Select Clr-g Expression on Activated Dendritic Cells Facilitates Cognate Interaction with a Minor Subset of Splenic NK Cells Expressing the Inhibitory Nkrp1g This information is current as Receptor of September 26, 2021. Miriam E. Friede, Stefan Leibelt, Diana Dudziak and Alexander Steinle J Immunol published online 20 December 2017 http://www.jimmunol.org/content/early/2017/12/20/jimmun Downloaded from ol.1701180

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 20, 2017, doi:10.4049/jimmunol.1701180 The Journal of Immunology

Select Clr-g Expression on Activated Dendritic Cells Facilitates Cognate Interaction with a Minor Subset of Splenic NK Cells Expressing the Inhibitory Nkrp1g Receptor

Miriam E. Friede,* Stefan Leibelt,* Diana Dudziak,† and Alexander Steinle*

Natural killer gene complex–encoded immunomodulatory C-type lectin-like receptors include members of the NKRP1 and C-type lectin-like 2 (CLEC2) gene families, which constitute genetically linked receptor-ligand pairs and are thought to allow for NK cell– mediated immunosurveillance of stressed or infected tissues. The mouse C-type lectin-like receptor Nkrp1g was previously shown to form several receptor-ligand pairs with the CLEC2 proteins Clr-d, Clr-f, and Clr-g, respectively. However, the physiological expression of Nkrp1g and its CLEC2 ligands as well as their functional relevance remained poorly understood. Recently, we demonstrated a gut-restricted expression of Clr-f on intestinal epithelial cells that is spatially matched by Nkrp1g on subsets of Downloaded from intraepithelial lymphocytes. In this study, we investigated expression and ligand interaction of Nkrp1g in the splenic compartment, and found an exclusive expression on a small subset of NK cells that upregulates Nkrp1g after cytokine exposure. Whereas transcripts of Clr-d and Clr-f are virtually absent from the spleen, Clr-g transcripts were abundantly detected throughout different leukocyte populations and hematopoietic cell lines. However, a newly generated anti–Clr-g mAb detected only residual Clr-g surface expression on splenic monocytes, whereas many hematopoietic cell lines brightly display Clr-g. Clr-g surface expression was strongly upregulated on splenic CD8a+ conventional dendritic cells (DCs) and plasmacytoid DCs upon TLR- http://www.jimmunol.org/ mediated activation and detectable by Nkrp1g, which dampens NK cell effector functions upon Clr-g engagement. Hence, different to the intestinal tract, in the spleen, Nkrp1g is selectively expressed by a subset of NK cells, thereby potentially allowing for an inhibitory engagement with Clr-g-expressing activated DCs during immune responses. The Journal of Immunology, 2018, 200: 000–000.

atural killer cells are cytotoxic innate lymphocytes that NKRP1 family members are expressed by NK cells and T cells, eliminate transformed or infected cells upon recognition the expression pattern of their CLEC2 ligands is more diverse (10, through a broad variety of germline-encoded inhibitory 11). In humans, these NKC-encoded C-type lectin-like receptor-

N by guest on September 26, 2021 and activating receptors, but also secrete cytokines such as IFN-g ligand pairs include activation-induced C-type lectin (AICL, (1–3). A major group of NK cell receptors are C-type lectin-like CLEC2B) and the activating NK receptor NKp80 (12, 13), receptors encoded by the NK gene complex (NKC) in both man keratinocyte-associated C-type lectin (KACL, CLEC2A), and the and mouse (4–7). These include NKG2D and CD94/NKG2x re- activating receptor NKp65 (14, 15), as well as lectin-like tran- ceptors, but also various members of the NKRP1 and C-type script 1 (LLT1, CLEC2D) engaging the inhibitory receptor NKR- lectin-like 2 (CLEC2) families that have been shown to estab- P1A/CD161 (16, 17). In mice, nonhomologous but structurally lish genetically linked receptor-ligand pairs (8–10). Whereas related members of the NKRP1 and CLEC2 gene families also constitute several receptor-ligand pairs (8, 18). Of these, the in- hibitory receptor Nkrp1b and its ligand Clr-b have been studied *Institute for Molecular Medicine, Goethe-University Frankfurt/Main, 60590 Frank- most extensively. Clr-b is broadly expressed by hematopoietic as furt am Main, Germany; and †Laboratory of Dendritic Cell Biology, Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander University Erlan- well as nonhematopoietic tissues and its expression is downreg- gen-Nurnberg,€ 91052 Erlangen, Germany ulated on infected, stressed, or malignant cells (19–21). Reduced ORCIDs: 0000-0001-9358-134X (D.D.); 0000-0001-5081-8503 (A.S.). Clr-b surface expression renders such cells more susceptible to Received for publication August 15, 2017. Accepted for publication November 13, NK cell cytolysis due to diminished inhibitory signals provided by 2017. Nkrp1b (19, 22). Hence, this receptor-ligand pair has then pro- This work was supported in part by grants to D.D. from the German Research posed to represent another MHC class I–independent missing-self € Foundation (CRC1181-TPA7) and the Interdisziplina¨res Zentrum fur Klinische recognition system (19, 23–26). This hypothesis was recently Forschung (IZKF-A65). supported by the finding that the murine CMV (MCMV) glyco- The microarray data presented in this article have been submitted to ArrayExpress (https://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-6223. protein m12 acts as a Clr-b substitute assuring inhibitory Nkrp1b Address correspondence and reprint requests to Dr. Alexander Steinle, Institute for ligation and viral immunoevasion from NK cell recognition even Molecular Medicine, Goethe-University Frankfurt am Main, Theodor-Stern-Kai 7, when Clr-b is downregulated in the course of MCMV infection 60590 Frankfurt am Main, Germany. E-mail address: [email protected] (27). Of note, the viral immunoevasin m12 also binds to the ac- The online version of this article contains supplemental material. tivating receptors Nkpr1a and Nkrp1c that have remained orphan Abbreviations used in this article: cDC, conventional DC; CHO, Chinese hamster receptors (27). In contrast, several CLEC2 family members have ovary; CLEC2, C-type lectin-like 2; DC, dendritic cell; IEC, intestinal epithelial cell; been reported as ligands for the related Nkrp1f and Nkrp1g re- IEL, intestinal intraepithelial lymphocyte; IFNAR, type I IFN receptor; LAK, lymphokine-activated killer; MCMV, murine CMV; NKC, NK gene complex; ceptors: both Nkrp1 receptors are capable of engaging Clr-d and pDC, plasmacytoid DC; poly(I:C), polyinosinic-polycytidylic acid; qPCR, quantita- Clr-g, whereas Nkrp1f binds in addition to Clr-c, and Nkrp1g to tive PCR; SFI, specific fluorescence index. Clr-f (8, 18, 28, 29). With regard to Clr-f, we recently reported a Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 pronounced tissue-specific expression on intestinal epithelial cells

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701180 2 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN

