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Core DomainApoptotic Cells Is Mediated by the Annexin The Tolerogenic Function of Annexins on

Peter H. Krammer and Heiko WeydAlexandra Kurz, Andrea Mahr, Sandra Pfrang, Linda Linke, Björn Linke, Lucie Abeler-Dörner, Veronika Jahndel,

http://www.jimmunol.org/content/194/11/5233doi: 10.4049/jimmunol.1401299April 2015;

2015; 194:5233-5242; Prepublished online 27J Immunol 

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9.DCSupplemental.htmlhttp://www.jimmunol.org/content/suppl/2015/04/25/jimmunol.140129

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The Journal of Immunology

The Tolerogenic Function of Annexins on Apoptotic Cells IsMediated by the Annexin Core Domain

Bjorn Linke, Lucie Abeler-Dorner,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

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: 5233–5242.

Onecrucial mechanism of peripheral tolerance induction isthe uptake, processing, and presentation of apoptotic cell(AC)-derived Ags by professional phagocytes (1). In the

steady-state, immature dendritic cells (DCs) continuously engulfACs and present self-Ags from peripheral tissues in the draininglymph nodes (2, 3). In the absence of inflammation or infection, thepresentation of self-Ags to naive T cells induces tolerance (4–6).Defects in AC uptake, AC processing, or tolerization of phagocytesafter AC uptake have been implicated in the development of au-toimmune diseases and chronic inflammatory disorders (7–10).Although a plethora of eat-me signals and their uptake receptors onphagocytes are well characterized, little is known about moleculeson ACs that mediate DC tolerization. Recently, we identified the

early exposure of the cytosolic protein annexin (Anx)1 (AnxA1) asa tolerogenic signal on the surface of ACs (11).AnxA1 belongs to an evolutionarily conserved family of proteins

that binds to negatively charged phospholipids in a Ca2+-dependentmanner (12). Lipid binding is mediated by the C-terminal coredomain, which is highly conserved among all Anx family mem-bers (12, 13). The N-terminal domain is unique for each Anx andis proposed to mediate specific functions of individual Anxs (12,14). Peptides corresponding to the AnxA1 N terminus were shownto bind to members of the N-formyl peptide receptor (FPR) family,resulting in a reduction of neutrophil transmigration in severalmodels of acute and chronic inflammation (15–18). Downstreamsignaling induced by binding of AnxA1 N-terminal peptides to FPRfamily members causes activation of ERK but not of p38 or JNK (19,20). Additional anti-inflammatory properties of AnxA1 have beenattributed to the inhibition of cytosolic phospholipase A2 (cPLA2)and secretory phospholipase A2 (sPLA2) activity (21–23). Inhibitionof sPLA2 is mediated by the antiflammin-2 (AF-2) sequence, whichalso negatively regulates polymorphonuclear leukocyte adhesion toactivated endothelium by binding to human FPR2 (24).The presence of multiple Anx family members in all higher

eukaryotes suggests a fundamental role for Anxs in cell biology.However, mice deficient in individual Anx family members haveno severe phenotype, suggesting that several Anxs have (partially)overlapping functions (12, 25). In fact, functional redundancy ofAnxs was proved in the context of membrane trafficking, inhibitionof phospholipase A2 activity, and blood coagulation (12). Whethertolerance induction, which we showed to be mediated by AnxA1, isa property shared by several Anx family members and, therefore,represents an additional redundant function of Anxs is unknown (11).In this article, we describe two novel tolerogenic signals on the

surface of ACs, AnxA5 and AnxA13, promoting the development ofDCs with a tolerogenic phenotype (i.e., DCs with an impairedupregulation of costimulatory molecules and with low proin-flammatory cytokine secretion upon TLR stimulation). Theseeffects were mediated by the Anx core domain and were inde-pendent of N-terminal peptides of AnxA1 or the AF-2 sequence. Inline with this, AnxA1, AnxA5, and AnxA13 inhibit the induction

Division of Immunogenetics, Tumor Immunology Program, German Cancer Re-search Center, 69120 Heidelberg, Germany

1Current address: Peter Gorer Department of Immunobiology, King’s College Lon-don, London, U.K.

2Current address: BioNTech, Mainz, Germany.

3Current address: immatics biotechnologies, T€ubingen, Germany.

4Current address: Division of Pediatric Neurooncology, German Cancer ResearchCenter, Heidelberg, Germany.

Received for publication May 20, 2014. Accepted for publication March 30, 2015.

This work was supported in part by the Helmholtz Alliance on Immunotherapy.

Address correspondence and reprint requests to Dr. Heiko Weyd and Dr. Peter H.Krammer, Division of Immunogenetics, Tumor Immunology Program, German CancerResearch Center, Im Neuenheimer Feld 280, Heidelberg, Baden-W€urttemberg 69120,Germany. E-mail addresses: h.weyd@dkfz.de (H.W.) and p.krammer@dkfz.de (P.H.K.)

The online version of this article contains supplemental material.

Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AC, apoptoticcell; AF-2, antiflammin-2; Anx, annexin; aS2 AnxA1, AnxA1-expressing apoptoticS2 cell; aS2 AnxA5, AnxA5-expressing apoptotic S2 cell; aS2 AnxA13, AnxA13-expressing apoptotic S2 cell; aS2 mock, mock-transfected apoptotic S2 cell; aS2mOVA, apoptotic, membrane-anchored OVA–expressing S2 cell; BM, bone marrow;BMDC, BM-derived DC; cPLA2, cytosolic phospholipase A2; DC, dendritic cell;FPR, N-formyl peptide receptor; lpr, lymphoproliferation; m, mouse; mOVA,membrane-anchored OVA; o/n, overnight; PD-L, programmed cell death ligand;sPLA2, secretory phospholipase A2; S2, Schneider 2; WT, wild-type.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

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of Ag-specific CD8+ T cell responses in vivo. Importantly, miceimmunized with AnxA5- or AnxA13-expressing ACs showeda significantly reduced population of CD8+ T cells specific for AC-associated Ags and became refractory to an antigenic challenge.Based on these results, we propose that several Anxs contribute toAC-induced immunosuppression in a functionally redundant man-ner and, thus, contribute to peripheral tolerance induction.

