LST1: A Gene with Extensive Alternative Splicing and Immunomodulatory Function Ingrid Rollinger-Holzinger, Brigitte Eibl, Marc Pauly, Ute Griesser, François Hentges, Bernhard Auer, Georg Pall, This information is current as Peter Schratzberger, Dietger Niederwieser, Elisabeth H. of September 28, 2021. Weiss and Heinz Zwierzina J Immunol 2000; 164:3169-3176; ; doi: 10.4049/jimmunol.164.6.3169 http://www.jimmunol.org/content/164/6/3169 Downloaded from References This article cites 21 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/164/6/3169.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 28, 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. LST1: A Gene with Extensive Alternative Splicing and Immunomodulatory Function1 2 † † ‡ Ingrid Rollinger-Holzinger, * Brigitte Eibl, Marc Pauly*, Ute Griesser, Franc¸ois Hentges, Bernhard Auer,§ Georg Pall,† Peter Schratzberger,† Dietger Niederwieser,¶ Elisabeth H. Weiss,ʈ and Heinz Zwierzina† The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3؅ end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be Downloaded from observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology. The Journal of Immunology, 2000, 164: 3169–3176. he leukocyte-specific transcript (LST1)3 gene is encoded whereas TNFd4 was negatively associated with rejection of renal http://www.jimmunol.org/ on the short arm of chromosome 6 within the designated transplants, implicating a role of LST1 in the immune response (9, T MHC class IV in the TNF complex (1–4). The MHC class 10). IV region spans the telomeric part of the former MHC III complex, Northern blot analysis has shown constitutive expression of the and it contains a high concentration of genes that may play a role LST1 cDNA in T cells, macrophages, and U937 cells and strong in various aspects of stress, inflammation, or infection (4). The induction of transcription by stimulation of monocytic cell lines LST1 gene is encoded ϳ9 kb centromeric of the TNF-␣ gene with IFN-␥ (2). Transcription was also detected in human tonsil, (TNFA), and it is flanked telomerically at a distance of 4 kb by lung, and placenta, the liver cell lines Hep G2 and Hep 3B, and by ␤ LTB, which codes for lymphotoxin and forms a heterotrimer means of expression-tagged sequences in fetal liver/spleen and by guest on September 28, 2021 with TNF-␤ (2, 5). Centromerically, the LST1 gene is flanked by adult brain. Because the hybridization signal of ϳ800 nucleotides the 1C7 gene which is located immediately adjacent to LST1, en- is very broad in the Northern blot analysis, a variation in length of coded by the opposite DNA strand such that the 3Ј ends of the two the LST1 mRNA has been suspected. In fact, previous studies have mRNA templates come within a few bases of overlapping (6). identified four protein-encoding exons and five alternative noncod- Several polymorphisms have been identified thus far within the ing exons 1 leading to eight different transcripts expressed in var- LST1 gene: intron 4 encompasses the polymorphic microsatellites ious cell lines which encode five different proteins (2, 11). TNFd and TNFe; and a polymorphic PvuII site is located down- To characterize the complex LST1 expression pattern, we ana- stream of the LST1 polyadenylation signal that is linked to 1C7 (2, lyzed LST1 transcription and protein expression in freshly isolated 7, 8). The d3 allele of TNFd has been associated with severe grade PBMC, T cells, and B cells and after incubation of these cells with acute graft-vs-host disease in HLA-identical sibling transplants, various cytokines. We describe essential parts of the biological function of two LST1 protein isoforms representing the two groups of LST1 polypeptides. *Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Luxembourg, Luxembourg; †Department of Internal Medicine, Innsbruck University Hospital, Inns- bruck, Austria; ‡Department of Immunology and Allergology, Centre Hospitalier, Materials and Methods Luxembourg, Luxembourg; §Institute for Biochemistry, University of Innsbruck, Innsbruck, Austria; ¶Department of Hematology/Oncology, University