(IEC) that is spatially matched by the selective intestinal expres- All media contained 10% FCS (Biochrom, Berlin, Germany), 2 mM sion of Nkrp1g on a subset of intestinal intraepithelial L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 mg/ml lymphocytes (IEL) facilitating immunosurveillance of the gut streptomycin (all from Sigma-Aldrich). epithelium by certain gd IEL (30). Similarly, we recently de- Isolation of primary cells from mouse tissue scribed an exclusive expression of the orphan CLEC2 molecule Clr-a on mouse gut epithelium, supporting the idea that CLEC2 Single-cell suspensions of splenocytes were generated by passing the spleen family members may fulfill tissue-specific immune functions (31, through a 100 mm nylon mesh (Corning, Corning, NY) and washed with PBS supplemented with 3% FCS and 2 mM EDTA. Following lysis of 32). Tissue-specific expression may also provide a rationale for erythrocytes using Pharm Lyse buffer (BD Biosciences, Heidelberg, Ger- the redundancy of Nkrp1g ligands. Whereas little is known about many) for 3 min at room temperature, splenocytes were washed and finally the expression of Clr-d, several studies detected Clr-g transcripts passed through a 40 mm nylon mesh (Corning). For analysis of DCs or in hematopoietic cells, including lymphokine-activated killer monocytes, the spleen was put into a gentleMACS C-Tube (Miltenyi (LAK) cells (5), bone marrow–derived dendritic cells (DCs), and Biotec, Bergisch-Gladbach, Germany) containing 2 ml HBSS buffer with Ca2+ and Mg2+ (Thermo Fisher Scientific, Waltham, MA) supplemented macrophages (8), B cells, and T cells (33), as well as preferentially with 2% FCS, 2 mM MgCl2, 0.5 mg/ml Collagenase D (Roche, Mannheim, in hematopoietic organs (30, 31). Germany), and 3000 U/ml DNase I (PanReac Applichem, Darmstadt, NK cells are known to sculpt immune responses by interacting Germany), and dissociated using the gentleMACS Octo Dissociator with DCs (34–41). In mice, there are three splenic DC populations, (Miltenyi Biotec) according to the manufacturer’s protocol. The resulting + 2 cell suspensions were then passed through a 100 mm nylon mesh and namely CD8a and CD8a conventional DCs (cDCs) and plas- processed as described above for a single-cell suspension of splenocytes. macytoid DCs (pDCs) (42, 43). These subtypes exert different For isolation of blood lymphocytes, blood was collected from hearts of functions and express nonredundant pattern recognition receptors, euthanized mice, and single-cell suspensions processed as described for Downloaded from allowing them to specifically react to insults. CD8a+ cDCs are the splenocytes. NK cells were isolated from splenocytes using the mouse NK main population expressing TLR3, thereby sensing dsRNA, Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s + 2 protocol. LAK cells were prepared as described (49). Briefly, splenocytes CD8a and CD8a cDCs equally express TLR4 recognizing LPS, were applied to a nylon wool column and nonadherent cells collected by and pDCs mainly express TLR7 and 9, which sense ssRNA or washing the column with complete RPMI 1640 and cultured in complete CpG DNA, respectively (44–46). TLR-activated DCs can shape RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1 mM so- dium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin, 50 mM innate immune responses through activation of NK cells. Con- http://www.jimmunol.org/ 2-ME, 13 MEM nonessential amino acids, and 1000 U/ml recombinant versely, the NK cells can also regulate adaptive immune responses human IL-2 (Proleukin S; Novartis Pharma, Basel, Schweiz). Cells were through impacting on maturation and activation of DCs, both by passaged at days 7, 10, and 13, and nonadherent cells analyzed in all cell-cell contact or soluble factors (47). experiments. Isolation of intestinal cells was performed with modifications In this study, we report the exclusive expression of the inhibitory as described previously (50). Briefly, after removal of Peyer’s patches and immunoreceptor Nkrp1g in the spleen on a minor subset of mouse longitudinal sectioning, the small intestine was washed with ice-cold PBS, cut into small pieces, and then incubated three times for 20 min at 37˚C in NK cells that is spatially matched by expression of the Nkrp1g- HBSS (Sigma-Aldrich) containing 5% FCS, 5 mM EDTA, and 1 mM DTT. ligand Clr-g on activated splenic DCs. Therefore, Nkrp1g on ac- Each purification step was passed through a 100 mm nylon mesh, diluted tivated NK cells may dampen NK responses toward splenic DCs in with PBS followed by sedimentation of crypt cells for 10 min. A final the course of immune responses. processing through a 40 mm nylon mesh and a washing step with all by guest on September 26, 2021 pooled purification steps generated a single-cell suspension of intestinal cells. Unless stated otherwise, mice from 6 to 15 wk were used for all experiments. Materials and Methods Animals Cytokine treatment, CFSE labeling, and TLR stimulation BALB/c, C57BL/6, C3H, 129, and CD-1 ISG mice were purchased from In vitro cytokine treatment was performed with MACS-purified NK cells in Envigo (Horst, the Netherlands) or Charles River Laboratories (Sulzfeld, complete RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, m Germany). LOU/C rats were from Envigo. NOD SCID (NOD.Cg- 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin, scid scid 50 mM 2-ME and 13 MEM nonessential amino acids. Cells were seeded Prkdc /J) and NOD SCID GAMMA mice (NOD.Cg-Prkdc 6 tmlWjl at a density of 1 3 10 per ml and cytokines were added at the following Il2rg /SzJ) were kindly provided by Martin Zo¨rnig (Georg-Speyer- Haus, Frankfurt, Germany). Animals were housed at the local animal concentrations: 1000 U/ml (in combination with IL-15) or 10,000 U/ml (when used alone) recombinant human IL-2 (Proleukin S; Novartis facility. High m.w. polyinosinic-polycytidylic acid [poly(I:C)] (Invivo- Gen, Toulouse, France) was injected i.p. at 15 mg per g body weight and Pharma), 50 ng/ml recombinant human IL-15 (Miltenyi Biotec), 2 ng/ml mice euthanized 16 h later for isolation of splenocytes. Animal experi- recombinant mouse IL-12 (PeproTech, Rocky Hill, NJ), or 50 ng/ml recombinant mouse IL-18 (MBL Pharma, Karachi, Pakistan). Stimula- ments were approved by the local authorities (Regierungspra¨sidium Darmstadt, Germany; permit numbers F146/Anz03, F146/Anz04, and tion was performed for the indicated time periods and cells were routinely FU/Anz1035) and performed in full compliance with the respective na- passaged with fresh medium and cytokines at day 3 and/or the day before tional guidelines. analysis. In long-term culture of NK cells with IL-15, the cells were passaged with fresh medium and cytokines at days 3 and 7, and then weekly. For analysis of cell proliferation, NK cells were labeled directly 6 Cell lines and transfectants after purification with CFSE. Briefly, 1.5 3 10 cells were diluted in PBS/ 0.1% BSA (PAN Biotec, Aidenbach, Germany) at 1 3 106 per ml, CFSE Cell lines were from the Deutsche Sammlung von Mikroorganismen und (Thermo Fisher Scientific) was added at 0.5 mM and cells were incubated Zellkulturen (Braunschweig, Germany). DC2.4 (48) and BWN.3G-Nkrp1g for 10 min at 37˚C. The reaction was terminated by adding 53 v/v ice-cold (18) reporter cells were kindly provided by Andreas Diefenbach (Mainz, complete RPMI 1640 and cells were subsequently washed three times. Germany) and Lise Kveberg (Oslo, Norway), respectively, and maintained Labeled NK cells were cultured for 7 d with IL-15 as described above and in RPMI 1640 supplemented either with 50 mM 2-ME, 13 MEM nones- analyzed on days 0, 3, and 7 by flow cytometry. For in vitro TLR stimu- sential amino acids, and 10 mg/ml G418 (Carl Roth, Karlsruhe, Germany) lation, single-cell suspensions of splenocytes were generated by digestion (DC2.4), or with 0.5 mg/ml hygromycin B (Enzo Life Sciences, Lo¨rrach, with DNase I and Collagenase D as described above. Cells were seeded at Germany) and 1 mg/ml G418 (BWN.3G-Nkrp1g). Chinese hamster ovary a density of 1 3 106 per ml and cultured in complete RPMI 1640 sup- (CHO) transfectants and NK-92MI transductants were generated and plemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, cultured as described elsewhere (30). B16F10, MC38, and RAW309 cells 100 U/ml penicillin, and 100 mg/ml streptomycin, 50 mM 2-ME, and were cultured in complete DMEM (Sigma-Aldrich, Steinheim, Germany) 13 MEM nonessential amino acids. TLR agonists were used as follows: supplemented with 50 mM 2-ME and 13 MEM nonessential amino acids. 10 mg/ml poly(I:C) (InvivoGen), 10 mg/ml LPS, and 10 mg/ml R848 (both All other cell lines were cultured in RPMI 1640 (Sigma-Aldrich). Sigma-Aldrich) for 4–6 h. The Journal of Immunology 3

RT-PCR and quantitative real-time PCR using a BD FACSAria III cell sorter (BD Biosciences). For sorting of DC populations and monocytes in the resting or poly(I:C)-activated state, RNA was isolated from tissue samples using peqGOLDTriFast (Peqlab, B cells were depleted from the single-cell suspension of splenocytes with Erlangen, Germany) and the gentleMACS Octo Dissociator for tissue mouse CD19 MicroBeads (Miltenyi Biotec) according to the manufac- dissociation or RNAqueous-Micro Kit (Thermo Fisher Scientific). RNAwas turer’s protocol. converted into cDNA with M-MLV RT (H-) (Promega, Mannheim, Ger- many) according to the manufacturer’s protocol after treatment with DNase Co-immunoprecipitation and immunoblotting I (kit component; Promega, Madison, WI) for 30–45 min at 37˚C. For quantitative PCR (qPCR), cDNA was amplified on the real-time PCR Briefly, 1 3 108 BALB/c NK cells that were expanded with IL-15 for up to system StepOnePlus (Applied Biosystems, Foster City, CA) using SYBR 28 d were resuspended in 100 ml of PBS per stimulation and rested for 30 Green technology (Roche) for detection, as well as the following nucleo- min at 4˚C. Then cells were prewarmed for 1 min at 37˚C and stimulated tides: Clr-d forward 59-TCCCCCAACATGGGTGTAAC-39, Clr-d reverse with 10 ml pervanadate mix (PBS with 20 mM Na3VO4,67mMH2O2) for 59-CCACTGGTGAACCTGAGACC-39, Clr-f forward 59-GAATATAGCA- 5 min at 37˚C or left untreated for 5 min at 37˚C. The reaction was stopped ACTTGGTTCTC-39, Clr-f reverse 59-GGATTTACAACTACTGACAAAC- by addition of ice-cold lysis buffer (50 mM Tris/HCl [pH 8], 150 mM 39, Clr-g forward 59-GGGATTTTGATTGCTGGATTG-39, Clr-g reverse 59- NaCl, 1% Nonidet P-40, 5 mM iodoacetamide, complete phosphatase in- GCTCAGGTAGGCATATCTTC-39, Nkrp1f forward 59-GCCATTTAGG- hibitor [Roche]), and lysis was performed for 20 min on ice and cleared by TGTCCAGGGTA-39, Nkrp1f reverse 59-CCAAATGATGCCAGTGTGGG-39, centrifugation. For immunoprecipitation of Nkrp1g, protein A/G magnetic Nkrp1g forward 59-TTGGAAGTGGATGAATGGTTC-39, Nkrp1g reverse 59- beads (Thermo Fisher Scientific) were loaded with 10 mg of mAb 8A10 or ACTTCTGTTTGTGAGATGAGG-39, 18S rRNA forward 59-CGGCTACCA- respective isotype control per 25 ml of beads and, after washing with PBS- CATCCAAGGAA-39,and18SrRNAreverse59-GCTGGAATTACCGCGGCT-39. T (0.1% Tween 20; PanReac Applichem), beads and cleared lysates were Copy numbers were normalized with the DD cycle threshold method incubated overnight at 4˚C on a rotator. Beads were washed five times with using 18S rRNA. RT-PCR for assessment of Clr-g isoform expression was wash buffer [50 mM Tris/HCl (pH 8), 150 mM NaCl, 5 mM EDTA] and performed with Phusion High-Fidelity DNA Polymerase (New England proteins were eluted by addition of 20 ml23 nonreducing Laemmli buffer Downloaded from Biolabs, Frankfurt, Germany) according to the manufacturer’s protocol. (20 mM Tris/HCl [pH 6.8], 1.7% glycerol, 0.3% SDS, 8 3 1024% cDNA was used for the PCR and was amplified in 35 cycles on a standard bromphenol blue) and boiling at 95˚C for 10 min. Proteins were separated thermocycler. PCR products were visualized on a 5% agarose gel sup- by SDS-PAGE, followed by wet blotting onto a nitrocellulose membrane plemented with ethidium bromide. (GE Healthcare, Little Chalfont, U.K.). Blots were blocked with TBS-T (0.1%) and 5% milk powder, and then incubated with anti–SHP-1 Ab Soluble ectodomains and Clr-f/Clr-g–specific mAb 1H7 (1:1000, clone 52/PTP1C/SHP1; BD Biosciences) overnight at 4˚C. Sec- ondary HRP-conjugated goat anti-mouse IgG Ab (1:10,000; Jackson