Materials and MethodsMice

Tlr42/2, AnxA12/2, wild-type (WT), OT-I, and lymphoproliferation (lpr)mice (all on a C57BL/6 background) were maintained and bred underspecific pathogen–free conditions. Tlr42/2 mice were kindly provided byS. Akira (Osaka University, Osaka, Japan), S. Uematsu (Osaka University),and L. Gissmann (German Cancer Research Center). AnxA12/2 mice werepurchased from B & K Universal. OT-I mice were kindly providedby N. Garbi (University of Bonn, Bonn, Germany) and G. Hammerling(German Cancer Research Center).

Preparation of bone marrow–derived dendritic cells

Bone marrow (BM)-derived DCs (BMDCs) were prepared from male orfemale mice between 6 and 12 wk of age, as described (26, 27). In brief, BMcells were flushed from tibias and femurs, and RBCs were lysed by briefexposure to 0.168 M NH4Cl. Cells were washed twice with RPMI 1640.For differentiation of BM precursors to BMDCs using recombinant murineGM-CSF, 1 3 106 cells were seeded at a density of 1 3 106 cells/ml inRPMI 1640 complete medium (10% FCS, 10 U/ml penicillin/streptomycin,300 mg/l L-glutamine, 20 ng/ml GM-CSF [Immunotools]) in a 24-well plate.After 2 d, the medium was replaced by fresh medium. After 4 d, half of themedium was removed and replaced by fresh medium. Experiments wereconducted 7–8 d after differentiation. For differentiation of BM precursors toBMDCs using recombinant human Flt3L, cells were seeded at a density of 33106 cells/ml in RPMI 1640 complete medium (10% FCS, 10 U/ml penicillin/streptomycin, 300 mg/l L-glutamine, 300 ng/ml Flt3L [eBioscience]) in a 100-mm petri dish for 8–10 d. Additional Flt3L was added to the culture at day 5 or6. Differentiation of BM precursors to BMDCs was monitored by flow cyto-metric analysis of cells using mAbs against CD11c and MHC class II.

Flow cytometry

mAbs and reagents used for FACS were purchased from BD Biosciences(anti-CD3–FITC, anti-CD4–FITC, anti-CD8–PE, anti-CD8–allophycocyanin,anti-CD40–PE, anti-CD45R–PerCP–Cy5.5, anti-CD86–PE, anti-Thy1.1–PE,and streptavidin-allophycocyanin), eBioscience (anti-CD14–allophycocyanin,anti-CD44–PerCP–Cy5.5, anti-CD80–FITC, anti-PD-L1–PE, anti-PD-L2–FITC), Caltag (anti-CD11c–PE, anti-MHC class II–FITC), Immuno-tools (anti-CD62L–PE), or QIAGEN (anti-His–biotin). Anti-human AnxA1(DAC5) and anti-MHC class I–biotin (Y3) Abs were generated in our labo-ratory and the laboratory of G. Hammerling (11). Appropriate isotype controlswere purchased from BD Biosciences or eBioscience. Phosphatidylserineexposure and cellular membrane integrity were analyzed by staining withAnxV-FITC (Immunotools) and 7-aminoactinomycin D (7-AAD; Sigma-Aldrich), respectively. Stained cells were analyzed on a FACSCanto (BDBiosciences) with FlowJo software (TreeStar). For detection of FPR familymembers, cells were stained with the fluorescently labeled FPR ligandWKYMVM-FAM (Phoenix Pharmaceuticals) or Formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (Life Technologies).

Abs and reagents

Anti-murine AnxA1 (8D10) and anti-human AnxA1 (DAC5) Abs weregenerated in our laboratory (11). Commercially available Abs for detection ofMAPK, a-tubulin, and b-actin were purchased as follows: AnxA5 (AF399;R&D Systems), AnxA13 (AF4149; R&D Systems), p-ERK (E10; CellSignaling), ERK1 (MK12; BD), p-JNK (G9; Cell Signaling), JNK1/3 (C17;Santa Cruz Biotechnology), p-p38 (D3F9; Cell Signaling), p38a (5F11; CellSignaling), a-tubulin (B-5-1-2; Sigma-Aldrich), and b-actin (AC-15;Abcam). Cytokine concentrations in supernatants were determined byELISA for murine TNF-a, IL-12p40, IL-6, and IL-10 (PeproTech),according to the manufacturers’ instructions. Boc-1 and Boc-2 were pur-chased from MP Biomedicals. fMLF was purchased from Sigma-Aldrich.

Plasmid constructions, cell culture, and transfections

The mouse (m)AnxA1-pET41a, mAnxA1 mAF-2-pET41a, mAnxA1 DN-pET41a, mAnxA1 DN mAF-2-pET41a, mAnxA5-pET41a, and mAnxA13-pET41a plasmids were generated by cloning of mAnxA1, mAnxA1 mAF-2,

mAnxA1 DN, mAnxA1 DN mAF-2, mAnxA5, or mAnxA13, respectively,into a modified version of pET41a harboring a C-terminal FLAG tag, aPreScission Protease cleavage site, and a protein A tag. Recombinant proteinswere expressed in the Escherichia coli strain BL21 (DE3) pLysS (Promega).Removal of LPS during protein purification was achieved by washing withTBS containing 0.1% Triton X-114 (Sigma-Aldrich). Residual LPS concen-trations in the protein preparations were determined by the LimulusAmebocyte Lysate Assay (Lonza). The mAnxA1-pAc5.1 V5/HisA, mAnxA5-pAc5.1 V5/HisA, and mAnxA13 pAc5.1/V5-HisA plasmids were gen-erated by cloning mAnxA1, mAnxA5, and mAnxA13, respectively,into pAc5.1/V5-HisA (Life Technologies). Drosophila melanogasterSchneider 2 (S2) cells were transfected with Ca3(PO4)2, according to themanufacturer’s instructions (Life Technologies). To generate stablytransfected S2 cell lines, cotransfection with pCoHygro was performed,and cells were selected using Hygromycin B (PAA). D. melanogaster S2cells were cultured in Schneider’s insect medium (Sigma-Aldrich) sup-plemented with 10% FCS. Human Jurkat T cells and the human T-ALLcell line CEM were cultured in RPMI 1640 medium (Sigma-Aldrich)supplemented with 10% FCS.

