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Home , Geotrichum candidum, Malassezia, Malassezia furfur, Malassezia sympodialis

C-type lectin Mincle is an activating receptor for pathogenic ,

Sho Yamasakia,1, Makoto Matsumotob, Osamu Takeuchic,d, Tetsuhiro Matsuzawae, Eri Ishikawaa, Machie Sakumaa, Hiroaki Tatenof, Jun Unoe, Jun Hirabayashif, Yuzuru Mikamie, Kiyoshi Takedag,h, Shizuo Akirac,d, and Takashi Saitoa,i,1

aLaboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, ; bDepartment of Immunology and Medical Zoology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan; cDepartment of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; dLaboratory of Host Defence, hLaboratory of Mucosal Immunology, and iLaboratory of Cell Signaling, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan; eMedical Mycology Research Center, Chiba University, Chiba 260-8673, Japan; fResearch Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8568, Japan; and gLaboratory of Immune Regulation, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan

Edited by Max D. Cooper, Emory University, Atlanta, GA, and approved December 15, 2008 (received for review May 29, 2008) Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor Results and Discussion expressed in activated phagocytes. Recently, we have demon- Specific Recognition of Malassezia Species by Mincle. We first ex- strated that Mincle is an FcR␥-associated activating receptor that amined whether Mincle act as a receptor for living fungi by senses damaged cells. To search an exogenous ligand(s), we establishing cell-based indicator system. To discriminate puta- screened pathogenic fungi using cell line expressing Mincle, FcR␥, tive Mincle ligands from possible contaminating TLR ligands in and NFAT-GFP reporter. We found that Mincle specifically recog- fungi, we used nonmyeloid T cell hybridomas as host cells and nizes the Malassezia species among 50 different fungal species used an NFAT-GFP reporter as a specific detector of ITAM- tested. Malassezia is a that causes skin diseases, mediated signals (10). Crosslinking of Mincle with immobilized such as and , and fatal sepsis. anti-Mincle mAb induced strong GFP expression in an indicator However, the specific receptor on host cells has not been identi- cell line expressing NFAT-GFP reporter (10), Mincle and FcR␥ fied. Mutation of the putative -binding motif within (Fig. 1A, Inset) (2). We therefore used this reporter cell line to C-type lectin domain of Mincle abrogated Malassezia recognition. screen reactive species from more than 50 species of pathogenic Analyses of glycoconjugate microarray revealed that Mincle selec- fungi (Table 1). Intriguingly, only Malassezia fungi induced tively binds to ␣-mannose but not mannan. Thus, Mincle may profound NFAT activation (Fig. 1A, lane 31–39). Although recognize specific geometry of ␣-mannosyl residues on Malassezia structurally similar Dectin-1, Dectin-2, and DC-SIGN are re- species and use this to distinguish them from other fungi. Malasse- ported to recognize or species (5, zia activated macrophages to produce inflammatory cytokines/ 11–13), at least three strains of C. albicans (serotype A) or chemokines. To elucidate the physiological function of Mincle, Aspergillus species tested did not activate Mincle-expressing cells Mincle-deficient mice were established. Malassezia-induced cyto- (Fig. 1A, lane 18–20). Some species, which were negative for kine/chemokine production by macrophages from Mincle؊/؊ mice GFP but partially affected the viability