The soluble recombinant ectodomains of Nkrp1g (sNkrp1g: Pro66 through http://www.jimmunol.org/ Val214) and Clr-f (sClr-f: Pro78 through Val218) were described previously ImmunoResearch Laboratories) was incubated for 1.5 h at room temper- (30). The Clr-f/Clr-g–specific mAb 1H7 was generated by immunizing ature and blots developed by ECL using HRP-Juice Plus (PJK, Kleinbit- LOU/C rats (Envigo) with the soluble ectodomain of Clr-f and resulting tersdorf, Germany) and a Fusion SL machine (Vilber Lourmat, hybridoma were screened for specific reactivity against CHO-Clr trans- Eberhardzell, Germany). fectants. For visualization of Nkrp1g protein in immunoblot, BALB/c or NOD SCID NK cells were stimulated for 14 d with IL-15 and then lysed using ice- cold lysis buffer. Lysis was performed for 20 min on ice and cleared by Flow cytometry and cell sorting centrifugation. Then 250 mg of BALB/c, 330 mg of NOD SCID or 10 mgof NK-92MI-Nkrp1g protein lysate were deglycosylated with PNGaseF (New For analysis of cell surface expression, cells were resuspended in ice-cold England Biolabs) for 1.5 h at 37˚C under native conditions by only adding PBS with 3% FCS and 2 mM EDTA. For primary cells, Fc receptors were G7 buffer and PNGaseF. Lysates were then separated under nonreducing by guest on September 26, 2021 blocked with 30 mg/ml mouse IgG (Thermo Fisher Scientific) and 10 mg/ml conditions by SDS-PAGE, followed by wet blotting onto a nitrocellulose anti-CD16/32 (clone 93 and clone 2.4G2; BioLegend, London, U.K.) for membrane. Blocking was performed with TBS-T (0.1%) and 5% milk 20 min at 4˚C. Cells were then stained with the respective primary and powder. Primary mAb 8A10-bio (30 mg/ml) was incubated overnight at secondary Abs for 20 min at 4˚C and washed twice. Flow cytometry 4˚C, followed by HRP-conjugated streptavidin (1:25,000; Jackson analysis was performed with a FACSCanto II (BD Biosciences) and data ImmunoResearch Laboratories) for 1.5 h at room temperature and de- were analyzed using FlowJo (Tree Star, Ashland, OH). Primary Abs were veloped as described above. as follows: anti–CD3ε-PerCP (clone 145-2C11; BioLegend), anti–CD8a- allophycocyanin/Cy7 (clone 53-6.7; BioLegend), anti–CD11b-BV510 Degranulation and cytotoxicity (clone M1/70; BioLegend), anti–CD11c-FITC (clone HL3; BD Biosci- ences), anti–CD11c-PE/Cy7 (clone N418; eBioscience), anti–CD14-FITC Degranulation of NK-92MI-Nkrp1g transductants was quantified by flow (Sa14-2; BioLegend), anti–CD19–Pacific Blue and -PerCP (clone 6D5; cytometric detection of cell surface CD107a upon 1.5 h coculture with CHO BioLegend), anti–CD27-PE (clone LG.7F9; eBioscience), anti–CD49b- transfectants at a 1:1 E:T ratio in the presence or absence of mAb 1H7. FITC (clone DX5; BioLegend), anti–human CD56-PE (clone N901; Stimulation with 25 ng/ml PMA and 1 mM ionomycin (both Sigma- Beckman Coulter, Brea, CA), anti–CD69-PE (clone H1.2F3; BioLegend), Aldrich) served as positive control. NK-92MI cells were gated as CD56+ anti–human CD107a-allophycocyanin (clone H4A3; BD Biosciences), cells. Cytotoxicity was assessed by a 4 h 51Cr release assay. CHO trans- anti-FLAG (M2; Sigma-Aldrich), anti–I-A/I-E-FITC (clone M5/114.15.2; fectants (targets) were labeled with 51Cr for 2 h at 37˚C and cocultured BioLegend), anti–IFN-g–FITC (clone XMG1.2; BioLegend), anti–Ly-6C– with NK-92MI transductants (effectors) at 37˚C for 4 h at indicated E:T PerCP/Cy5 (clone HK1.4; BioLegend), anti–Ly-6G–Pacific Blue (clone ratios. Coculture supernatants were mixed with OptiPhase Supermix 1A8; BioLegend), anti–Ly49C/I-FITC (clone 5E6; BD Biosciences), anti– scintillation mixture (PerkinElmer, Waltham, MA) in an IsoPlate-96 and NKp46-PE (clone 29A1.4; BioLegend), anti–NKp46-V450 (clone 29A1.4; measured with a MicroBeta2 plate counter (PerkinElmer). Percent specific BD Biosciences), anti–Nkrp1f-A647 (clone C8; BioLegend and self- lysis was calculated as follows: 100 3 (experimental release 2 sponta- conjugated) anti–PDCA-1–allophycocyanin (Miltenyi Biotec), anti–Clr-f neous release)/(maximum release 2 spontaneous release). [clones 10A6 and 10A8 (30)], anti–Clr-f/Clr-g (clone 1H7; current study), and anti-Nkrp1g [clone 8A10 (30)]. Unless stated otherwise, all IFN-g assay Abs are against mouse Ags. Isotype controls: mouse IgG1 (clone N1G9), rat IgG1, and rat IgG1-FITC (clone RTK2071; BioLegend), rat IgG2a (clone BALB/c NK cells were MACS purified and stimulated in vitro with IL-15 RTK2758; BioLegend), rat IgM (clone RTK2118; BioLegend). Secondary for 5 d. Before induction of IFN-g by IL-12, IL-18, and IL-15, cells were reagents: goat anti–mouse-allophycocyanin F(ab9)2 (Jackson Immuno- blocked with 30 mg/ml mIgG (Thermo Fisher Scientific) and 10 mg/ml Research, West Grove, PA) for staining of anti-FLAG mAb M2, mouse anti-CD16/32 (clone 93; BioLegend) for 20 min at room temperature. 5 anti–rat-A647 F(ab9)2 (Jackson ImmunoResearch) for staining of anti–Clr- Cells were seeded at 2–3 3 10 per 96 wells and incubated for 1 h at 37˚C. f mAb 10A6, mouse anti-rat IgG1-bio (clone RG11/39.4; BD Biosciences) GolgiStop (1:1500; BD Biosciences) was added to the culture. Cells were for staining of anti-Nkrp1g mAb 8A10 and anti–Clr-f mAb 10A8, mouse further incubated for 3 h and finally analyzed for intracellular IFN-g ex- anti-rat IgM-bio (BD Biosciences) for staining of anti–Clr-f/Clr-g mAb pression by flow cytometry. Permeabilization and fixation was done with 1H7, streptavidin-allophycocyanin and -PE (Jackson ImmunoResearch) for BD Cytofix/Cytoperm (BD Biosciences). As control, cells were only in- staining of biotinylated secondary Abs. All gatings included a singlet cubated with IL-15. The specific fluorescence index (SFI) of Nkrp1g+/2 gating and a viability staining. Single-cell suspensions of splenocytes or NK cells was calculated as follows: mean fluorescence intensity IFN-g NK cells were used for sorting indicated cell populations (purity .98%) (IL-12, IL-18, IL-15) – mean fluorescence intensity IFN-g (IL-15). 4 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN

Reporter cell assay pattern similar to splenic NK cells, indicating there is a non–tissue- + BWN.3G-Nkrp1g reporter cells (1 3 104) were cocultured with poly(I:C)- resident Nkrp1g NK cell population (Fig. 1D). Flow cytometric activated and FACS-sorted DC populations or monocytes at a ratio of 1:2 analyses of NK cell maturation stages as defined by CD27 and for 18 h at 37˚C in the presence or absence of mAb 1H7 in 96-well round- CD11b (51) did not reveal a pronounced association of Nkrp1g bottom plates. Coculture with CHO transfectants or stimulation with expression with NK cell maturation although there was a minor, but 25 ng/ml PMA and 1 mM ionomycin served as controls. Subsequently cells significant, increase of Nkrp1g abundance subsequent to stage I were washed and BWN.3G-Nkrp1g cells analyzed for GFP expression by 2 2 flow cytometry. (CD27 CD11b ) (Fig. 1F). Microarray analysis Nkrp1g is upregulated on cytokine-stimulated NK cells NK cells were purified with mouse CD49b MicroBeads (Miltenyi Biotec) Recently, for rat Nkrp1b and Nkrp1g receptors, a modulation of and NK cells from spleens of eight mice were sorted using FACS according surface expression upon stimulation with cytokines has been to Nkrp1g expression as detected with mAb 8A10. RNA was isolated from + 2 2 2 + reported (52). Hence, we addressed modulation of Nkrp1g on sorted Nkrp1g and Nkrp1g splenic NK cells (CD19 CD3 CD49b splenic NK cells by cytokines. To this end, we isolated splenic NK NKp46+ lymphocytes), respectively, and subjected to microarray analysis (ATLAS Biolabs, Berlin, Germany) using an Affymetrix GeneChip Mouse cells and stimulated them in vitro with various cytokines for 7 d, Gene 2.0 ST array system. Three biological replicates of each cell type and subsequently analyzed Nkrp1g expression by flow cytometry were analyzed. Bioinformatics analysis was performed by ATLAS Biolabs. (Fig. 2A). Of note, Nkrp1g expression was strongly increased on Microarray data (accession number E-MTAB-6223) were deposited at the surface of NK cells upon treatment with either IL-2 or IL-15, ArrayExpress (https://www.ebi.ac.uk/arrayexpress). with an expression 4–5 fold higher as compared with unstimulated

Statistical analysis NK cells (Fig. 2A, 2B). Nkrp1g expression was not further in- Downloaded from Statistical analyses are detailed in the figure legends and were performed creased upon combined IL-2/IL-15 treatment, but was not any- with GraphPad Prism 5 (GraphPad Software, San Diego, CA). more detectable upon incubation with inflammatory cytokines IL-12 and IL-18 (Fig. 2A). Flow cytometry staining of Nkrp1g Results by mAb 8A10 was again corroborated by FACS sorting of the Splenic Nkrp1g expression is confined to a small subpopulation respective NK cell populations and subsequent qPCR analysis of of NK cells Nkrp1g transcripts, revealing that Nkrp1g transcripts exclusively http://www.jimmunol.org/ reside in the NK cell subpopulation stained by 8A10 (Fig. 2C). We recently reported that expression of Nkrp1g receptors in the Unexpectedly, increased Nkrp1g expression on cytokine-activated gastrointestinal tract is restricted to a subset of IEL and that this NK cells was not reflected in increased relative levels of Nkrp1g IEL-specific Nkrp1g expression is spatially matched by a selective transcripts in Nkrp1g+ cytokine-activated NK cells versus expression of the Nkrp1g-ligand Clr-f on IEC (30). A tissue screen Nkrp1g+ resting NK cells, indicating regulation by posttranscrip- of BALB/c mice repeatedly exhibited in addition appreciable tional mechanisms (Fig. 2C). amounts of Nkrp1g transcripts in the spleen (Fig. 1A), which prompted us to investigate the yet undefined Nkrp1g receptor Nkrp1g homodimers are inherently expressed by a small subset of NK cells expression in the spleen. Analysis of sorted splenic lymphocyte by guest on September 26, 2021 populations by qPCR clearly attributed Nkrp1g transcripts to the The C-type lectin-like CLEC2 and NKRP1 family members have NK cell population with transcripts being absent from B cells and been described as homo- or heterodimeric disulfide-linked gly- almost undetectable in T cells (Fig. 1B). To corroborate these coproteins (6). Accordingly, we detected homodimers of Nkrp1g results, we made use of two immune-deficient mouse strains: the in nonreducing immunoblots of lysates of IL-15–stimulated NK NOD SCID mouse, which lacks B and T cells, and the NOD SCID cells from spleens of both BALB/c and NOD SCID mice as well GAMMA mouse, which lacks B, T, and NK cells. In line with as in lysates of human NK-92MI cells transduced with Nkrp1g previous findings, splenocytes from BALB/c and NOD SCID mice (NK-92MI-Nkrp1g), which were included for control (Fig. 2D). contained substantial amounts of Nkrp1g transcripts (Fig. 1C). In Of note, there were also higher molecular forms (∼130 kDa) of contrast, spleens of NOD SCID GAMMA mice were fully devoid Nkrp1g multimers in lysates of splenic NK cells, but not in NK- of Nkrp1g transcripts, consistent with the absence of NK cells in 92MI-Nkrp1g controls, possibly corresponding to Nkrp1g tetra- these mice. To further define the physiological expression of mers or Nkrp1g heteromers. Next, we addressed inherent stability Nkrp1g in the spleen, which had been remained undefined mainly of Nkrp1g expression by NK cells. NK cells were isolated from due to the lack of specific mAb, we assayed the expression on NK spleens of BALB/c mice, stimulated with IL-15 for 7 d in vitro, cells by flow cytometry using the recently reported Nkrp1g- and then sorted via FACS according to Nkrp1g expression using specific mAb 8A10 (Fig. 1D) (30). A small NK cell subpopula- mAb 8A10. Subsequently, the Nkrp1g+ and Nkrp1g2 populations tion (0.5–1%) from the spleens of BALB/c mice was stained by were separately stimulated for another 7 d with IL-15 and then mAb 8A10 and this staining was blocked by preincubation with analyzed for Nkrp1g expression by flow cytometry (Fig. 2E). soluble Nkrp1g supporting specificity (Fig. 1D). The flow cyto- Remarkably, the Nkrp1g2 NK cell fraction remained fully nega- + metric staining of Nkrp1g NK cells by mAb 8A10 was further tive for Nkrp1g, whereas the Nkrp1g+ mostly maintained the ini- corroborated by using FACS to sort the 8A10-stained NK cell tial Nkrp1g expression (96.9% after sorting; 87.2% after 7 d IL-15). population followed by qPCR analysis for Nkrp1g transcripts A differential proliferation of both subsets was addressed by a CFSE + (Fig. 1E). Sorted Nkrp1g gd T cells from the small intestine (30) proliferation assay (Fig. 2F), which did not reveal any significant served as comparison. Abundance of Nkrp1g transcripts perfectly difference in proliferation of Nkrp1g+ versus Nkrp1g2 NK cells after matched stainings of mAb 8A10 in splenic NK cells sorted with 3 or 7 d, respectively. In conclusion, Nkrp1g expression appears to be + FACS with levels of Nkrp1g transcripts among splenic Nkrp1g stably associated with a small subset of splenic NK cells, pointing to + NK cells almost identical to intestinal Nkrp1g gd T cells an inherent Nkrp1g expression phenotype of these NK cells. (Fig. 1E). Additionally, NK cells that were not stained by mAb 8A10 also were nearly devoid of Nkrp1g transcripts, conclusively Strain-specific expression of Nkrp1g on BALB/c NK cells demonstrating specificity of 8A10 staining for Nkrp1g. Further- Intestinal Nkrp1g expression on IELs has been reported to be strain more, NK cells from peripheral blood showed an Nkrp1g expression specific, because there are ∼10% Nkrp1g+ gd IEL in BALB/c The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Nkrp1g is expressed on a small population of splenic NK cells. Relative abundance of Nkrp1g transcripts (A) in various tissues of female BALB/c mice, (B) in FACS sorted subpopulations of splenic lymphocytes from female BALB/c mice, (C) in spleens of immunodeficient mouse strains, and (E) in intestinal gd T cells (CD3+CD103+gdTCR+ lymphocytes), and splenic NK cells (CD192CD32CD49b+NKp46+ lymphocytes) from female BALB/c mice, FACS sorted according to their staining with mAb 8A10 (Nkrp1g+/2). (A–C and E) Assessment of relative abundance of Nkrp1g transcripts was performed by qPCR using primers Nkrp1g forward/Nkrp1g reverse. All data were normalized to 18S rRNA and arbitrarily set relative to transcript levelsin samples marked by a cross (3). One representative of at least two independent experiments with at least three mice per group is shown. Bars represent mean 6 SD. ND, not detectable. (D) Representative dot plots showing flow cytometry analysis with mAb 8A10 (aNkrp1g) on resting NK cells (CD192 CD32CD49b+NKp46+ lymphocytes) from spleen and blood of female BALB/c mice. Preincubation of mAb 8A10 or respective isotype control with the soluble C-type lectin-like domain of Nkrp1g was used for blocking. One representative of at least two independent experiments is shown. (F) Percentage of Nkrp1g+ splenic NK cells (CD192CD32CD49b+NKp46+ lymphocytes) among the different NK cell maturation stages as defined by CD27 and CD11b. Stage I: CD272CD11b2, stage II: CD27+CD11b2, stage III: CD27+CD11b+, stage IV: CD272CD11b+. Shown is the mean 6 SD of 18 experiments. One- way ANOVA was performed with a Tukey posttest (*p , 0.05) for comparison. mice, whereas only ∼1% of gd IEL of C57BL/6 mice express regulatory mechanism may differentially control Nkrp1g surface Nkrp1g at the cell surface (30). In this study, we observed a expression in BALB/c versus C57BL/6 mice. Low frequencies of similar strain-associated bias for Nkrp1g expression by splenic Nkrp1g+ NK cells (,1%) were also found among splenic NK NK cells with frequencies of Nkrp1g+ NK cells being about four cells from inbred mouse strains C3H and 129 as well as from times higher among BALB/c NK cells as compared with C57BL/6 outbred mouse strain CD-1 (data not shown), suggesting that NK cells (∼0.8% versus 0.2%) (Fig. 3A). However, Nkrp1g ex- rareness of Nkrp1g+ splenic NK cells is a general feature of Mus pression in C57BL/6 mice does not appear to be restricted tran- musculus. scriptionally, as Nkrp1g transcript expression is five times higher + in splenocytes of C57BL/6 mice compared with splenocytes of Characterization of Nkrp1g NK cells BALB/c mice (Fig. 3B), although frequencies of NK cells in To more precisely define the Nkrp1g+ NK cell subset, we per- C57BL/6 spleens were lower than in BALB/c (BALB/c: ∼3.2% formed flow cytometry stainings for several NK cell markers and NK cells, C57BL/6: ∼1.6% NK cells). Amino acid sequences of receptors and found that Nkrp1g+ NK cells are more skewed to- Nkrp1g differ in both mouse strains by a conservative substitution ward expressing the inhibitory Ly49C receptor on their surface of the carboxyterminal amino acid (Ile in BALB/c versus Val in (Fig. 4A, 4B). Further characterization of Nkrp1g+ NK cells was C57BL/6) (Fig. 3C), which is unlikely to explain the marked carried out by microarray analysis of FACS sorted Nkrp1g+ versus differences in Nkrp1g+ NK subsets by altered protein expression Nkrp1g2 splenic NK cells. We found 214 differentially regulated or a differential recognition by mAb 8A10. Rather, an additional genes that were at least 2-fold up- or downregulated (38 upregu- 6 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Nkrp1g is upregulated on cytokine-stimulated NK cells. NK cells from spleens of female BALB/c mice were MACS purified and stimulated with indicated cytokines for several days. (A) Representative dot plots showing flow cytometry analysis with mAb 8A10 (aNkrp1g) on resting NK cells directly after purification and on cytokine-stimulated NK cells after 7 d (NK cells: CD192CD32CD49b+NKp46+ lymphocytes). (B) SFI of geometric mean fluorescence intensities for Nkrp1g on Nkrp1g+ NK cells shown in (A). Compiled data from two experiments are shown with means and SDs. (C) Relative abundance of Nkrp1g transcripts in splenic NK cells (CD192CD32CD49b+NKp46+ lymphocytes) stimulated with IL-2 and IL-15 for 7 d and FACS sorted according to mAb 8A10 staining (Nkrp1g+/2). Data for spleen, MACS purified NK cells, and Nkrp1g+/2 resting NK cells were included for comparison. Relative abundance of Nkrp1g transcripts was determined by qPCR using primers Nkrp1g forward/Nkrp1g reverse. Data were normalized to 18S rRNA and arbitrarily set relative to transcript levels in the sample marked by a cross (3). Bars represent mean 6 SD. (D) Nkrp1g glycoproteins in lysates of BALB/c or NOD SCID NK cells, cytokine stimulated for 14 d in vitro, were detected with mAb 8A10-bio in immunoblotting. Lysates of NK-92MI-Nkrp1g transductants served as control (left). Lysates were deglycosylated by PNGaseF and separated by SDS-PAGE under nonreducing conditions. (E) Stable Nkrp1g expression on splenic NK cells (CD192CD32CD49b+NKp46+ lymphocytes). Splenic NK cells were MACS purified and cytokine stimulated for 7 d and then subjected to FACS sorting according to Nkrp1g expression (mAb 8A10) (left). Purity after sorting is shown. FACS (Figure legend continues) The Journal of Immunology 7 lated versus 176 downregulated). A selection of differentially From these qPCR-based tissue screens, Clr-g was singled out expressed immune-related genes is displayed in Fig. 4C. Among as putative ligand of splenic Nkrp1g+ NK cells, because both Clr-d the downregulated genes were transcription factors T-bet and and Clr-f transcripts were virtually absent from the spleen. The Rxra, the CLEC2 family member and Nkrp1b ligand Clr-b splenic compartment was further fractionated by FACS sorting (Clec2d) as well as S1pr1. However, no significant differences into various leukocyte populations, which were analyzed by qPCR were detected for transcription factors Eomes, GATA3, and for Clr-g transcripts. It turned out that Clr-g transcripts are broadly RORgT, indicating that Nkrp1g+ NK cells are bona fide NK cells. present in both splenic lymphocytes (T cells, B cells, NK cells) Genes upregulated in Nkrp1g+ NK cells were Ly49e and the decay and myeloid cells (monocytes, DCs) at similar levels (Fig. 5E). accelerating factor CD55. The latter was also found upregulated in Next, we assessed the abundance of Clr-g transcripts in various Nkrp1g+ gd IEL (30), linking expression of Nkrp1g and CD55 in a mouse cell lines (Fig. 5F). All tested T cell lines (YAC-1, RMA, yet unknown fashion. GO enrichment analysis of downregulated RMA-S, EL4) and B cell lines (Ba/F3, SP2/0) contained Clr-g genes revealed several enriched gene ontology terms, most nota- transcripts at levels roughly equivalent to splenic lymphocytes. bly innate immune response in mucosa and lymphocyte migra- Of note, the NK sensitive T cell line YAC-1 contained the lowest tion, which may possibly indicate a less migratory property levels of Clr-g transcripts, whereas both B cell lines exhibited the (Supplemental Table I). Nkrp1f shares two ligands with Nkrp1g highest levels in line with highest transcript levels found in splenic (Clr-d and Clr-g), and therefore has been considered as the B cells. However, nonlymphocytic hematopoietic cell lines (P815, complementary receptor for Nkrp1g. Hence, coexpression of RAW309, DC2.4) as well as cell lines of nonhematopoietic origin Nkrp1f on Nkrp1g+ NK cells was of particular interest. Analyzing (B16F10, MC38) were fully devoid of Clr-g transcripts (Fig. 5F).