RNA isolation and quantitative RT-PCR

RNAwas isolated from cells using the RNAqueous-Micro Kit, according tothe manufacturer’s instructions (Ambion). RNA was quantified by detec-tion of SYBR Green incorporation using the ABI Prism 7500 sequencedetector system (Applied Biosystems). Expression levels were normalizedto GAPDH. The following primer sequences were used: Fpr1 59-GCCGTC ACC ATG CTC ACT GTC A-39 (fwd) and 59-AAC CCG CAA AGGACG GCT GG-39 (rev); Fpr2 59-AGC TGG TTG TGC AGA CAA AATGGA-39 (fwd) and 59-TGC CCA GCA CAC CAA GGA AG-39 (rev); Fpr359-CCT TCC CGA GTT CTT ACA GG-39 (fwd) and 59-CAC TAA ACTGCATCT CTT TGAG-39 (rev); and GAPDH 59-ACT CCACTC ACG GCAAAT TCA-39 (fwd) and 59-GCC TCA CCC CAT TTG ATG TT-39 (rev).

Isolation of murine primary neutrophils and splenocytes

Murine neutrophils were isolated from BM by positive selection using anti-Ly6G MicroBeads (Miltenyi Biotec), according to the manufacturer’sinstructions. Neutrophils were cultured in RPMI 1640 medium supple-mented with 10% FCS at a density of 2 3 106 cells/ml. The purity of cellswas controlled by flow cytometric analysis using anti-Gr-1–FITC (Milte-nyi Biotec). Cells were .90% positive for Gr-1. Murine splenocytes wereisolated from spleens and filtered through a 40-mm cell strainer (BDBiosciences). Splenocytes were cultured in RPMI 1640 medium supple-mented with 10% FCS.

Preparation of ACs

To generate early ACs, Jurkat T cells or CEM cells were UV-C irradiated(50 mJ/cm2, Stratalinker 1800; Stratagene) in six-well plates at a cell densityof 1 3 106 cells/ml. Subsequently, cells were cultured at 37˚C for 2–6 h. Togenerate early apoptotic splenocytes, cells were UV-C irradiated (15 mJ/cm2)in six-well plates at a cell density of 13 106 cells/ml. Subsequently, cells werecultured in media at 37˚C for 3 h. Primary murine neutrophils spontaneouslyundergo apoptosis in cell culture and show characteristics of early ACs after1 d. S2 cells were irradiated with 250 mJ/cm2 at a cell density of 13 106 cells/ml in 100-mm petri dishes and used after overnight (o/n) incubation.

In vivo experiments

For analysis of OVA-specific, endogenous T cells, a mixture of 5 3 106

apoptotic, membrane-anchored OVA (mOVA)-expressing S2 cells (aS2mOVA) together with 5 3 106 AnxA1-, AnxA5-, or AnxA13-expressing,apoptotic S2 cells (aS2 AnxA1, aS2 AnxA5, and aS2 AnxA13, respectively)or mock-transfected apoptotic S2 cells (aS2 mock) were injected i.v. intofemale C57BL/6 WT mice. Six to eight days after immunization, single-cellsuspensions of mesenteric lymph nodes were analyzed for OVA-specificCD8+ T cells using PE-labeled Kb/SIINFEKL pentamers, according to themanufacturer’s instructions (ProImmune). For the analysis of OVA-specificT cell proliferation and activity, 1 3 106 CFSE-labeled OT-I T cells wereinjected i.v. into female C57BL/6 WT mice. The next day, mice wereimmunized i.v. with a mixture of 0.5 3 106 aS2 mOVA, together with1 3 106 aS2 AnxA5, aS2 AnxA13, or aS2 mock. After 12–13 d, mice werechallenged i.p. with 50 mg OVA protein (InvivoGen) in 125 ml PBSemulsified in 125 ml IFA (Sigma-Aldrich). On day 6 after challenge,splenic cell suspensions were analyzed by flow cytometry and preparedfor ELISPOT assays in vitro. For determination of absolute OT-I T cellnumbers/spleen, we analyzed an exact aliquot of each spleen usingTrucount Beads (Becton Dickinson), according to the manufacturer’sinstructions.

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ELISPOT analysis

IFN-g ELISPOT assays were performed with 1.5 3 106 splenic cells/welland 1 mM the OVA-derived, MHC class I–dependent peptide SIINFEKL(Axxora). Splenic cell suspensions were stimulated o/n, and IFN-g ELI-SPOTs were developed according to the manufacturer’s instructions (BDPharmingen).

Coculture of BMDCs and ACs or pretreatment withrecombinant proteins

A total of 1 3 105 BMDCs was incubated with recombinant protein (125–500 nM) or with apoptotic Jurkat T cells (2–4 3 105 cells), apoptoticneutrophils (13 105–13 106 cells), apoptotic splenocytes (13 105–13 106

cells), or apoptotic S2 cells (5 3 105–1 3 106) for 6–8 h. Depending on themouse background, BMDCs were subsequently stimulated with LPS(Sigma-Aldrich), CpG 1668, or CpG 2395 (InvivoGen). Cytokine concen-trations in the supernatants were analyzed by ELISA 12–16 h after TLRstimulation. Surface molecule expression was analyzed 48 h after TLRstimulation by flow cytometry.

Statistical analysis

Statistical analysis of data was performed by one-way ANOVA, followed bythe Bonferroni posttest for multiple comparisons. If not otherwise indicated,

the significance of the difference compared with CpG-stimulated cells isdepicted. The p values , 0.05 were considered statistically significant.

