IMMUNOLOGY ORIGINAL ARTICLE Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen-presenting cells and promotes resistance to Salmonella infection Timothy J. Wilson,1,2 Summary Simon Clare,3 Joseph Mikulin,1 Signalling lymphocyte activation molecule family member 9 (SLAMF9) is Christopher M. Johnson,4 an orphan receptor of the CD2/SLAM family of leucocyte surface pro- Katherine Harcourt,3 Paul A. teins. Examination of SLAMF9 expression and function indicates that Lyons,2,5 Gordon Dougan3,5 and SLAMF9 promotes inflammation by specialized subsets of antigen-pre- Kenneth G. C. Smith2,5 1 senting cells. Within healthy liver and circulating mouse peripheral blood Department of Microbiology, Miami + À low 2 mononuclear cells, SLAMF9 is expressed on CD11b , Ly6C , CD11c , University, Oxford, OH, USA, Department low + + of Medicine, Cambridge Institute for Medical F4/80 , MHC-II ,CX3CR1 mononuclear phagocytes as well as plasma- Research, University of Cambridge, cytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal Cambridge, 3Wellcome Trust Sanger Institute, B1 cells and small (F4/80low), but not large (F4/80high), peritoneal macro- 4 Hinxton, Cambridge, MRC Laboratory of phages. Upon systemic challenge with Salmonella enterica Typhimurium, À/À Molecular Biology, Cambridge, and Slamf9 mice were impaired in their ability to clear the infection from 5Cambridge Institute for Therapeutic Immunology and Infectious Disease, the liver. In humans, SLAMF9 is up-regulated upon differentiation of Department of Medicine, University of monocytes into macrophages, and lipopolysaccharide stimulation of Cambridge, Cambridge, UK PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte–macrophage colony-stimu- doi:10.1111/imm.13169 lating factor, tumour necrosis factor-a, and interleukin-1b. Taken Received 16 September 2019; revised 16 together, these data implicate SLAMF9 in the initiation of inflammation December 2019; accepted 20 December 2019. and clearance of bacterial infection. Correspondence: Timothy J. Wilson, Keywords: dendritic cells; inflammation; mononuclear phagocytes; Department of Microbiology, Miami Salmonella; SLAMF9. University, 700 E. High Street, Oxford, OH 45056, USA. Email: [email protected] Senior author: Kenneth G. C. Smith Introduction adapters by SLAM family receptors promotes stable intercellular adhesion and cellular effector function,3 SLAM family receptors are a family of cell surface pro- whereas ITSM signalling in the absence of these adapters teins with differential expression across all leucocyte sub- can inhibit cellular activation.4–8 types. In humans and mice, they comprise nine members The best characterized functions of SLAM family recep- based on protein ectodomain structure and gene phy- tors are their contributions to intercellular adhesion,3,9,10 logeny; SLAM, CD48, Ly9, 2B4, CD84, NTB-A, CRACC, leucocyte migration11 and cellular cytotoxicity.7,8,12–15 In BLAME and Signalling lymphocyte activation molecule addition to these functions, SLAM family receptors have family member 9 (SLAMF9). Of these, six contain one or been directly implicated in pathogen recognition and more cytosolic immunoreceptor tyrosine-based switch clearance by macrophages. For example, SLAM con- motifs (ITSMs) capable of interacting with the signalling tributes to recognition of Gram-negative bacteria,16 and adapter proteins SAP and EAT-2.1,2 Recruitment of these Abbreviations: cDC, conventional dendritic cell; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte–macrophage colony-stimulating factor; ITSM, immunoreceptor tyrosine-based switch motif; LPS, lipopolysaccharide; M-CSF, macrophage col- ony-stimulating factor; pDC, plasmacytoid dendritic cell; PMA, phorbol-12-myristate-13-acetate ª 2019 The Authors. Immunology published by John Wiley & Sons Ltd., Immunology, 159, 393–403 393 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Wilson et al. BLAME regulates the production of reactive oxygen spe- immunized twice by intraperitoneal injection with soluble cies.17 human SLAMF9-huIgG1-Fc fusion protein (R&D Sys- The mRNA and amino acid sequences of SLAMF9 were tems, Minneapolis, MN) in alum and once in the absence first reported in 2001,18–20 but the protein has remained of adjuvant. After fusion, 736 hybridoma clones were minimally characterized since then. SLAMF9 is struc- screened by ELISA against the antigen as well as human turally similar to the other SLAM family receptors. It IgG1. Thirteen clones were found to be specifically reac- comprises an N-terminal immunoglobulin V-type tive to the SLAMF9-Fc fusion protein and not human domain, a C2-type immunoglobulin superfamily