Nkrp1f transcripts in subsets of lymphocytes and DCs clearly Downloaded from Converse Clr-g surface expression on cell lines and splenic confines Nkrp1f expression to NK cells (Fig. 4D). However, leukocytes Nkrp1f surface expression on Nkrp1g+ NK cells did not signifi- cantly vary from the previously described broad expression of Detection of abundant Clr-g transcripts in leukocytes as well as in T Nkrp1f on the vast majority of splenic NK cells (Fig. 4E) (28). and B cell lines then prompted us to characterize Clr-g surface Finally, to address functional differences between Nkrp1g+ and expression by flow cytometry. By immunizing rats with 2

Nkrp1g NK cells, we assessed degranulation and IFN-g secre- recombinant soluble Clr-f we had generated mAb 1H7 that detects http://www.jimmunol.org/ tion. We consistently observed that IL-15–stimulated Nkrp1g+ NK both Clr-f and Clr-g, but does not cross-react with Clr-a, Clr-b, or cells are producing significantly more IFN-g after exposure to Clr-c, respectively (Supplemental Fig. 2). Hence, we performed IL-12 plus IL-18 than Nkrp1g2 NK cells (Fig. 4F), whereas no flow cytometry with mAb 1H7 always in parallel with the Clr-f differences were observed with regard to degranulation in re- specific mAb 10A8 to address a potential confounding effect of sponse to several target cells (data not shown). Clr-f expression. However, mAb 10A8 did not bind to any of the cell lines tested nor to any splenic subpopulation well in line with the complete lack of Clr-f transcripts (Supplemental Fig. 1). Differential Nkrp1g ligand expression in the splenic In contrast, mAb 1H7 strongly stained all T cell lines (YAC-1, compartment RMA, RMA-S, EL4) and B cell lines (Ba/F3, SP2/0) shown to by guest on September 26, 2021 Nkrp1g ligates three CLEC2 members, namely Clr-d, Clr-f, and abundantly contain Clr-g transcripts, whereas all other cell lines, Clr-g (18, 29). A qPCR-based tissue screen of BALB/c mice for which are devoid of Clr-g transcripts (P815, RAW309, DC2.4, Clr-f transcripts revealed abundant amounts of Clr-f transcripts in B16F10, MC38), also did not bind mAb 1H7, establishing a the small intestine, and, at lower levels, in the colon and kidney, as perfect match between 1H7 binding and abundance of Clr-g previously described (30). However, Clr-f transcripts were barely transcripts (Fig. 6A). detectable or undetectable in all other tissues analyzed including We next analyzed splenic B cells, T cells, NK cells, pDCs, spleen and lymph nodes (Fig. 5A). Because a previous study had CD8a+/2 cDCs, and Ly6Chi/lo monocytes for Clr-g surface ex- described Clr-f transcripts in LAK cells (5), we stained various pression by flow cytometry. Unexpectedly, mAb 1H7 did not de- splenic leukocyte populations, cytokine-stimulated NK cells, and tect any surface Clr-g on B cells, T cells, NK cells, or DC LAK cells with the Clr-f-specific mAb 10A6 and 10A8 (30), but populations, despite Clr-g transcript levels comparable to T and could not detect any Clr-f surface expression (Supplemental Fig. 1). B cell lines. Only monocytes weakly bound mAb 1H7 (Fig. 6B). Further, we assessed levels of Clr-f transcripts in LAK cells and Previously, we described that Clr-f is upregulated on IECs after IL-15–stimulated NK cells and found that they were more than challenge with poly(I:C) (30). Therefore, we wondered whether 10,000-fold lower than in the small intestine, explaining the lack Clr-g surface expression may become detectable upon in vivo of cell surface expression (Fig. 5B). Subsequently, we performed challenge with poly(I:C). Although monocytes and lymphocytes qPCR-based tissue screens of BALB/c mice for transcripts of the from spleens of poly(I:C)-treated mice were not stained, there was other Nkrp1g-ligands Clr-d and Clr-g, respectively. Although Clr- a strong and specific induction of Clr-g surface expression on d transcripts are most abundant in the eye, and at lower levels CD8a+ cDCs (Fig. 6C). To validate these results, we analyzed detectable in lung, small intestine, colon, thymus, and kidney, they FACS sorted subsets of splenic DCs and monocytes for Clr-g were hardly detectable in the spleen (Fig. 5C). In stark contrast, transcripts. In line with the results above, Clr-g transcripts were Clr-g transcripts are most abundant in lymphoid organs such as the detected in all subsets of DCs and monocytes from naive mice at spleen and the thymus, strongly arguing for a preferential ex- roughly similar levels (Fig. 6D). However, analysis of these sub- pression by hematopoietic cells (Fig. 5D). sets from poly(I:C)-treated mice, revealed a pronounced selective

sorted NK cells were recultured with IL-15 for another 7 d and then reanalyzed for Nkrp1g expression by flow cytometry staining with mAb 8A10 (right). (F) Assessment of proliferation of Nkrp1g+/2 NK cells by CFSE staining. NK cells were MACS purified, stained with CFSE on day 0, and then cultured with IL-15 for up to 7 d. CFSE intensity of Nkrp1g+ (solid lines) and Nkrp1g2 (shaded histograms) NK cells (CD192CD32NKp46+ lymphocytes) was determined on the indicated days. (A–F) One representative of at least two independent experiments with at least three mice per group is shown. 8 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN

