The Tolerogenic Function of Annexins on Apoptotic Cells Is Mediated by the Annexin Core Domain This information is current as Björn Linke, Lucie Abeler-Dörner, Veronika Jahndel, of September 28, 2021. Alexandra Kurz, Andrea Mahr, Sandra Pfrang, Linda Linke, Peter H. Krammer and Heiko Weyd J Immunol published online 27 April 2015 http://www.jimmunol.org/content/early/2015/04/25/jimmun ol.1401299 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/04/25/jimmunol.140129 Material 9.DCSupplemental 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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2015, doi:10.4049/jimmunol.1401299 The Journal of Immunology The Tolerogenic Function of Annexins on Apoptotic Cells Is Mediated by the Annexin Core Domain Bjo¨rn Linke, Lucie Abeler-Do¨rner,1 Veronika Jahndel,2 Alexandra Kurz, Andrea Mahr,3 Sandra Pfrang, Linda Linke,4 Peter H. Krammer, and Heiko Weyd Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow–derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Downloaded from Anx-expressing ACs prevented induction of Ag-specific CD8+ T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders. The Journal of Immunology, 2015, 194: 000–000. ne crucial mechanism of peripheral tolerance induction is AnxA1 belongs to an evolutionarily conserved family of proteins http://www.jimmunol.org/ the uptake, processing, and presentation of apoptotic cell that binds to negatively charged phospholipids in a Ca2+-dependent O (AC)-derived Ags by professional phagocytes (1). In the manner (12). Lipid binding is mediated by the C-terminal core steady-state, immature dendritic cells (DCs) continuously engulf domain, which is highly conserved among all Anx family mem- ACs and present self-Ags from peripheral tissues in the draining bers (12, 13). The N-terminal domain is unique for each Anx and lymph nodes (2, 3). In the absence of inflammation or infection, the is proposed to mediate specific functions of individual Anxs (12, presentation of self-Ags to naive T cells induces tolerance (4–6). 14). Peptides corresponding to the AnxA1 N terminus were shown Defects in AC uptake, AC processing, or tolerization of phagocytes to bind to members of the N-formyl peptide receptor (FPR) family, after AC uptake have been implicated in the development of au- resulting in a reduction of neutrophil transmigration in several toimmune diseases and chronic inflammatory disorders (7–10). models of acute and chronic inflammation (15–18). Downstream by guest on September 28, 2021 Although a plethora of eat-me signals and their uptake receptors on signaling induced by binding of AnxA1 N-terminal peptides to FPR phagocytes are well characterized, little is known about molecules family members causes activation of ERK but not of p38 or JNK (19, on ACs that mediate DC tolerization. Recently, we identified the 20). Additional anti-inflammatory properties of AnxA1 have been early exposure of the cytosolic protein annexin (Anx)1 (AnxA1) as attributed to the inhibition of cytosolic phospholipase A2 (cPLA2) a tolerogenic signal on the surface of ACs (11). and secretory phospholipase A2 (sPLA2) activity (21–23). Inhibition of sPLA2 is mediated by the antiflammin-2 (AF-2) sequence, which also negatively regulates polymorphonuclear leukocyte adhesion to Division of Immunogenetics, Tumor Immunology Program, German Cancer Re- search Center, 69120 Heidelberg, Germany activated endothelium by binding to human FPR2 (24). 1 The presence of multiple Anx family members in all higher Current address: Peter Gorer Department of Immunobiology, King’s College Lon- don, London, U.K. eukaryotes suggests a fundamental role for Anxs in cell biology. 2Current address: BioNTech, Mainz, Germany. However, mice deficient in individual Anx family members have 3Current address: immatics biotechnologies, Tubingen,€ Germany. no severe phenotype, suggesting that several Anxs have (partially) 4 overlapping functions (12, 25). In fact, functional redundancy of Current address: Division of Pediatric Neurooncology, German Cancer Research Center, Heidelberg, Germany. Anxs was proved in the context of membrane trafficking, inhibition Received for publication May 20, 2014. Accepted for publication March 30, 2015. of phospholipase A2 activity, and blood coagulation (12). Whether Address correspondence and reprint requests to Dr. Heiko Weyd and Dr. Peter H. tolerance induction, which we showed to be mediated by AnxA1, is Krammer, Division of Immunogenetics, Tumor Immunology Program, German Cancer a property shared by several Anx family members and, therefore, Research Center, Im Neuenheimer Feld 280, Heidelberg, Baden-Wurttemberg€ 69120, represents an additional redundant function of Anxs is unknown (11). Germany. E-mail addresses: [email protected] (H.W.) and [email protected] (P.H.K.) In this article, we describe two novel tolerogenic signals on the The online version of this article contains supplemental material. surface of ACs, AnxA5 and AnxA13, promoting the development of Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AC, apoptotic DCs with a tolerogenic phenotype (i.e., DCs with an impaired cell; AF-2, antiflammin-2; Anx, annexin; aS2 AnxA1, AnxA1-expressing apoptotic S2 cell; aS2 AnxA5, AnxA5-expressing apoptotic S2 cell; aS2 AnxA13, AnxA13- upregulation of costimulatory molecules and with low proin- expressing apoptotic S2 cell; aS2 mock, mock-transfected apoptotic S2 cell; aS2 flammatory cytokine secretion upon TLR stimulation). These mOVA, apoptotic, membrane-anchored OVA–expressing S2 cell; BM, bone marrow; effects were mediated by the Anx core domain and were inde- BMDC, BM-derived DC; cPLA2, cytosolic phospholipase A2; DC, dendritic cell; FPR, N-formyl peptide receptor; lpr, lymphoproliferation; m, mouse; mOVA, pendent of N-terminal peptides of AnxA1 or the AF-2 sequence. In membrane-anchored OVA; o/n, overnight; PD-L, programmed cell death ligand; line with this, AnxA1, AnxA5, and AnxA13 inhibit the induction sPLA , secretory phospholipase A ; S2, Schneider 2; WT, wild-type. 2 2 of Ag-specific CD8+ T cell responses in vivo. Importantly, mice Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 immunized with AnxA5- or AnxA13-expressing ACs showed www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401299 2 ANNEXINS ARE REDUNDANT TOLEROGENIC SIGNALS ON ACs a significantly reduced population of CD8+ T cells specific for AC- into a modified version of pET41a harboring a C-terminal FLAG tag, a associated Ags and became refractory to an antigenic challenge. PreScission Protease cleavage site, and a protein A tag. Recombinant proteins Based on these results, we propose that several Anxs contribute to wereexpressedintheEscherichia coli strain BL21 (DE3) pLysS (Promega). Removal of LPS during protein purification was achieved by washing with AC-induced immunosuppression in a functionally redundant man- TBS containing 0.1% Triton X-114 (Sigma-Aldrich). Residual LPS concen- ner and, thus, contribute to peripheral tolerance induction. trations in the protein preparations were determined by the Limulus Amebocyte Lysate Assay (Lonza). The mAnxA1-pAc5.1 V5/HisA, mAnxA5- pAc5.1 V5/HisA, and mAnxA13 pAc5.1/V5-HisA plasmids were gen- Materials and Methods erated by cloning mAnxA1, mAnxA5, and mAnxA13, respectively, Mice into pAc5.1/V5-HisA (Life Technologies). Drosophila melanogaster Tlr42/2, AnxA12/2, wild-type (WT), OT-I, and lymphoproliferation (lpr) Schneider 2 (S2) cells were transfected with Ca3(PO4)2,accordingtothe mice (all on a C57BL/6 background) were maintained and bred under manufacturer’s instructions (Life Technologies). To generate