of Leipzig, Cell separation Leipzig, Germany; and ʈInstitute for Anthropology and Human Genetics, Munich, Germany PBMC were isolated from buffy coats of healthy volunteer donors by den- sity gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). T Received for publication January 6, 1999. Accepted for publication January 6, 2000. cells, monocytes, and B cells were separated from PBMC by positive se- The costs of publication of this article were defrayed in part by the payment of page lection with the immunomagnetic bead system using the respective Dyna- charges. This article must therefore be hereby marked advertisement in accordance beads M-450 (CD4), M-450 (CD8), M-450 (CD14), and M-450 (CD19) with 18 U.S.C. Section 1734 solely to indicate this fact. (Dynal, Oslo, Norway) as described previously (12–14). After selection, 1 This work was partly supported by the Fondation de Recherche Cancer et Sang and the cells were treated with Detachabeads (Dynal) to remove the CD4, CD8, by the O¨ sterreichischer Fonds zur Fo¨rderung der Wissenschaftlichen Forschung FWF and CD19 Abs from the membrane receptors (15). The purities of the P-9671. recovered cells were 98% for CD4ϩ cells, 99% for CD8ϩ cells, 99.6% for ϩ ϩ 2 Address correspondence and reprint requests to Dr. I. Rollinger-Holzinger, Recher- CD19 cells, and 80–90% for CD14 cells. che sur le Cancer et les Maladies du Sang, Centre Universitaire, Avenue de la Faı¨encerie 162A, L-1511 Luxembourg, Luxembourg. E-mail address: Culture of dendritic cells [email protected]. 3 Abbreviations used in this paper: LST1, leukocyte-specific transcript 1; TPO, throm- Dendritic cells were generated from PBMC as described (16–18). Briefly, bopoietin; DHFR, dihydrofolate reductase; ORF, open reading frame. mononuclear cells were isolated from leukocyte-enriched buffy coats by Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 3170 LST1: COMPLEXLY SPLICED GENE WITH IMMUNOMODULATORY FUNCTION Downloaded from http://www.jimmunol.org/ FIGURE 1. LST1 expression and the control vectors. The vector pQE-16 was used for expression of the control protein DHFR. The clone ⌬LST1/C- DHFR was created by cloning ⌬LST1/C cDNA into the BglII and HindIII restriction sites of the expression vector pQE-40, which contains the N-terminal part of the DHFR protein. The expression vector pQE-30 was used to generate the ⌬LST1/A clone by cloning the BglII-restricted cDNA into the BamHI site of pQE-30. All three expression vectors encode an N-terminal histidine tail (6ϫ His). The DNA and amino acid sequences of ⌬LST1/A and ⌬LST1/C are indicated. standard density gradient centrifugation on Ficoll-Paque (Pharmacia, Upp- RNA isolation and RT-PCR by guest on September 28, 2021 sala, Sweden) and resuspended in complete medium (Biological Industries, Beth Haemek, Israel; RPMI 1640 supplemented with 10% heat-inactivated With the use of Trizol, total RNA samples were prepared from cells at each FCS (30 min, 56°C), 50 U/ml penicillin, 50 ␮g/ml streptomycin, 2.5 ␮g/ml time indicated, according to the manufacturer’s instructions. To remove ␮ Fungizone, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM nonessential traces of contaminating DNA, 2 g of total RNA were digested with 1 IU amino acids, 1 mM pyruvate, and 5 ϫ 10Ϫ5 M 2-ME), and 5.0 ϫ 107 DNase (Promega, Madison, WI) for 1 h, phenolized, and precipitated as PBMCs were allowed to adhere in 75-cm2 cell culture flasks (2 h, 37°C). described previously (19). One reverse transcription reaction for every cell ␮ Nonadherent cells were removed, and the adherent fraction of the mono- stimulation was performed with 2 g RNA using avian myeloblastosis nuclear cells was allowed to detach during overnight incubation in com- virus reverse transcriptase, oligo(dT)15, and RNasin (all purchased from plete medium in the absence of exogenous cytokines. These cells were Promega) for1hat37°C (19). The revealed cDNA was used for all PCR replated at high cell density (5 ϫ 106 cells/ml) resulting in rapid readher- amplifications of the respective cell stimulations and control amplifications ence.