Clr-g surface expression on CD8a+ cDCs is type I IFN receptor dependent CD8a+ cDCs represent the major DC population expressing TLR3 and are hence directly responsive to stimulation with poly(I:C), a mimic of viral dsRNA. As we had observed a poly(I:C)-induced surface expression of Clr-g on CD8a+ cDCs in vivo after 16 h, we wondered if this could be recapitulated in vitro. To this end, we stimulated splenocytes in vitro with different TLR ligands [poly(I:C), LPS, R848] for 4 h (Fig. 7A). Interestingly, only R848, a ligand of TLR7/8, was able to induce Clr-g surface expression on the pDC population in vitro after this short timeframe. To further analyze the kinetic of poly(I:C)-induced Clr-g expression on CD8a+ cDCs, we treated mice with poly(I:C) and harvested spleens at different time points to stain for Clr-g expression (Fig. 7B). Clr-g surface expression on CD8a+ cDCs could be only detected after 16 h but not at earlier time points, suggesting that Clr-g might not be directly induced upon TLR3 stimulation. A previous study has shown that signaling by the type I IFN receptor (IFNAR) plays an indispensable role in inducing immunogenicity of DCs after poly Downloaded from (I:C) treatment by analyzing gene expression of wild-type DCs or IFNAR2/2 DCs upon poly(I:C) injection (53). Analyzing this available dataset for Clr-g expression (gene Clec2i) revealed that induced Clr-g expression by DCs upon poly(I:C) treatment was strongly dependent on IFNAR (Fig. 7C), supporting the assertion of Clr-g being secondarily induced upon TLR3 stimulation. http://www.jimmunol.org/

Nkrp1g and Clr-g interaction is inhibitory To address functional consequences of an interaction between Nkrp1g and Clr-g, we performed a degranulation assay using NK- 92MI-Nkrp1g transductants together with CHO-Clr-g transfectants or control-transfected CHO cells (Fig. 8A). Degranulation of NK- 92MI-Nkrp1g cells in cocultures with CHO–Clr-g cells was strongly reduced as compared with cocultures with CHO-mock FIGURE 3. Strain-specific expression of Nkrp1g in BALB/c mice. by guest on September 26, 2021 (A) Percentage of Nkrp1g+ splenic NK cells (CD192CD32CD49b+ cells, but could partially be restored when Clr-g–specific mAb NKp46+ lymphocytes) in 47 BALB/c and 10 C57BL/6 mice, respectively. 1H7 was added to the culture (Fig. 8A). In chromium release Shown is the mean 6 SD. Unpaired t test was performed (****p , 0.0001) assays, CHO–Clr-g and CHO-mock cells were similarly lysed by for comparison. (B) Relative abundance of Nkrp1g transcripts in single-cell control-transduced NK-92MI cells, whereas cytolysis of CHO– suspensions from indicated organs of BALB/c and C57BL/6 mice. Single- Clr-g by NK-92MI-Nkrp1g was selectively abrogated as com- cell suspensions of BALB/c mice contained on average 3.2% NK cells and pared with CHO-mock cells (Fig. 8B). These experiments showed 2.2% IELs. Single-cell suspensions of C57BL/6 mice contained on average that Nkrp1g engagement by Clr-g effectively inhibits effector 1.6% NK cells and 1.3% IELs. Nkrp1g transcripts were determined by qPCR using primers Nkrp1g forward/Nkrp1g reverse. Data were normalized to 18S functions of NK-92 cells. This is likely mediated through ITIM rRNA and arbitrarily set relative to transcript levels in the sample marked by signaling as Nkrp1g bears an ITIM motif in its cytoplasmic tail across(3). Bars represent mean 6 SD. One experiment with three mice per (29). Inhibitory receptors bearing an ITIM in their cytoplasmic tail group is shown. One-way ANOVA with a Tukey posttest was performed become phosphorylated by Src kinases upon activation and allow (***p , 0.001) for comparison. (C) Alignment of Nkrp1g amino acid se- recruitment of phosphotyrosine phosphatases such as SHP-1 as quences from BALB/c and C57BL/6 mouse strains. Differences are high- shown for inhibitory Nkrp1b (54, 55). We therefore attempted to lighted in bold and are boxed. coimmunoprecipitate SHP-1 with Nkrp1g from IL-15–expanded BALB/c NK cells (Fig. 8C). Immunoblotting detected SHP-1 in induction of Clr-g transcripts in CD8a+ cDCs (∼35-fold) well in Nkrp1g-immunoprecipitates, independently of a pretreatment with line with the observed upregulation of Clr-g on the cell surface. pervanadate, pointing to a constitutive association of SHP-1 with We considered the possibility that alternative splicing and ex- Nkrp1g. pression of different Clr-g isoforms in splenic lymphocytes versus Finally, we addressed whether Clr-g induced on CD8a+ cDCs T and B cell lines may account for the observed discrepancy be- upon poly(I:C) challenge can be recognized via Nkrp1g. To this 2 tween Clr-g transcript levels and Clr-g surface expression. There aim, pDCs, CD8a+ cDCs, CD8a cDCs, and monocytes were are four isoforms of differentially spliced Clr-g transcripts FACS sorted from splenocytes of poly(I:C)-treated mice and (Supplemental Fig. 3A). Using a PCR-based assay distinguishing cultured together with BWN-Nkrp1g transfectants in the absence all four isoforms, we found that Clr-g transcript isoform 2 is or presence of mAb 1H7. BWN-Nkrp1g reporter cells specifically uniformly and predominantly expressed in all tested DCs, respond to CHO–Clr-g cells, but not to CHO-mock controls, and monocytes, and lymphocytes, as well as in Clr-g–expressing cell the response to CHO–Clr-g can be specifically blocked by addition lines (Supplemental Fig. 3B, 3C), suggesting that alternative of mAb 1H7 to the cocultures (Fig. 8D). Although BWN-Nkrp1g splicing of Clr-g transcripts is not accountable for the deficient reporter cells did not specifically react in cocultures with pDCs, 2 Clr-g surface expression on lymphocytes. CD8a cDCs, and monocytes, they specifically responded to The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 4. Characterization of Nkrp1g+ NK cells. (A) Representative histogram of Ly49C surface expression on splenic Nkrp1g+/2 NK cells (CD192 CD32NKp46+ lymphocytes). Flow cytometry staining with mAb 8A10 and 5E6 is shown. One representative of three independent experiments is shown. (B) Percentage of Ly49C+ NK cells among Nkrp1g+/2 cell populations. Unpaired t test was performed (***p , 0.0001) for comparison. Data compiled from three independent experiments are shown. (C) Microarray analysis of RNA isolated from FACS sorted Nkrp1g+ versus Nkrp1g2 splenic NK cells (CD192CD32CD49b+NKp46+ lymphocytes) was performed by ATLAS Biolabs using an Affymetrix GeneChip Mouse Gene 2.0 ST array system. A by guest on September 26, 2021 selection of differentially expressed immune-related genes is shown. (D) Relative abundance of Nkrp1f transcripts in FACS sorted leukocytes from female BALB/c mice. Nkrp1f transcripts were determined by qPCR using primers Nkrp1f forward/Nkrp1f reverse. Data were normalized to 18S rRNA and ar- bitrarily set relative to transcript levels in the sample marked by a cross (3). Bars represent mean 6 SD. One experiment with at least three mice per group is shown. (E) SFI of geometric mean fluorescence intensities of Nkrp1f on resting or IL-15–stimulated Nkrp1g+/2 NK cells (CD192CD32NKp46+ lymphocytes) determined by flow cytometry staining. Shown is the mean 6 SD. One representative of two independent experiments with two mice is shown. (F) Relative SFI of geometric mean fluorescence intensities of IFN-g in Nkrp1g+/2 NK cells (CD192CD32NKp46+ lymphocytes) determined by intracellular FACS staining. Splenic NK cells were stimulated for 5 d with IL-15 and IFN-g production was then triggered by a combination of IL-12, IL-15, and IL-18 for 4 h. Shown is the mean 6 SD. Compiled data from five independent experiments with at least three mice per experiment. Unpaired t test was performed (****p , 0.0001) for comparison.