Ethics statement

All animal studies were approved by the veterinary authorities (Regier-ungsprasidium Karlsruhe) of Baden-W€urttemberg (G-96 06, G-173/11).

ResultsThe tolerogenic effect of AnxA1 is mediated by its core domain

We previously showed that AnxA1 on the surface of ACs negativelyregulates DC activation, as evidenced by low expression of co-stimulatory molecules and low proinflammatory cytokine se-cretion upon TLR stimulation (11). To investigate the extent towhich known anti-inflammatory sequences of AnxA1—the N ter-minus and the AF-2 sequence—contribute to this tolerogenic effect,we generated recombinant full-length AnxA1 and AnxA1 mutants(Fig. 1A, Supplemental Fig. 1A–D). LPS was carefully removedduring purification of recombinant proteins by addition of TritonX-114, and all in vitro experiments were performed using BMDCsfrom Tlr42/2 mice to exclude effects induced by endotoxin toler-

FIGURE 1. The core domain mediates the tolerogenic effect of AnxA1. (A) Full-length AnxA1 and the AnxA1 mutants AnxA1DN, AnxA1 mAF-2, and

AnxA1DN mAF-2. (B) GM-CSF–differentiated BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein. Six to eight

hours later, cells were stimulated with 40 nM CpG o/n. TNF-a concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three

independent experiments. (C) Quantification of mRNA levels of FPR1, FPR2, and FPR3 relative to GAPDH in neutrophils (NF) and GM-CSF–differentiated

BMDCs by quantitative RT-PCR. (D) Surface expression of FPR1, FPR2, and FPR3 on transiently transfected 293T or BMDCs was detected using the fluo-

rescently labeled FPR ligands fMLF-fluorescein and WKYMVM-FAM. (E) GM-CSF–differentiated BMDCs from Tlr42/2mice were incubated with the indicated

concentrations of recombinant AnxA1 in the presence or absence of the FPR antagonists Boc-1 and/or Boc-2 or apoptotic Jurkat T cells (aJ, UV-C-irradiation

50 mJ/cm2, 4 h). Six to eight hours later, cells were stimulated with 40 nM CpG o/n. TNF-a concentrations in the supernatants were determined by ELISA. Data

are mean6 SEM of three independent experiments. (F) Immunoblot analysis of MAPK activation of BMDCs after incubation with 500 nM of recombinant AnxA1

for the indicated times. Lysates of CpG- and LPS-stimulated BMDCs from Tlr42/2 and WT mice, respectively, served as positive controls. Data are representative

of three independent experiments. (G) Immunoblot analysis of MAPK activation of BMDCs after incubation with fMLF for the indicated times. Lysates of CpG-

and LPS-stimulated BMDCs from Tlr42/2 and WT mice, respectively, served as positive controls. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

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ance. In addition, a control protein, serpin 8b, purified in an iden-tical fashion as AnxA1, was inactive in our functional assays (11).Surprisingly, deletion of the N-terminal domain, mutation of theAF-2 sequence, or a combination of both did not affect AnxA1-induced inhibition of proinflammatory cytokine secretion uponstimulation with the TLR9 ligand CpG (Fig. 1B, Supplemental Fig.1E). This was not due to an absence of FPR expression, becauseFPR family members involved in AnxA1 recognition are expressedin BMDCs on the mRNA level and on the protein level (Fig. 1C,1D). To further study the involvement of FPR family members inAnxA1-induced DC tolerization, the pan-specific FPR antagonistsBoc-1 and Boc-2 were used in subsequent experiments. The presenceof Boc-1 and Boc-2 did not abrogate inhibition of TLR-inducedTNF-a secretion by AnxA1 (Fig. 1E, Supplemental Fig. 1F, 1G).In line with this, the prototypic FPR ligand fMLF, but not AnxA1, ledto activation of MAPK (Fig. 1F, 1G). In conclusion, FPR familymembers do not contribute to the recognition of AnxA1 by BMDCsin the context of suppression of DC activation upon TLR stimulation.Instead, the AnxA1 core domain is sufficient to mediate DCtolerization, independent of the AF-2 sequence and the AnxA1N-terminal domain.

Several Anx family members translocate to the cell surfaceupon induction of apoptosis

The Anx core domain is highly conserved among Anx familymembers, and redundancy was reported for various functions ofAnxs (12, 25, 28–31). Therefore, we hypothesized that severalAnxs might act as tolerogenic signals on the surface of ACs. Totest this hypothesis, we first investigated whether other Anxs, inaddition to AnxA1, translocate to the cell surface upon apoptosisinduction. We focused on AnxA5, because it is upregulated indifferent tissues of AnxA12/2 mice and might compensate for theloss of AnxA1 (32), as well as on AnxA13, which represents thefounding member of the family (33). The surface translocation ofAnxs was first studied by ectopic expression of murine Anxs inD. melanogaster S2 cells. Stably transfected S2 cells showed surfaceexposure of AnxA1, AnxA5, or AnxA13 during early apoptosiswhen membrane integrity was still intact (Fig. 2A, SupplementalFig. 2). Next, we induced apoptosis in the human T cell lineCEM and monitored translocation of endogenous Anxs. In line withthe findings observed in stably transfected S2 cells, AnxA5 andAnxA13, in addition to AnxA1, translocated to the surface ofapoptotic CEM cells (Fig. 2B, 2C). Of note, protein amounts ofAnxs in the cytosolic fraction remained almost unchanged, in-dicating that only a minor fraction of the cytosolic pool of Anxstranslocates to the cell surface during apoptosis (Fig. 2B). Insummary, AnxA1, as well as AnxA5 and AnxA13, are exposedon early ACs.

