derived imental autoimmune encephalomyelitis (EAE) in C57BL/6 mice dendritic cells (MoDCs), CD83 is up-regulated after stimulation (19). Latest studies suggest that soluble CD83 is present in the with LPS, TNF-␣, or CD40L (6–8). Addition of high doses of serum of healthy donors (20–22) and it was proposed that the IL-4, GM-CSF, and TNF-␣ induces the expression of CD83 on mechanism for the generation of soluble CD83 is shedding of cell by guest on September 23, 2021 monocytes, granulocyte-precursor cells, and myelocytes (9, 10). It surface-associated CD83. However, soluble forms of cell surface was also shown that CD83 is expressed on polymorphonuclear proteins can also result from alternative splicing as reported for neutrophils (11) and on murine thymus epithelial cells (12). Fur- several members of the Ig superfamily, e.g., CD80 (B7-1), CD86 thermore, CD83 is expressed on Hodgkin cells (13) and EBV- (B7-2), CTLA-4, or CD28 (23–28). transformed lymphoblastoid cell lines (2, 14). In this study, we report that alternative splicing generates soluble The function of CD83 remains largely unknown. CD83 knock- forms of CD83. Stimulation of freshly isolated PBMCs with PHA, out mice showed a block in the development of CD4ϩ single- LPS, or TNF-␣/IL-1␤ up-regulates transcripts encoding TM CD83 positive T cells (12). CD83 expression on mature dendritic cells and down-regulates transcripts coding for alternatively spliced prod- and activated lymphocytes (1, 2, 10, 15) suggests that CD83 might ucts of CD83. These putative soluble forms of CD83 proteins are be involved in activating immune effector cells. Along these lines, characterized by a partial deletion of the extracellular and the TM melanoma cells that were engineered to express cell surface CD83 domain. Soluble CD83 displayed inhibitory effects in MLRs and, therefore, might have important immunoregulatory functions in vivo. *Institute of Clinical Molecular Biology and Tumor Genetics, GSF National Research Materials and Methods Center (GSF) for Environment and Health and †Clinical Cooperation Group of Pe- Isolation of human PBMCs and DCs diatric Oncology, GSF and Children’s Hospital of the Technical University, Munich, Germany Human PBMCs were isolated by centrifugation on Ficoll-Paque (Amer- sham Biosciences). Isolated PBMCs were cultured in complete medium Received for publication September 15, 2004. Accepted for publication March (RPMI 1640 medium; Invitrogen Life Technologies) supplemented with 15, 2005. 10% FCS (Dynacyte), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1 mM The costs of publication of this article were defrayed in part by the payment of page sodium-pyruvate, 2 mM L-glutamine (all from Invitrogen Life Technolo- charges. This article must therefore be hereby marked advertisement in accordance gies) and were left either unstimulated or stimulated with PHA (1 ␮g/ml), with 18 U.S.C. Section 1734 solely to indicate this fact. LPS (1 ␮g/ml), or a mix of TNF-␣ (2.5 ng/ml; Sigma-Aldrich), IL-1␤ (1 1 This work was supported by a grant from Deutsche Forschungsgemeinschaft ng/ml; Sigma-Aldrich), and PGE2 (1 ng/ml; Sigma-Aldrich) for the indi- (SFB455; to F.N.). G.W.B. was supported by Fonds der Chemischen Industrie. cated time. Human DCs were generated as described (4) and matured with 2 ␣ ␤ Address correspondence and reprint requests to Dr. Diana Dudziak at the current LPS (100 ng/ml) or TNF- /IL-1 /PGE2. address: Laboratory of Molecular Immunology, The Rockefeller University, Box 220, 1230 York Avenue, New York, NY 10021. E-mail address: [email protected] Cloning and sequencing of the CD83 mRNA splice products 3 Current address: Laboratory of Molecular Genetics and Immunology, The Rock- Total RNA of PBMCs was extracted using the RNeasy Midi kit (Qiagen). efeller University, 1230 York Avenue, New York, NY 10021 RNA from human tissues was kindly provided by J. Mautner (Clinical 4 Abbreviations used in this paper: TM, transmembrane; MoDC, monocyte-derived Cooperation Group of Pediatric Oncology, GSF, Munich, Germany). Sin- dendritic cell; ORF, open reading frame. gle-stranded cDNA was synthesized from 5 ␮g of total RNA by reverse Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6673 transcription using Superscript reverse transcriptase (Invitrogen Life Tech- Results nologies) and an oligo-dT primer (Amersham Biosciences) at 42°C. CD83 Identification of alternatively spliced variants of the CD83 mRNA cDNA was amplified with primers designed to amplify the entire coding sequence of CD83 (5Ј-GCGGGGGAATTCCTCGAGATGTCGCGCGG Recently, it was shown that soluble CD83 variants are present in CCTCCAGCTTC-3Ј and 5Ј-CCCCGGGCGGCCGCTCATACCAGTTCT the serum of healthy individuals and that they are enriched in leu- GTCTTGTGAGGAGTC-3Ј; Metabion). The PCR was performed using PFU DNA Polymerase (Promega) as follows: 94°C for 4 min, than 35 kemia patients (20–22). To examine whether soluble CD83 forms cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min, followed by are generated by alternative splicing, we isolated total RNA from a final extension step at 72°C for 10 min (PerkinElmer). The amplified PBMCs that were either left untreated or treated for 4 or 16 h with fragments were separated on a 2% agarose gel and visualized by PHA (Fig. 1A) or for 16 h with LPS or TNF-␣/IL-1␤, respectively ethidium bromide. After excision from the gel, resulting products were (Fig. 1B). Stimulation of PBMCs with PHA was followed by cloned into the pcDNA3.1 vector. Cloned cDNAs were verified by se- quencing (Sequiserve). RNA integrity and cDNA synthesis was proven FACS analysis with an anti-human CD83-PE-labeled Ab showing by amplifying GAPDH cDNA (5Ј-ACCACAGTCCATGCCATCAC-3Ј a strong induction of CD83 on the cell surface of PBMCs 4 h after and 5Ј-TCCACCACCCTGTTGCTGTA-3Ј). PHA addition (Fig. 1C). The amplification of the CD83 coding sequence by RT-PCR using primers flanking the ATG initiation Generation of His-tagged soluble CD83 codon and the translation termination codon revealed the consti- tutive expression of four transcripts by nonactivated PBMCs, with CD83 splice variants were tagged with a C-terminal histidine hexapep- tide.