of the reporter line, were was significantly impaired. In vivo inflammatory responses against also confirmed at a nontoxic concentration (data not shown). Malassezia was also impaired in Mincle؊/؊ mice. These results Microscopic analysis revealed that only Malassezia-bound cells indicate that Mincle is the first specific receptor for Malassezia were GFP positive (Fig. 1B). The activation was dose-dependent ␥ species to be reported and plays a crucial role in immune responses of Malassezia (Fig. 1C). Reporter cells expressing only FcR did to this fungus. not respond to Malassezia (Fig. 1C), demonstrating that intro- duced Mincle, but not other endogenous receptors associated ␥ ITAM ͉ macrophages ͉ signal transduction with FcR , is responsible for NFAT activation. Furthermore, IMMUNOLOGY soluble anti-Mincle mAb blocked Malassezia-induced NFAT activation in a dose-dependent manner (Fig. 1D). These results incle (also called as Clec4e and Clecsf9) is a C-type lectin indicate that Mincle directly recognizes Malassezia species. Mreceptor expressed in activated macrophages (1). We have ␥ recently demonstrated that Mincle is associated with FcR Mincle Recognizes Mannosyl Residue. Next, we addressed the struc- chain, an immunoreceptor tyrosine-based activation motif ture of Malassezia species recognized by Mincle. Although (ITAM)-containing adaptor, and acts as an activating receptor several Malassezia proteins are known as major antigens for IgE that senses damaged cells (2). Mincle is genetically mapped to a in atopic dermatitis patient (8), recombinant Mal f2 (14) and Mal cluster on mouse chromosome 6F2 and human chromosome f6 (15) did not induce NFAT activation in the Mincle reporter 12p31 (1, 3). Among these clusters, Dectin-1 and Dectin-2 are cells (data not shown). Heat-killed Malassezia retained the ITAM-coupled C-type lectin receptors that directly recognize stimulatory activity, suggesting that Mincle recognizes the non- specific fungi (4, 5). Given that Mincle possesses a similar protein determinant of Malassezia (data not shown). Since structure to these receptors, it is possible that Mincle also Mincle possesses a typical carbohydrate recognition domain recognizes nonself ligand such as specific fungi (6). Malassezia species are ubiquitous residents of but are associated with several diseases, such as tinea versicolor, Author contributions: S.Y. designed research; S.Y., M.M., O.T., T.M., E.I., M.S., H.T., J.U., J.H., Y.M., and K.T. performed research; M.M., O.T., T.M., J.U., Y.M., K.T., and S.A. contributed , and atopic dermatitis (7, 8). In neonate, invasive new reagents/analytic tools; S.Y. analyzed data; and S.Y. and T.S. wrote the paper. infection by Malassezia often causes lethal sepsis (9). Despite the The authors declare no conflict of interest. important role played by Malassezia in multiple diseases, little is This article is a PNAS Direct Submission. known about the molecular mechanism involved in host cell 1To whom correspondence may be addressed. E-mail: [email protected] or recognition. [email protected]. In this study, we show that Mincle is a specific receptor for This article contains supporting information online at www.pnas.org/cgi/content/full/ Malassezia species and plays a crucial role in immune responses 0805177106/DCSupplemental. to this fungus. © 2009 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805177106 PNAS ͉ February 10, 2009 ͉ vol. 106 ͉ no. 6 ͉ 1897–1902 Downloaded by guest on September 29, 2021 A 80 Immobilized mAb Cont. IgG anti-Mincle 70