CD8a+ cDCs in a Clr-g–specific manner as the response could be ligands on DCs reportedly shape the DC repertoire (62–67). It has blocked by the addition of mAb 1H7 (Fig. 8D). also been shown that Clr-b and Nkrp1b play a role in NK-DC interactions (8, 68). Discussion In this study we report an exclusive expression of the C-type Understanding immune cell activation and regulation is indis- lectin-like receptor Nkrp1g on a minor subset of splenic NK pensable when it comes to the treatment of infectious diseases or cells, which is matched by selective expression of the Nkrp1g cancer. NK cells in this study play an important role by eradicating ligand Clr-g on activated splenic DCs. Previous studies indicated infected or transformed cells, which is mediated through a plethora the presence of Nkrp1g transcripts in the intestine, spleen, and of germline-encoded activating and inhibitory receptors (3, 56, 57). thymus of various strains of mice (30, 31), but cellular Nkrp1g However, NK cells not only eliminate dangerous cells through expression as well as splenocytes expressing Nkrp1g receptors cytotoxic activity, they also shape the adaptive immune response remained undefined. Recently, we showed that in the small in- by killing activated lymphocytes and engaging in a mutual cross- testine, Nkrp1g is expressed on subsets of IEL, particularly on gd talk with DCs (58, 59). Both soluble factors and cell-cell contacts IEL (30). In contrast, we find Nkrp1g in the spleen exclusively contribute to this NK-DC cross-talk. For example, cytokines such expressed by a minor subset of NK cells, which is also present in as IL-12, -15, and -18, and type I IFNs from activated DCs can the periphery (Fig. 1D). Nkrp1g expression on resting NK cells is stimulate IFN-g secretion and cytotoxicity of NK cells (47, 60, rather weak, but is markedly upregulated upon exposure to cyto- 61). In turn, TNF or IFN-g secreted by NK cells promote the kines IL-2 and/or IL-15. This is well in line with a previous study maturation of DCs and thereby influence the priming of T cell on rat NK cells showing upregulation of Nkrp1g in response responses. In addition, interactions of NK cell receptors such as to IL-2 treatment (52). This study also reported an upregulation NKp30, CD94/Nkg2a, Nkg2d, and CD27 with their respective of Nkrp1g on rat NK cells upon IL-18 treatment. However, we 10 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Nkrp1g ligand expression in the splenic compartment. Various tissues of female BALB/c mice were assessed for abundance of (A) Clr-f, (C) Clr-d, and (D) Clr-g transcripts. (B) Relative abundance of Clr-f transcripts in purified LAK or IL-15–stimulated NK cells from female BALB/c mice. The intestine was included for comparison. (E and F) Clr-g transcript abundance in sorted splenic leukocytes (E) and murine cell lines (F). T cells, B cells, and spleen were used for comparison. (A–F) Relative abundance of transcripts was determined by qPCR using specific primers Clr-f forward/Clr-f reverse, Clr-d forward/Clr-d reverse, or Clr-g forward/Clr-g reverse. All data were normalized to 18S rRNA and arbitrarily set relative to transcript levels in samples marked by a cross (3). One representative of at least two independent experiments with at least three mice per group is shown. Bars represent mean 6 SD. ND, not detectable. (E) Gating strategy: NK cells (CD192CD32NKp46+), B cells (CD19+), T cells (CD192NKp462CD3+), monocytes (CD192CD32 NKp462CD14+), cDCs (CD192CD32NKp462CD11c+). observed a loss of Nkrp1g+ mouse NK cells following exposure to Characterization of Nkrp1g+ NK cells revealed an increased IL-18, pointing to a differential regulation of Nkrp1g in rat and coexpression of the inhibitory Ly49C, indicating a population of mouse. In further contrast to mice, Nkrp1g is expressed more NK cells that is licensed and controlled by MHC class I–specific broadly in rats, with an expression on 10% NK cells and 13% NK- receptors. Of note, we also found that Nkrp1g expression in both like T cell subsets (52). Nkrp1g expression of mouse NK cells IEL and splenic NK cells is linked to an enhanced expression of appears to be quite stable and limited to a small subset, pointing to CD55, which is protective against lysis by the complement sys- an epigenetically fixed expression pattern. However, Nkrp1g sur- tem. Nkrp1f, which shares ligands Clr-d and Clr-g with Nkrp1g, face expression is regulated in a strain-specific manner, as was not differentially expressed between Nkrp1g+ and Nkrp1g2 Nkrp1g+ IELs and Nkrp1g+ splenic NK cells are almost absent in cells in line with the findings in rats, where Nkrp1f is also coex- C57BL/6 mice as compared with BALB/c mice, although Nkrp1g pressed on NK cells (52). transcripts are present at similar levels. This points to a differential By qPCR-based tissue screens we singled out Clr-g as a putative posttranscriptional regulation of Nkrp1g expression in these splenic Nkrp1g ligand, as Clr-d and Clr-f transcripts were almost mouse strains. Similarly, Nkrp1g transcript levels in cytokine- absent from the spleen. Our data further support the idea that most stimulated Nkrp1g+ NK cells were lower than in resting Clr molecules are differentially expressed in a tissue-specific or Nkrp1g+ NK cells despite high Nkrp1g surface expression on tissue-biased manner, and thus may be involved in a tissue-specific activated NK cells, suggesting an additional level of regulation. immunosurveillance (30, 31). The only exception thus far is Clr-b, The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Clr-g surface expression on cell lines, lymphocytes, monocytes, and DC subsets. Representative histograms of (A) various murine cell lines, (B)resting,or(C) poly(I:C)-activated splenic leukocyte populations from female BALB/c mice stained with anti–Clr-g mAb 1H7. One repre- sentative of at least two independent experiments is shown. (B and C) Gating strategy: NK cells (CD192CD32CD49b+NKp46+), B cells (CD19+), Tcells(CD192NKp462CD3+), cDCs CD8a+ (CD192Ly-6G2NKp462I-A/I-E+CD11c+CD11b2CD8a+), cDCs CD8a2 (CD192Ly-6G2NKp462I-A/I- E+CD11c+CD11b+CD8a2), pDCs (CD192Ly-6G2NKp462CD11cintPDCA-1+), monocytes Ly6Chi/lo (CD192Ly-6G2NKp462CD11c2CD11b+SSC- AsmallLy6Chi/lo). (D) Relative abundance of Clr-g transcripts in FACS sorted resting or poly(I:C)-activated DCs or monocytes. Abundance of transcripts was determined by qPCR using specific primers Clr-g forward/Clr-g reverse. All data were normalized to 18S rRNA and arbitrarily set relative to transcript levels in the sample marked by a cross (3). Bars represent mean 6 SD. ND, not detectable. One experiment with three mice per group is shown. 12 Nkrp1g AND Clr-g EXPRESSION IN THE SPLEEN Downloaded from

FIGURE 7. Clr-g surface expression on cDCs is IFNAR dependent. +/2

(A) Clr-g surface expression on CD8a cDCs or pDCs after 4 h of in vitro http://www.jimmunol.org/ stimulation with indicated TLR agonists. Histograms of flow cytometry staining with mAb 1H7 are depicted. Gating strategy: cDCs CD8a+ (CD192 Ly-6G2NKp462I-A/I-E+CD11c+CD11b2CD8a+)cDCsCD8a2 (CD192 Ly-6G2NKp462I-A/I-E+CD11c+CD11b+CD8a2), pDCs (CD192Ly-6G2 NKp462CD11cintPDCA-1+). One representative of at least two independent experiments is shown. (B) Clr-g surface expression on CD8a+ cDCs (CD192 2 2 2 FIGURE 8. Nkrp1g and Clr-g interaction is inhibitory. (A) Degranula- Ly-6G NKp46 I-A/I-E+CD11c+CD11b ) upon in vivo poly(I:C) challenge tion of NK-92MI-Nkrp1g cells after coculture with CHO cells stably for indicated time points determined by flow cytometry staining with mAb expressing Clr-g at an E:T ratio of 1:1. Mock transfectants and PMA/ 1H7. One representative of three independent experiments is shown. (C)