AnxA5 and AnxA13 inhibit TLR-induced proinflammatorycytokine secretion by BMDCs

Because AnxA5 and AnxA13 met the prerequisite to act as a tol-erogenic signal, surface translocation during early apoptosis, we nextstudied whether they also, like AnxA1, regulate TLR-induced DCactivation. For this, either purified recombinant proteins or stablytransfected S2 cells exposing the respective Anx upon apoptosisinduction were used (Fig. 2A, Supplemental Fig. 3A, 3B). Incu-bation of BMDCs with recombinant AnxA1, AnxA5, or AnxA13significantly reduced the secretion of the proinflammatory cyto-kines TNF-a, IL-12p40, and IL-6 by TLR-stimulated BMDCs ina concentration-dependent manner (Fig. 3A–D). Importantly,this effect was independent of altered cell viability of BMDCs(Supplemental Fig. 3C). Similar to the results obtained usingrecombinant Anxs, aS2 AnxA1 or aS2 AnxA5 reduced TLR-induced TNF-a secretion. In contrast, aS2 mock did not modu-late DC activation (Fig. 3E). Flow cytometric analysis did notreveal differences in phosphatidylserine externalization, indi-cating that apoptosis kinetics were comparable in mock- andAnx-transfected S2 cells (Supplemental Fig. 3D). The tolero-genic effect of Anxs was concentration dependent, because theinhibition of TLR-induced proinflammatory cytokine secretionincreased with the ratio of apoptotic S2 cells/BMDCs and cor-related with Anx expression levels in S2 cells (Fig. 3E, 3F,Supplemental Fig. 3E). CD8a+ DCs, which can be generated viadifferentiation of BM precursors using Flt3L in vitro, playa critical role in the presentation of self-Ags derived from ACs(34). Addition of recombinant Anxs to Flt3L-derived BMDCsreduced the TLR-induced secretion of TNF-a in a concentration-dependent manner, whereas the secretion of the anti-inflammatorycytokine IL-10 was not affected (Fig. 3G, 3H). These effectswere recapitulated with Flt3L-derived BMDCs incubatedwith aS2 AnxA1 or aS2 AnxA5. Although the secretion of theproinflammatory cytokine TNF-a was reduced, Anxs had no in-fluence on the secretion of IL-10 (Supplemental Fig. 3F, 3G). Takentogether, our data show that AnxA5 and AnxA13 inhibit the TLR-induced secretion of proinflammatory cytokines by DCs, includingCD8a+ DCs, whereas secretion of the immune-regulatory cytokineIL-10 was not affected.

AnxA5- and AnxA13-treated BMDCs show impairedupregulation of costimulatory molecules upon TLR-inducedDC activation

ACs and AnxA1 also regulate surface expression of costimulatorymolecules on DCs (11, 35, 36). Therefore, the effect of recombinantAnxA5 and AnxA13 on the expression of coregulatory and MHCmolecules was investigated 2 d after TLR stimulation. Incubation ofBMDCs with AnxA1, AnxA5, or AnxA13 did not influence the

FIGURE 2. AnxA5 and AnxA13 translocate to the cell

surface upon induction of apoptosis. (A) Flow cytometric

analysis of aS2. Sixteen hours after UV-C irradiation (250

mJ/cm2), mock- or AnxA1/5/13-transfected aS2 cells were

early apoptotic (AnxV+/7-AAD2) and stained using biotin-

conjugated anti-6xHis streptavidin-allophycocyanin. (B)

Immunoblot analysis of cytosolic and membrane (EDTA

wash) fractions of viable (0 h) or early UV-C–irradiated

apoptotic CEM cells (50 mJ/cm2, 2–6 h). (C) Quantification

of cell viability of UV-C–irradiated early apoptotic CEM

cells in (B) by flow cytometry using AnxV-FITC/7-AAD.

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surface expression of MHC class I molecules, but it slightly in-creased the expression of MHC class II molecules (Fig. 4A, 4B).Remarkably, AnxA1, AnxA5, and AnxA13 inhibited upregulationof the costimulatory molecules CD40, CD80, and CD86 uponTLR stimulation (Fig. 4C–E). In contrast, surface expression ofthe coinhibitory molecules programmed cell death ligand (PD-L)1 and PD-L2, as well as the cell viability of BMDCs, were notaffected (Fig. 4F–H). In summary, AnxA1, AnxA5, and AnxA13impair TLR-induced upregulation of costimulatory moleculeswithout affecting surface expression of coinhibitory and MHCmolecules.

FPR family members are not required for DC tolerization byAnxA5 and AnxA13

The phenotypes of BMDCs incubated with AnxA5 or AnxA13 re-semble the phenotype induced by AnxA1. Therefore, we next testedwhether recognition of the AnxA5, AnxA13, or the AnxA1 coredomain is also independent of FPR family members. Indeed, analysisof MAPK activation revealed that MAPK was not activated inBMDCs after incubation with AnxA5, AnxA13, or the AnxA1 core

domain (Fig. 5A). Furthermore, the pan-specific FPR antagonistBoc-2 did not abrogate inhibition of TLR-induced proinflammatorycytokine secretion by AnxA5, AnxA13, or the AnxA1 core domain(Fig. 5B). In conclusion, AnxA1, AnxA5, and AnxA13 induce thedevelopment of DCs with a tolerogenic phenotype, as characterizedby low expression of costimulatory molecules and low proinflam-matory cytokine secretion independent of FPR signaling.

AnxA5 and AnxA13 suppress CD8+ T cell immune responsesin vivo

Several Anxs translocate to the surface of ACs to promote thedevelopment of tolerogenic DCs, and Anx single-knockout micelack a severe phenotype. Therefore, we hypothesized that Anxs areredundant tolerogenic signals in vivo. Thus, deletion of AnxA1

should neither impair suppressive effects of ACs on AC-treated

BMDCs nor provoke autoimmunity against self-Ags derived

from ACs. In fact, the absence of AnxA1 does not lead to an altered

phenotype of BMDCs after AC engulfment. Apoptotic neutrophils

or splenocytes from WT and AnxA12/2 mice suppressed TLR-

induced TNF-a secretion by BMDCs to a comparable extent.