domain, IgG1. These 13 clones were screened by flow cytometry and a transmembrane domain. In contrast to the SLAM on human SLAMF9-transfected HEK-293T cells. Of these, family receptors that have long cytoplasmic tails with four (FC2, FC57, FC203 and FC354) were found to rec- multiple ITSMs, SLAMF9 has a short, lysine- and argi- ognize human SLAMF9 on the surface of transfected nine-rich cytoplasmic domain. Although the expression of 293T cells, but not untransfected controls. Clones FC2 SLAMF9 in mice and humans has remained largely unde- and FC203 were found to have superior signal-to-noise fined, recent studies have shown expression of SLAMF9 characteristics, and FC2 was expanded for purification among hepatic macrophages and tumour-associated and characterization. Direct conjugation of antibodies macrophages and have implicated SLAMF9 in macro- with biotin or Alexa Fluor 647 was performed using EZ- phage responses to lipopolysaccharide (LPS) and the reg- Link NHS-Sulfo-LC-Biotin and Alexa Fluor 647 NHS ulation of Toll-like receptor 4 (TLR4) expression.21–23 In Ester (Thermo Fisher, Waltham, MA) respectively accord- this study, we use novel monoclonal and polyclonal anti- ing to the manufacturer’s instructions. bodies to define the expression patterns of SLAMF9 in human peripheral blood mononuclear cells (PBMCs) and À À Generation of Slamf9 / mice mouse circulating and tissue-resident leucocytes. We À À show that SLAMF9 differentially regulates pro-inflamma- Slamf9 / mice (C57BL/6N-Atm1Brd Slamf9tm1a(EUCOMM)Wtsi/ tory cytokine production in response to LPS, and that WtsiOulu) were generated by homologous recombination SLAMF9 is necessary for clearance of Salmonella from the at the Wellcome Trust Sanger Institute according to pre- liver during systemic infection. viously reported protocols.24,25 Knockout-first (tm1a) alleles were used for most studies reported in this manu- script. Replication of S. Typhimurium infection and tis- Materials and methods sue expression of mouse SLAMF9 were performed using the reporter tagged deletion (tm1b) allelic form. The Generation of SLAMF9-specific antibodies absence of SLAMF9 protein in both targeted alleles was Polyclonal rabbit antisera were raised against mouse confirmed by flow cytometry and Western blotting. SLAMF9 C-terminal peptide RVRKLKRNRIKLRKKGKSG Routine genotyping of mice was performed using the fol- coupled to keyhole-limpet haemocyanin by Pacific lowing primer sets: 50-CAGCTTGTGTTTCCACAGCC - Immunology (Ramona, CA). Peptide-specific antibodies forward; 50-ATCAAGGATCTGGGAGGGG - wild-type from serum 9318 were then affinity-purified by liquid reverse; 50-TCGTGGTATCGTTATGCGCC - cassette chromatography over peptide-coupled agarose. Mono- reverse. All animal procedures were performed according clonal anti-mouse SLAMF9 (M349) was generated by to UK Home Office regulations and Miami University À À immunizing Slamf9 / C57BL/6N mice intraperitoneally Institutional Animal Care and Use Committee-approved twice with soluble mouse SLAMF9-CD4-His fusion pro- protocols. tein in alum, and once with soluble SLAMF9 in the absence of adjuvant. Three days after the final immuniza- Analysis of SLAMF9 expression by flow cytometry tion, splenocytes were fused with SP2/0 myeloma cells and placed under hypoxanthine-aminopterin-thymidine Human PBMCs were isolated from peripheral blood or selection. Supernatants from 480 of the resulting clones leucocyte cones of healthy donors by density gradient were screened for reactivity by enzyme-linked centrifugation using Histopaque 1077 (Sigma, St Louis, immunosorbent assay (ELISA) and by flow cytometry on MO). Ethical approval was obtained from the NRES SLAMF9-transfected HEK-293T cells and mouse bone- Committee, East of England-Cambridge Central. Cells marrow-derived macrophages. Thirty-two clones were were stained with the following antibodies from Miltenyi selected for isotyping and further screening. Clone M349 Biotech (Bergisch Gladbach, Germany), BD Biosciences (ms IgG1-j) was selected for its specificity and reactivity (Franklin Lakes, NJ), eBioscience (San Diego, CA), and À À against wild-type but not Slamf9 / bone-marrow- Sigma: CD14-FITC (TUK4), CD3 (OKT3), CD16 (B73Á1), derived macrophages by flow cytometry. Monoclonal CD19-V450 (HIB19), and IgG1 isotype control anti-human SLAMF9 (FC2; ms IgG1) was generated in a (MOPC21). Anti-SLAMF9 (FC2) was generated in our manner similar to M349. Briefly, C57BL/6N mice were laboratory for this study. Cell viability was determined 394 ª 2019 The Authors. Immunology published by John Wiley & Sons Ltd., Immunology, 159, 393–403 SLAMF9 in APCs and Salmonella resistance using the Fixable