ionomycin served as control. mAb 1H7 was used to block interaction by guest on September 26, 2021 Abundance of Clr-g transcripts in indicated DC populations extracted from between Nkrp1g and Clr-g. Percentages of CD107a+ NK-92MI-Nkrp1g microarray data generated by Pantel et al. (53). The microarray data are cells (CD56+) are shown. One-way ANOVA with a Tukey posttest was available through the National Center for Biotechnology Information Gene used for comparison (***p , 0.001). (B) Chromium release from CHO- Expression Omnibus under accession number GSE46478. One-way ANOVA mock–Clr-g target cells after coculture with NK-92MI-mock or -Nkrp1g with a Tukey posttest was used for comparison (***p , 0.001). effector cells. Specific lysis of target cells by effectors at indicated E:T ratios is depicted. (A and B) One representative of three independent ex- which is expressed rather ubiquitously like MHC class I (30, 31), periments is shown. (C) Coimmunoprecipitation of SHP-1 with Nkrp1g and ligates the inhibitory receptor Nkrp1b presumably acting as an from protein lysates of 22–28 d IL-15–stimulated BALB/c NK cells. Cells MHC class I–independent missing-self detection system (19, 23, were stimulated with pervanadate or left untouched and subsequently 25, 26). In contrast, expression of the three Clr molecules ligated lysed. The lysates were incubated with protein A/G magnetic beads coated by the inhibitory Nkrp1g receptor is strongly biased toward spe- with rIgG1 or 8A10 to precipitate Nkrp1g. Precipitated proteins were separated by SDS-PAGE under nonreducing conditions, blotted, and pro- cific cell types: Clr-f expression is restricted to the gastrointestinal bed with aSHP-1 mAb. Input fractions of the respective immunoprecipi- epithelium and the kidney, Clr-d is preferentially expressed in the tations served as control for SHP-1 presence in lysates. SHP-1 is marked eye, whereas Clr-g expression appears to be associated with cells with an arrowhead. Two out of three independent experiments are shown. of hematopoietic origin. Such a tissue-biased expression pattern of (D) BWN reporter assay with BWN.3G-Nkrp1g transfectants cocultured Clr molecules might explain the multiple ligand specificities of with indicated poly(I:C)-activated and FACS sorted splenic leukocytes. Nkrp1g and Nkrp1f, respectively, by specifically regulating the CHO-Mock and CHO–Clr-g transfectants as well as PMA/ionomycin activity of NK and/or T cells depending on the tissue context. served as control. Shown is the percentage of eGFP+ BWN.3G-Nkrp1g Analyzing Clr-g transcripts in mouse splenocytes revealed a reporter cells responding to the coculture with indicated targets. To block broad presence in all tested subpopulations, whereas mouse cell the interaction between Nkrp1g and Clr-g, the targets were preincubated lines showed a more restricted Clr-g expression primarily by mouse with mAb 1H7. One representative of two independent experiments is shown. (B and D) Two-way ANOVA with a Bonferroni posttest was used T and B cell lines. Using a novel anti–Clr-g mAb, we could show for comparison (****p , 0.0001). that the presence of Clr-g transcripts in these cell lines perfectly matches Clr-g surface expression. In contrast, no Clr-g molecules only residually expressed on human monocytes despite abundant were detectable on splenic lymphocytes or DCs, despite transcript transcripts in all leukocyte populations (12). Indirect evidence for levels comparable to T and B cell lines, with only residual Clr-g a low or absent Clr-g expression on primary splenocytes was al- expression on splenic monocytes. This points to an additional ready provided by an early study, where Nkrp1f reporter cells did posttranscriptional mechanism stringently controlling Clr-g sur- not respond to mouse splenocytes (8). In this study, Nkrp1f re- face expression in lymphocytes that is absent in tumor cell lines. porter cells selectively responded against cultured bone marrow– This is also reminiscent of the restricted surface expression of the derived DC and bone marrow–derived macrophages, and the human CLEC2 family member and NKp80 ligand AICL, which is response was abrogated upon LPS treatment, suggesting LPS-mediated The Journal of Immunology 13 downregulation of the respective Nkrp1f ligand. However, it Acknowledgments remained unclear which Clr molecule is recognized by Nkrp1f in We thank Christian Lehmann for critical advice in DC phenotyping, Tobias this context. In contrast, we observed a marked upregulation of Zo¨ller for help in cytokine detection, and Praveen Mathoor for cell sorting. Clr-g on the surface of splenic DCs upon challenge with TLR + ligands. We found that Clr-g is upregulated on CD8a cDCs Disclosures upon in vivo challenge with poly(I:C), which is in line with an The authors have no financial conflicts of interest. upregulation of Clr-g transcripts in this cell population. Such in vivo–induced Clr-g expression on DCs could be sensed by Nkrp1g as shown by Nkrp1g-expressing reporter cells. In vitro, References we observed that Clr-g surface expression can be rapidly induced 1. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274. 2. Vivier, E., D. H. Raulet, A. Moretta, M. A. Caligiuri, L. Zitvogel, L. L. Lanier, on pDCs upon treatment with R848, which is in line with the W. M. Yokoyama, and S. Ugolini. 2011. Innate or adaptive immunity? The expression of TLR7/9 by pDCs. However, we were unable to example of natural killer cells. Science 331: 44–49. recapitulate the poly(I:C)-induced Clr-g induction on CD8a+ 3. Long, E. O., H. S. 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Innate immune response in mucosa Lymphocyte migration Hist1h2b,f,l,k,g,e,c Tnfsf14 Clec2d Myo1g Tcf3 Padi2 Sp110 Tbx21 Tnk2 S1pr1 Polr3e Tap2 Mir26a-2 Shmt2 Upf1 Unc13d Sec14l1 Pde2a

Enriched GO terms in significantly downregulated genes of Nkrp1g+ NK cells. Microarray analysis of RNA isolated from FACsorted Nkrp1g+ versus Nkrp1g- splenic NK cells was performed by ATLAS Biolabs (Berlin, Germany) using an Affymetrix GeneChip Mouse Gene 2.0 ST array system. Gene ontology and enrichment analysis was carried out with Metscape (http://metascape.org). Shown are two enriched GO terms from significantly downregulated genes with the respective gene candidates.

FRIEDE et al. A B IgG1 10A8 IgG2a 10A6 IL-2 LAKs d0 105 105 5 5 0.1 0.2 10 10 4 4 0.2 0.3 10 10 104 104

3 3 10 10 103 103

2 2 10 10 102 102 0 0 0 0 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 IL-15 LAKs d7 105 105 105 105 0.1 0.1 0.1 0.1 104 104 104 104

103 103 103 103 NKp46 102 102 102 102 0 0 0 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 IL-2+12

CD69 LAKs d10 105 105 105 105 0.2 0.3 0.2 0.3 104 104 104 104

103 103 103 103

102 102 102 102 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 IL-2+18 Fluorescence 105 105 0.2 0.2 104 104

103 103

102 102 0 0

0 102 103 104 105 0 102 103 104 105 Fluorescence C YAC-1 RMA RMA-S EL4 Ba/F3 SP2/0 100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 rIgG1 40 40 40 40 40 40 10A8

% of Max 20 20 20 20 20 20

0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Fluorescence D E B cells T cells NK cells B cells T cells NK cells 100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60

40 40 40 40 40 40

20 20 20 20 20 20

0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 pDCs cDCs CD8α+ cDCs CD8α- pDCs cDCs CD8α+ cDCs CD8α- 100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60

40 40 40 40 40 40

% of Max 20 20 20 % of Max 20 20 20

0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Monocytes Ly6Chi Monocytes Ly6Clo Monocytes Ly6Chi Monocytes Ly6Clo 100 100 100 100

80 80 80 80

60 60 60 60 rIgG1 rIgG1 40 40 10A8 40 40 10A8 20 20 20 20

0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Fluorescence Fluorescence SUPPLEMENTAL FIGURE 1. No Clr-f surface expression on splenocytes or murine cell lines. Repre- sentative dot plots of (A) cytokine-stimulated NK cells and (B) LAK cells detected with Clr-f-specific mAb 10A8 (A) or 10A6 (B). Gating strategy: (A, B) NK cells/LAK cells (CD19-CD3-CD49b+NKp46+). One rep- resentative of two independent experiments is shown. (C-E) Representative histograms of (C) murine cell lines, (D) resting splenocytes, or (E) poly(I:C)-stimulated splenocytes stained with Clr-f-specific mAb 10A8. One representative of two independent experiments is shown. (D, E) Gating strategy: NK cells (CD19-CD3-CD49b+NKp46+), B cells (CD19+), T cells (CD19-NKp46-CD3+), cDCs CD8α+ (CD19-Ly-6G- NKp46-I-A/I-E+CD11c+CD11b-CD8α+),cDCs CD8α- (CD19-Ly-6G-NKp46-I-A/I-E+CD11c+CD11b+CD8α-), pDCs (CD19-Ly-6G-NKp46-CD11c+PDCA-1+), monocytes Ly6Chi/lo (CD19-Ly-6G-NKp46-CD11c- CD11b+SSC-AsmallLy6Chi/lo). CHO mock CHO Clr-a CHO Clr-b CHO Clr-c CHO Clr-f CHO Clr-g 100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 mIgG1 40 40 40 40 40 40 M2

20 20 20 20 20 20

0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

100 100 100 100 100 100

% of Max 80 80 80 80 80 80

60 60 60 60 60 60 FMO 40 40 40 40 40 40 1H7

20 20 20 20 20 20

0 0 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Fluorescence SUPPLEMENTAL FIGURE 2. mAb 1H7 specifically detects Clr-g and Clr-f. Repre- sentative histograms of CHO cells stably transfected with different Clr proteins and stained with either mAb M2 or mAb 1H7. One representative of at least two independent experiments is shown. A Cyto TM CTLD Isoform 1: 246 aa Isoform 2: 217 aa Isoform 3: 221 aa

for Isoform 4: 156 aa rev

B hi lo hi pIClo pIC C

+ - y6C y6C + pIC- pIC y6C y6C α α α α

AC-1 cells MarkerY RMA RMA-SEL4 Ba/F3SP2/0 cDCs CD8cDCs pDCsCD8 MonocytesMonocytes LMarker cDCsL cDCsCD8pDCs CD8Monocytes pICMonocytes LMarker LB cellsT NK cells 300 300 250 250 ◄Isoform 3 200 ◄Isoform 3 200 ◄Isoform 2 ◄Isoform 2 150 150

100 100

SUPPLEMENTAL FIGURE 3. Predominant expression of Clr-g isoform 2 in subsets of splenocytes and cell lines. (A) Graphical scheme of the four Clr-g isoforms with num- ber of amino acids (aa) indicated and deletions shown by white boxes. Arrows indicate positions of the PCR primers used in (B, C). (B, C) PCR products of the various isoforms of Clr-g detected in (B) subsets of splenocytes and in (C) mouse B and T cell lines. Arrow- heads mark isoform 2 and 3, respectively. Sizes of the predicted isoforms: isoform 1 = 271 bp, isoform 2 = 184 bp, isoform 3 = 196 bp, isoform 4 = 113 bp. One representative of two independent experiments is shown.