FIGURE 3. AnxA5 and AnxA13 inhibit proinflammatory cytokine secretion of BMDCs upon TLR stimulation. (A) AnxA1, AnxA5, and AnxA13. The

N-terminal domain is unique for each Anx family member and is depicted in yellow. The highly conserved core domain is shown in blue. (B–D) GM-CSF–

differentiated BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein. Six to eight hours later, cells were stimulated

with 40 nM CpG o/n. TNF-a (B), IL-12p40 (C), or IL-6 (D) concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three

independent experiments. (E and F) GM-CSF–differentiated BMDCs from Tlr42/2mice were incubated with the indicated ratio of apoptotic Jurkat T cells (aJ) or

aS2 AnxA1, aS2 AnxA5, or aS2 mock. Four to six hours later, cells were stimulated with 40 nM CpG o/n. TNF-a concentrations in the supernatants were

determined by ELISA. Data are mean 6 SEM of four independent experiments. (G and H) Flt3L-differentiated BMDCs from Tlr42/2 mice were incubated with

the indicated concentrations of recombinant protein. Six to eight hours later, cells were stimulated with 40 nM CpG o/n. TNF-a (G) or IL-10 (H) concentrations in

the supernatants were determined by ELISA. Data are mean 6 SEM of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

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The level of suppression increased with an elevated ratio of ACs/

BMDCs, as seen with apoptotic Jurkat T cells, which served as

a positive control (Fig. 6A, Supplemental Fig. 4A, 4B). Impor-

tantly, neutrophils and splenocytes from WT and AnxA12/2 mice

showed no difference in apoptosis kinetics upon irradiation with

UV-C (Supplemental Fig. 4C).Next, AnxA12/2 mice were analyzed for signs of autoimmunity.

CD62L/CD44 surface expression of different splenic T cell pop-

ulations was comparable between WT and AnxA12/2 mice, indi-

cating that there was no change in the activation status of peripheral

T cells. In contrast, lpr mice, which served as a positive control

for the development of systemic autoimmunity, clearly showed

enhanced activation of splenic T cells (Supplemental Fig. 4D).Furthermore, splenomegaly and lymphadenopathy were absentin 5–6-mo-old AnxA12/2 mice (Supplemental Fig. 4E). For theabovementioned investigations (Supplemental Fig. 4D, 4E), cells

FIGURE 4. AnxA5 and AnxA13 specifically downregulate the expression of costimulatory molecules on BMDCs. (A–G) GM-CSF–differentiated

BMDCs from Tlr42/2 mice were incubated with 500 nM of the indicated recombinant protein. Six to eight hours later, cells were stimulated with 20 nM

CpG. After 2 d, cells were stained with Abs against MHC I (A), MHC class II (B), CD40 (C), CD80 (D), CD86 (E), PD-L1 (F), and PD-L2 (G). Depicted are

the mean fluorescence intensity (MFI) of individual mice and the group mean. (H) Quantification of cell viability of BMDCs of mouse 1 used in (A)–(G) by

flow cytometry using AnxV-FITC/7-AAD. *p , 0.05, ****p , 0.0001.

FIGURE 5. FPR family members are not involved in the recognition of AnxA5 and AnxA13 on BMDCs. (A) Immunoblot analysis of MAPK activation

of BMDCs after incubation with 500 nM of the indicated recombinant protein for the indicated times. Lysates of CpG- and LPS-stimulated BMDCs from

Tlr42/2 and WT mice, respectively, served as positive controls. Data are representative of three independent experiments. (B) GM-CSF–differentiated

BMDCs from Tlr42/2 mice were incubated with the indicated concentrations of recombinant protein in the presence or absence of the FPR antagonist Boc-2

or apoptotic Jurkat T cells (aJ). Six to eight hours later, cells were stimulated with 40 nM CpG o/n. IL-12p40 concentrations in the supernatants were

determined by ELISA. Data are mean 6 SEM of three independent experiments. *p , 0.05, ****p , 0.0001.

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from female and male mice were used, and no gender-specificdifferences were noticed. In conclusion, the lack of autoimmu-nity in AnxA12/2 mice and the unaltered suppressive capacity ofACs from AnxA12/2 mice indicated that AnxA5 and AnxA13might substitute for the loss of AnxA1 and might act as tolero-genic signals in vivo.Recently, our group showed that AnxA1 restrains the function and

reduces the frequency of Ag-specific CD8+ T cells upon immuni-zation of mice with xenogeneic ACs ectopically expressing AnxA1(11). To investigate whether AnxA5 and AnxA13 also regulate theinduction of Ag-specific T cell responses in vivo, aS2 mOVA wereinjected into mice, and the anti-OVAT cell response was monitored.Injection of aS2 mOVA induced a cell-mediated immune responseand led to the development of OVA-specific CD8+ T cells (Fig. 6B,Supplemental Fig. 4F). Notably, coinjection of apoptotic S2 cellsoverexpressing AnxA1, AnxA5, or AnxA13 strongly inhibited thedevelopment of OVA-specific CD8+ T cells (Fig. 6B). We furtheranalyzed the effect of Anxs on the surface of ACs with regard tothe stimulation and fate of Ag-specific CD8+ T cells in a trans-fer experiment. We injected CFSE-labeled OVA-specific OT-IT cells into C57BL/6 WT mice and followed proliferation and

expansion of this population with the help of the congenicmarker Thy-1.1. We detected no difference in proliferation orOT-I population size 6 d after immunization (data not shown).However, when challenging with OVA protein 12 d after theprimary immunization, we detected a significant reduction in theabsolute number of OT-I cells in mice coimmunized with aS2AnxA5 or aS2 AnxA13 (Fig. 6C). Importantly, the immune re-sponse in mice coimmunized with Anx-overexpressing apoptoticS2 cells was also functionally impaired, as evidenced by a severelyreduced number of IFN-g–secreting CD8+ T cells (Fig. 6D). Similarresults were obtained by ELISA (data not shown). Thus, AnxA5and AnxA13 negatively regulate the induction of Ag-specific CD8+

T cell responses and, therefore, act as tolerogenic signals on ACsin vivo.

