The Ubiquitin-like Modifier FAT10 Is Selectively Expressed in Medullary Thymic Epithelial Cells and Modifies T Cell Selection This information is current as Stefanie Buerger, Valerie L. Herrmann, Sarah Mundt, Nico of September 23, 2021. Trautwein, Marcus Groettrup and Michael Basler J Immunol published online 23 September 2015 http://www.jimmunol.org/content/early/2015/09/23/jimmun ol.1500592 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/09/23/jimmunol.150059 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 23, 2015, doi:10.4049/jimmunol.1500592 The Journal of Immunology The Ubiquitin-like Modifier FAT10 Is Selectively Expressed in Medullary Thymic Epithelial Cells and Modifies T Cell Selection Stefanie Buerger,* Valerie L. Herrmann,* Sarah Mundt,* Nico Trautwein,† Marcus Groettrup,*,‡ and Michael Basler*,‡ HLA-F adjacent transcript 10 (FAT10) is a cytokine-inducible ubiquitin-like modifier that is highly expressed in the thymus and directly targets FAT10-conjugated proteins for degradation by the proteasome. High expression of FAT10 in the mouse thymus could be assigned to strongly autoimmune regulator–expressing, mature medullary thymic epithelial cells, which play a pivotal role in negative selection of T cells. Also in the human thymus, FAT10 is localized in the medulla but not the cortex. TCR Vb- segment screening revealed a changed T cell repertoire in FAT10-deficient mice. Analysis of five MHC class I– and II–restricted Downloaded from TCR-transgenic mice demonstrated an altered thymic negative selection in FAT10-deficient mice. Furthermore, the repertoire of peptides eluted from MHC class I molecules was influenced by FAT10 expression. Hence, we identified FAT10 as a novel modifier of thymic Ag presentation and epitope-dependent elimination of self-reactive T cells, which may explain why the fat10 gene could recently be linked to enhanced susceptibility to virus-triggered autoimmune diabetes. The Journal of Immunology, 2015, 195: 000–000. http://www.jimmunol.org/ he maturation of T cells in the thymus is a highly or- Ubiquitin-like modifiers (ULM) posttranslationally modify chestrated process. Somatic TCR gene rearrangements of cellular targets in diverse biological pathways in analogy to the T several distinct gene segments called variable, joining, ubiquitin system. The covalent modification with the cytokine- and diversity leads to unique TCRs expressed on individual inducible ULM HLA-F adjacent transcript 10 (FAT10) tar- T lymphocytes. These developing T cells interact via their TCR gets proteins in a ubiquitin-independent manner for proteasomal with self-peptide–MHC complexes that are displayed by thymic degradation (3–5). FAT10 is conjugated to its substrates via APCs. In a process called positive selection, immature double- isopeptide-linkage mediated by an E1, E2, and possibly E3 positive (DP) thymocytes (CD4+CD8+) that express TCRs with enzyme cascade, in which UBA6 and UBA6-specific E2 en- intermediate avidity for self-peptide–MHC complexes are selected zyme (6, 7) serve as E1-type activating and E2-type conjugating by guest on September 23, 2021 to differentiate into mature single-positive (SP) thymocytes (CD4+ enzymes, respectively (6, 8, 9). The insight into the biological 2 2 CD8 or CD4 CD8+). Selected thymocytes that express TCRs function of FAT10 is still in its infancy. So far, FAT10 has been with high affinity for self-Ags and self-MHC are intrathymically implicated in multiple cellular processes like apoptosis (4, 10), eliminated during negative selection to produce a self-tolerant T cell spindle checkpoint control during mitotic cell cycle (11), and repertoire (1). Mature medullary thymic epithelial cells (mTECs) NF-kB activation (12). Basal FAT10 expression is most prom- contribute to self-tolerance through the promiscuous expression of inent in organs of the immune system, like thymus, fetal liver, tissue-specific Ags in the thymus, which is mainly controlled by the lymph nodes, and spleen (13, 14). In addition, expression of transcription factor autoimmune regulator (Aire) (2). FAT10 can be synergistically induced by the proinflammatory cytokines IFN-g and TNF-a (15), and it is upregulated during *Division of Immunology, Department of Biology, University of Konstanz, D-78457 dendritic cell (DC) maturation (14). The fusion of FAT10 to the Konstanz, Germany; †Department of Immunology, Interfaculty Institute for Cell N termini of two different viral Ags strongly accelerated their Biology, University of Tubingen,€ D-72076 Tubingen,€ Germany; and ‡Biotechnology Institute Thurgau at the University of Konstanz, CH-8280 Kreuzlingen, Switzerland degradation by the proteasome and enhanced the presentation of their T cell epitopes on MHC class I (MHC-I) molecules Received for publication March 10, 2015. Accepted for publication August 25, 2015. (16–18). These results suggest that linkage to FAT10 can feed This work was supported by German Research Foundation Grant GR 1517/10-2 and the Collaborative Research Center SFB969 (Project C01) (to M.G.). Ags into the MHC-I Ag processing pathway. Moreover, FAT10 Address correspondence and reprint requests to Dr. Michael Basler and Prof. Marcus binds to the autophagy adaptor p62 (19) and colocalizes in Groettrup, Division of Immunology, Department of Biology, University of Konstanz, cells with p62 and the autophagosome marker LC3B (20), Universitaetsstrasse 10, D-78457 Konstanz, Germany. E-mail addresses: michael. which opens the possibility that FAT10-conjugated proteins [email protected] (M.B.) and [email protected] (M.G.) may be processed for MHC class II (MHC-II)–mediated presen- The online version of this article contains supplemental material. tation by targeting them to autophagosomes. Analysis of FAT10- Abbreviations used in this article: Adig, autoimmune regulator–driven Igrp-Gfp; deficient mice demonstrated an enhanced sensitivity toward Aire, autoimmune regulator; cDC, conventional DC; cTEC, cortical thymic 2/2 epithelial cell; DC, dendritic cell; DN, double-negative; DP, double-positive; EpCAM, endotoxin challenge (13). Additionally, FAT10 mice have epithelial cellular adhesion molecule; FAT10, HLA-F adjacent transcript 10; GP, an extended lifespan and reduced adiposity compared with glycoprotein; HEK, human embryonic kidney; HPRT, hypoxanthine-guanine phosphoribosyltransferase; LCMV, lymphocytic choriomeningitis virus; MHC-I, wild-type mice (21). MHC class I; MHC-II, MHC class II; mTEC, medullary thymic epithelial cell; In this study, we could assign the previously recognized high pDC, plasmacytoid DC; SP, single-positive; TEC, thymic epithelial cell; tg, trans- FAT10 expression in the thymus (14) to terminally differenti- genic; ULM, ubiquitin-like modifier. ated mTECs. Thereby, FAT10 influenced the selection of thy- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 mocytes probably due to negative selection and altered peptide www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500592 2 FAT10 CODETERMINES THYMIC T CELL SELECTION presentation. Hence, we describe a previously unknown function temperature followed by incubation with primary Abs in 5% (w/v) BSA/ of FAT10 in modifying thymic T cell selection. PBS overnight at 4˚C. After washing three times with PBS/0.1% (v/v) Tween 20, the secondary Abs were diluted in 5% (w/v) BSA/PBS and incubated for 1 h at room temperature. Sections were mounted using Materials and Methods Kaiser’s glycerol gelatin (Merck) or Mowiol. All images were obtained Mice, viruses, and media using a point laser-scanning microscope Zeiss LSM 510 Meta (Carl Zeiss) at the Bioimaging Center of the University of Konstanz. Image analysis C57BL/6 mice (H-2b) were originally purchased from Charles River 2/2 was performed using ImageJ software (National Institutes of Health, Laboratories (Sulzfeld, Germany). FAT10 mice (13) were kindly pro- Bethesda, MD). The following reagents were used: monoclonal mouse vided by A. Canaan and S. M. Weissman (Yale University School of anti-human FAT10 (4F1) was purified from hybridoma supernatant Medicine, New Haven, CT). P14 mice (transgenic [tg] line 318) (22) were (19). Rat anti-human Aire (TM-724) was purchased from eBioscience obtained from Dr. Oliver Planz (Tubingen€ University, Tubingen,€ Ger- (Frankfurt, Germany). Polyclonal rabbit anti-Cytokeratin 5 was purchased many). OT-I (23), OT-II (24), and Smarta mice (25), were provided by the from Covance (purchased via Hiss Diagnostic, Freiburg, Germany). Mu- Swiss Immunological Mutant Mouse Repository. HY TCR-tg mice (26) rine thymic cortex was stained with mouse anti-cytokeratin 18 (RGE53;