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BDC 100 Mincle + FcRγ 16 FcRγ 14 80 12 %) 10 60 FP ( 8 G 40 6 AT- 4 NF NFAT-GFP (%) 20 2

0 0 3 4 4 5 5 6 6 7 7 7 (µg/ml) 0 0 0 0 0 0 0 0 1 3101 310 10 1 1 1 1 10 10 x1 x1 x10 x x x rat IgG anti-Mincle 3 1 3x 1 3x 1 3x 1 3x 3 Malassezia pachydermatis

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Fig. 1. Mincle recognizes Malassezia species and delivers activation signal. (A) Screening of pathogenic fungus for Mincle-ligand activity. Reporter cell line expressing Mincle, FcR␥, and NFAT-GFP was established and left unstimulated or stimulated with plate-coated anti-Mincle mAb (10 ␮g/ml) for 18 h (inset). Mincle-expressing NFAT-GFP reporter cells were cocultured with indicated pathogenic fungi (open, 1%; closed, 10%) for 18 h, or stimulated with plate-coated anti-Mincle as a positive control (gray). NFAT-GFP induction was analyzed by flow cytometry. Data are representative of 3 independent experiments. (B) Microscopic analysis. Mincle-expressing reporter cells were cocultured with M. pachydermatis (arrowhead) and analyzed with fluorescence microscopy (IX-81; Olympus). (Scale bars: 5 ␮m.) (C) Dose-dependent activation by M. pachydermatis. Reporter cells expressing Mincle and FcR␥ (closed) and only FcR␥ (open) were cocultured with indicated amount (cells/ml) of M. Pachydermatis for 18 h. (D) Anti-Mincle mAb blocked Malassezia-induced NFAT activation. Reporter cells were cultured with M. pachydermatis in the presence of rat IgG or anti-Mincle mAbs and GFP expression was determined by flow cytometry. (E) Critical role of Mannose-binding motif of Mincle for Malassezia recognition. MincleWT (WT), MincleE169Q/N171D (EPN -Ͼ QPD) were expressed together with FcR␥ in NFAT-GFP reporter cells. Cells were treated and analyzed as in (D). (F) Mincle specifically recognizes ␣-mannose. Mincle-Ig (1 ␮g/ml) precomplexed with Cy3-anti-human IgG (0.5 ␮g/ml) in TBS containing 1 mM CaCl2 or 10 mM EDTA was applied on the glycoconjugated microarray. After incubation at 20 °C for 3 h, binding was detected by an evanescent-field fluorescence-assisted scanner. Data were analyzed with the Array Pro analyzer ver. 4.5.

(CRD), it is possible that Mincle may recognize any specific into the QPD (Gln-Pro-Asp) motif that shows preference for carbohydrate structure of Malassezia species. (16). Mutant Mincle bearing E169Q/N171D (MincleEPN- Mincle CRD contains an EPN (Glu-Pro-Asn) motif (1), which ϾQPD) failed to respond to and Malassezia pachy- is a putative mannose-binding motif (16). To test whether dermatis (Fig. 1E, Left), while it did not affect anti-Mincle-induced Malassezia recognition by Mincle involves mannose or a related activation (Fig. 1E, Right). Thus, mannose recognition seems to be carbohydrate determinant, we mutated the EPN motif of Mincle crucial for Mincle to sense Malassezia species.

1898 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805177106 Yamasaki et al. Downloaded by guest on September 29, 2021 Table 1. IFM number of fungi used in this study A 11 kb Lane no. Strain IFM No.

1 Alternaria alternata 53969 exon 1 2354 6 ATG 2 mentagrophytes 5218 B E B B Wild-type 3 Aspergillus flavus 54306 allele 4 Aspergillus fumigatus 47439 B B 5 Aspergillus fumigatus 47450 B Targeting neo HSV-TK 6 Aspergillus fumigatus 49824 vector 7 Aspergillus nidulans 54308 B BB B 8 Aspergillus niger 54309 Targeted neo allele 9 Aspergillus parasiticus 42197 10 Aspergillus terreus 54310 B, BamHI; E, EcoRI 11 Aureobasidium pllulans 4802 12 Cladosporium cladosporioides 41450 B (+) primer (-) primer 13 Epidermophyton floccosum 46991 14 Exophiala dermatitidis 4826 Genotype: +/+ +/– –/– +/+ +/– –/– 15 54222 16 41134 17 Microsporum gypseum 53930 18 Candida albicans 54349 19 Candida albicans 40009 C 97.4 20 Candida albicans 54366 21 54350 Mincle+/+ 22 Candida guilliermondii 5787 23 Candida kefyr 5800

24 46839 F4/80 96.0 25 Candida parapsiolosis 51754 26 55047 Mincle–/– 27 var. grubii 5807

28 Cryptococcus albidus 5763 CD11b Mincle 29 Cryptococcus laurentii 50261 30 candidum 45995 D Mincle+/+ Mincle–/– 31 Malassezia pachydermatis 48586 32 51970 Malassezia 33 52993 Blot: anti-Mincle 34 53376 35 Malassezia slooffiae 48587 36 48588 anti-actin 37 Malassezia furfur 52635 Fig. 2. Generation of Mincle-deficient mice. (A) Genomic Mincle structure 38 Malassezia pachydermatis 48586 and targeting constructs with neomycin resistance (Neo) insertion. The Mincle 39 M. pachydermatis non oil 48586 exons are shown as black box. (B) Genomic PCR analysis of Mincle-targeted 40 asahii 48429 ϩ ϩ ϩ Ϫ Ϫ Ϫ