DiscussionUptake and processing of self-Ags derived from ACs by DCs inthe steady-state and subsequent presentation of self-peptides onMHC class I and MHC class II by tolerogenic DCs are importantmechanisms to induce CD4+ and CD8+ T cell tolerance in theperiphery (36, 37). Immunosuppression of phagocytes by ACs is

FIGURE 6. AnxA5 and AnxA13 suppress the induction of CD8+ T cell immune responses in vivo. (A) GM-CSF–differentiated BMDCs from WT mice

were incubated with the indicated ratio of apoptotic Jurkat T cells (aJ) or apoptotic neutrophils (aNF, after 1 d in vitro culture). Four hours later, cells were

stimulated with 1 ng/ml LPS o/n. TNF-a concentrations in the supernatants were determined by ELISA. Data are mean 6 SEM of three independent

experiments. (B) aS2 mOVA, together with aS2 AnxA1, aS2 AnxA5, or aS2 AnxA13 were injected i.v. into mice. Mice injected with aS2 mock served as

a negative control. After 8 d, the induction of OVA-specific CD8+ T cells in the mesenteric lymph nodes was analyzed by flow cytometry. Errors bars

represent mean6 SEM. (C and D) One day after transfer of 13 106 CFSE-labeled OT-I T cells, C57BL/6 WT mice were immunized i.v. with 0.5 3 106

aS2 mOVA together with 1 3 106 aS2 AnxA5, aS2 AnxA13, or aS2 mock. After 12–13 d, mice were challenged with 50 mg OVA protein in IFA, and

spleens were analyzed 6 d after challenge by flow cytometry for cell numbers of transferred OT-I cells in spleens (C) and IFN-g secreting CD8+ T cells

by ELISPOT (D). n = 18 (aS2 mOVA/aS2 mock), n = 9 (aS2 mOVA/aS2 AnxA5 and aS2 mOVA/aS2 AnxA13). *p , 0.05, **p , 0.01, ***p , 0.001,

****p , 0.0001.

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mediated by tolerogenic signals on the surface of ACs, includinggrowth arrest-specific gene 6, inactivated complement component3b, thrombospondin 1, and AnxA1 (10, 11, 38–40). In lymphoidorgans, the subsequent interaction of tolerogenic DCs and auto-reactive T cells induces anergy or deletion, or it may triggerconversion of naive CD4+ T cells into regulatory T cells (41–46).Because the knowledge of tolerogenic signals on the surface ofACs is limited, and functional redundancy has been reported fordifferent properties of Anxs, this study aimed at investigatingwhether Anxs act as redundant tolerogenic signals on ACs.Upon induction of apoptosis, AnxA1 translocates to the surface of

ACs and is recognized by phagocytosing DCs (11). Using a leuke-mia cell line and stably transfected apoptotic S2 cells, we showedthat translocation upon induction of apoptosis is a common featureof AnxA1, AnxA5, and AnxA13 (Fig. 2). To our knowledge, this isthe first report showing translocation of AnxA13 to the surface ofACs, whereas translocation of AnxA5 to the surface of rat car-diomyocytes upon treatment with staurosporine or H2O2 was re-ported previously (47). Externalization of AnxA5 also takes placeunder pathophysiological conditions like acute myocardial infarc-tion or glomerulonephritis (48, 49). In both cases, passive releasefrom necrotic cells cannot be ruled out. However, this study showsthat Anxs are externalized at an early stage of apoptosis precedingthe loss of membrane integrity (Fig. 2).AnxA5 and AnxA13 induce the development of a tolerogenic

DC phenotype, as we showed previously for AnxA1 (11). Solubleand membrane-bound AnxA5 and AnxA13 inhibit the TLR-induced secretion of proinflammatory cytokines (i.e., IL-12p40,IL-6, and TNF-a), whereas the secretion of IL-10 is not affected(Fig. 3B–H). TLR-induced upregulation of the costimulatory mol-ecules CD40, CD80, and CD86 was inhibited by Anx pretreatment,whereas surface levels of MHC class I and II molecules and coin-hibitory molecules were not altered (Fig. 4A–G). Thus, the phe-notype of DCs obtained after Anx incubation resembles the one ofDCs after uptake of ACs or upon incubation with AC-derived tol-erogenic signals (10, 38, 39, 50–52). Unaltered expression of MHCmolecules on DCs is a prerequisite for efficient presentation of self-peptides and, thus, is required for deletion or tolerization of auto-reactive T cells in the draining lymph nodes (2). In addition, lowexpression of costimulatory molecules, especially CD40, was de-scribed as a decisive feature of tolerogenic DCs (53, 54). Inhibitionof TLR-induced upregulation of costimulatory molecules wasobserved after incubation of BMDCs with the tolerogenic signalsgrowth arrest-specific gene 6 or inactivated complement component3b or upon ligation of receptors that recognize tolerogenic signals,including Mer tyrosine kinase, complement receptor 3, or CD36 (10,38, 39, 51). Importantly, deletion and functional inactivation of au-toreactive CD8+ T cells require the absence of CD40L-induced sig-naling, as well as the expression of PD-1 ligands PD-L1 and PD-L2,neither of which was affected by Anx pretreatment of BMDCs (46).Similar to AC uptake or ligation of Mer tyrosine kinase, CD36,

or complement receptor 3, preincubation of BMDCs with Anxsresults in inhibition of TLR-induced secretion of proinflammatorycytokines, including TNF-a, IL-6, and IL-12p40 (Fig. 3B–H) (10,38, 39, 50). The low secretion of the Th1-promoting cytokinesTNF-a and IL-12p40, as well as of the Th17-promoting cytokineIL-6, implies that Th1 and Th17 priming of naive CD4+ T cells byAnx-treated BMDCs is unlikely.Tolerance induction by AnxA1, AnxA5, and AnxA13 is inde-

pendent of DC apoptosis, recognition of Anxs by FPR familymembers, or inhibition of phospholipase A2 activity. cPLA2 wasdescribed to be the key enzyme for signal transduction of inflam-mation (55, 56). Inhibition of cPLA2 is mediated by specificinteraction of cPLA2 and aa 275–346 of AnxA1. Other Anxs,