allele. Genomic DNA isolated from / , / and / mice were amplified IMMUNOLOGY 41 Trichosporon asteroides 51965 with primer pairs for wild-type allele (ϩ) or targeted allele (Ϫ) as shown in 42 Trichosporon cutaneum 40066 arrowheads in (A). (C) Surface expression of Mincle. BMM␾ from WT 43 Trichosporon inkin 48551 (Mincleϩ/ϩ) and Mincle-deficient (MincleϪ/Ϫ) mice were stimulated with 1 44 Trichosporon mucoides 48611 ng/ml LPS for 18 h and stained with anti-rIgG1-biotin (thin line) or anti-Mincle- 45 Trichosporon ovoides 49887 biotin (thick line) and Streptavidine-APC. (D) Western blot analysis. Thiogly- ϩ/ϩ Ϫ/Ϫ 46 C. neoformans with oil 5807 colate-elicited peritoneal macrophages from Mincle and Mincle mice were left unstimulated (Ϫ) or stimulated with 1, 3, 10 ϫ 106 of M. pachyder- 47 Rhodotorula aurantiaca 40059 matis for 18 h. Cells were lysed and blotted with anti-Mincle and anti-actin 48 Saccharomyces cerevisiae 40022 mAbs as a control. 49 Scedosporium apiosermum 51940 50 55052 51 Chaetomium globosum 40872 These results suggest that Mincle may recognize specific geometry of ␣-mannosyl residues or any related carbohydrate on Malassezia species to distinguish them from other fungi. To We further examined glycan-binding specificity of Mincle by identify such specific determinant, anti-Malassezia monoclonal glycoconjugate microarray (Fig. S1) (17). Intriguingly, soluble antibodies which block MincleϪMalassezia interaction are now Mincle protein, Mincle-Ig, showed specific binding on the spots under development. of ␣-mannose-polyacrylaminde conjugates which are a highly multivalent form of ␣-mannose (Fig. 1F, Left, position 7C). Since EDTA completely blocked ␣-mannose binding to Mincle (Fig. Establishment of Mincle-Deficient Mice. Finally, to investigate the 1F, Right, position 7C), the binding required Ca2ϩ, suggesting role of Mincle in immune responses to Malassezia, we established Ϫ/Ϫ that CRD of Mincle is involved in this recognition. However, Mincle-deficient mice (Fig. 2A). Mincle mice were born Mincle-Ig showed no binding on the spots of mannan (18) (Fig. following Mendelian law and showed no obvious abnormalities, 1F, position 5E-6E). Consistently, soluble mannan did not block and their cellularity and subpopulation of thymus, spleen, lymph Mincle-mediated NFAT-activation induced by Malassezia (data node, and peritoneal cells were not altered (data not shown). not shown). BMM␾ showed normal development in the absence of Mincle

Yamasaki et al. PNAS ͉ February 10, 2009 ͉ vol. 106 ͉ no. 6 ͉ 1899 Downloaded by guest on September 29, 2021 8000 * 12000 5000 A Mincle+/+ * 300 Mincle–/– 10000 4000 6000 8000 3000 200

4000 * 6000 (pg/ml) 2000 α 4000 100 * 2000 *

MIP-2 (pg/ml) 1000 2000 TNF

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S san P o L LPS Malassezia ym Malassezia Z Zymosan

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20 KC 10 4 TNF α 10 IL-10 mRNA MIP-2 mRNA

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IL-6 (pg/ml) 200 0.4 0.4 CD11b 100 0.2 + 0.2 Peritoneal cells (x 10