including AnxA5, do not inhibit cPLA2 activity in vitro (57). Be-cause AnxA5 and AnxA13 promote the development of tolerogenicDCs (Figs. 3B–H, 4A–G), inhibition of cPLA2 is not required forsuppression of DC activation by Anxs. High amounts of AnxA1released into the inflammatory fluid accelerate apoptosis of neu-trophils and monocytes, which contributes to the resolution of in-flammation (58–60). AnxA5 can have proapoptotic or antiapoptoticeffects, depending on the cell type investigated (47, 61). Im-portantly, preincubation of BMDCs with AnxA1, AnxA5, orAnxA13 did not influence apoptosis progression, ruling out DCapoptosis as a mechanism for tolerance induction by Anxs (Fig. 4H,Supplemental Fig. 3C).N-terminal peptides and the AF-2 sequence in the core domain

were implicated in mediating anti-inflammatory effects of AnxA1(15, 21, 62, 63). However, several points make it unlikely thatrecognition by FPR family members contributes to the tolerogeniceffect of Anxs on BMDCs. First, the Anx core domain, which ishighly conserved among Anx family members, is sufficient toinhibit TLR-induced cytokine secretion of BMDCs (Fig. 1B).Second, mutation of the AF-2 sequence, the only other peptidesequence binding to FPR family members apart from the AnxA1N terminus, and ablation of the N-terminal domain of AnxA1 donot abrogate AnxA1-induced suppression of TLR-induced DCactivation (Fig. 1B) (24). Third, AnxA5 and AnxA13, which havenot been shown to bind to FPR family members, possess sup-pressive activity comparable to AnxA1 (Fig. 3B–D). Fourth, pan-specific antagonists of FPR family members do not abrogate thesuppressive effect elicited by Anxs (Figs. 1E, 5B). Finally, phos-phorylation of ERK, induced upon binding of N-terminal pep-tides of AnxA1 to FPR family members, could not be detected inBMDCs upon treatment with AnxA1, AnxA5, or AnxA13 (Figs. 1F,5A). The lower levels of FPR family members expressed on DCsand/or different signaling thresholds of DCs compared to neu-trophils may explain the differential effects noted for the individualdomains of Anxs, depending on the cell type or biological setting(Fig. 1C). The data presented in this article support the existence ofa different, hitherto unknown receptor involved in the recognition ofAnxs on DCs.Our results show that several Anx family members translocate to

the surface of ACs and induce tolerogenic signaling upon recog-nition by their cognate receptor on DCs. The absence of severephenotypes in mice deficient in individual Anx family membersand almost identical tissue-specific expression patterns of mostsimilar Anx genes suggest that Anxs have redundant functionsin vivo (12, 25, 28–31). In fact, redundant functions for differentAnxs have been reported in the context of membrane trafficking,inhibition of phospholipase activity, and blood coagulation in vitro(12). In a noninflammatory or noninfectious setting and on a mousebackground not prone to develop autoimmunity, AnxA12/2 micelack symptoms of autoimmune or chronic inflammatory diseases(Fig. 6). This is likely due to upregulation of other, equally sup-pressive Anx family members in AnxA12/2 mice (Fig. 6) (32). Byanalyzing AnxA1, AnxA5, and AnxA13 we chose representativeAnxs that are distributed equally through the phylogenetic tree ofvertebrate Anxs, including AnxA13 as the founding member of thisprotein family (13). As typical representatives of the Anx family,the core domains of murine AnxA1, AnxA5, and AnxA13 showa comparatively high mean amino acid similarity of 55–60% to allother Anx core domains. Therefore, we hypothesize that theimmune-suppressive activity residing in the Anx core domain, asidentified in our study, is likely shared by more, if not most,members of the Anx family. If so, the ubiquitous expression ofthe Anx family as a whole (13) could afford protection fromautoimmunity to virtually any tissue of the organism.

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By analyzing the fate of transferred Ag-specific T cells, weobserved that AnxA5 and AnxA13 exert a tolerogenic effect on thesurface of ACs, which leads to a significant reduction in thepopulation size and activity of Ag-specific CD8+ T cells. Re-garding CD8+ T cell activity, AnxA5 and AnxA13 recapitulate theeffect of AnxA1, as analyzed in our previous study (11). Moreover,these results further elucidate the Anx-mediated mechanism ofimmune suppression in vivo and suggest a mechanism similar todeletional tolerance, as described by Steinman and colleagues (64).A better understanding of tolerogenic signals on ACs provides

new opportunities to interfere with peripheral tolerance inductionfor the treatment of autoimmune diseases, chronic inflammatorydiseases, and cancer. The clearance of apoptotic tumor cells bytumor-associated macrophages or DCs is associated with the re-lease of anti-inflammatory cytokines and tolerance induction totumor Ags (65). The immunoregulatory environment establishedby the tumor and the presentation of tumor Ags in the absence ofdanger signals may account for the failure of most anticancerchemotherapies to promote curative T cell immunity (66). Onepowerful approach to counteract the tolerogenic properties of thetumor is to induce immunogenic cell death in which alarmins arereleased during apoptosis (67). However, the release of alarminshas severe side effects, including promotion of angiogenesis,resistance to chemotherapeutics, chronic inflammation, develop-ment of gout, and renal failure (65). Our data can guide thedevelopment of an alternative approach: interference with thepresentation of tolerogenic signals, such as Anxs, or blockade ofthose on dying tumor cells with the goal to maximize the impactof cancer immunotherapy.

AcknowledgmentsWe thank S. Akira, S. Uematsu, and L. Gissmann for providing Tlr42/2mice;

U. Rescher (University of M€unster, M€unster, Germany) for providing a

eukaryotic FPR1 overexpression plasmid; and N. Garbi and G. Hammerling

for providing OT-I mice and biotin-labeled Y3. We also thank H. Sauter for

excellent secretarial assistance.

DisclosuresThe authors have no financial conflicts of interest.

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