0 0.0 Gr1 0.0 WT KO WT KO WT KO WT KO WT KO

Malassezia Untreated Malassezia Untreated Malassezia

Fig. 3. Mincle deficiency failed to respond to Malassezia.(A) Malassezia-induced cytokine production. Mincleϩ/ϩ and MincleϪ/Ϫ BMM␾ were stimulated with 1, 3, 10 ϫ 106 of M. pachydermatis, 100 ␮g/ml zymosan or 1 ng/ml LPS for 18 h. *, P Ͻ 0.05. (B) Malassezia-induced cytokine transcription. BMM␾ was stimulated with 10 ϫ 106 M. pachydermatis or 100 ␮g/ml zymosan for 4 h, and mRNA expression was determined by real time PCR. Relative mRNA levels were expressed as fold induction. *, P Ͻ 0.05. (C) Mincleϩ/Ϫ (WT) and MincleϪ/Ϫ (KO) mice were injected with 4 ϫ 107 M. pachydermatis i.p. After 18 h, the peritoneal cavity was washed out with 5 ml of saline and cytokine concentration was determined by ELISA. Each symbol represents an individual mouse. Data are representativeof two independent experiments. *, P Ͻ 0.05. (D) Mincleϩ/ϩ or Mincleϩ/Ϫ (WT) and MincleϪ/Ϫ (KO) mice were injected with 2.5 ϫ 107 M. pachydermatis i.p. At 20 h after injection, peritoneal cells were stained with CD11b and Gr1 and analyzed by flowcytometry. Total number of peritoneal cells (Left) and CD11bϩGr1ϩ cells (Right) were indicated. Data are representative of two independent experiments. *, P Ͻ 0.05.

(Fig. 2B). However, surface Mincle expression was completely Crucial Role of Mincle in Malassezia-Mediated Immune Responses. We absent in MincleϪ/Ϫ mice even after LPS stimulation (Fig. 2C). also examined the immunostimulatory effect of Malassezia spe- Malassezia stimulation markedly up-regulated Mincle protein cies on macrophages. Coculture of increasing amount of M. expression in WT macrophages, suggesting that macrophages pachydermatis activated BMM␾ to produce MIP-2, TNF␣, KC, up-regulate a receptor for Malassezia after sensing the fungal and IL-10 in a dose-dependent manner (Fig. 3A). The cytokine bodies, possibly for the purpose of initiating immune response production was significantly impaired in MincleϪ/Ϫ macro- against this fungus (Fig. 2D). Importantly, the corresponding bands detected by anti-Mincle were completely lost in MincleϪ/Ϫ phages, whereas TLR-mediated stimuli such as zymosan and mice (Fig. 2D). LPS induced comparable responses (Fig. 3A). We confirmed that the induction of inflammatory cytokine/chemokine mRNA These results confirmed that Mincle protein expression was Ϫ Ϫ successfully disrupted in this mutant mouse, and also demon- expression was also significantly suppressed in Mincle / mice strated that Mincle is dispensable for the development of (Fig. 3B). These results indicate that Mincle is critical for macrophages as well as the hematopoietic lineage. macrophages to evoke inflammatory responses to Malassezia.

1900 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805177106 Yamasaki et al. Downloaded by guest on September 29, 2021 Since Malassezia species possess multiple cell-wall compo- filter-air laminar flow enclosure and provided standard laboratory food and nents (19), they might also stimulate cytokine production water ad libitum. All animal experiments were performed in compliance with through TLR engagement (20). Marginal induction of cytokines our institutional guidelines. by Malassezia observed in MincleϪ/Ϫ macrophages (Fig. 3 A-B) Cells. Thioglycolate-elicited peritoneal macrophages and bone marrow- may reflect cooperative contribution of Mincle and TLR to ␾ Malassezia response, as is shown between Dectin-1 and TLR2 for derived macrophages (BMM ) were prepared as described elsewhere (1). Cytokine production was determined by ELISA or Meso Scale Discovery assay the recognition of (21). The analysis of mice lacking both kit. Western blotting was performed as described previously (25). Mincle and MyD88 (22) may clarify this issue. In addition, inducible expression of Mincle may suggest that Mincle plays a Antibodies. Anti-Mincle mAbs were established by immunizing Wistar rats crucial role during late/chronic phase of the infection. with rat basophilic leukemia (RBL-2H3) cells expressing murine Mincle as To demonstrate a role of Mincle in host defense against described previously (2). Clone 1B6 (IgG1, ␬) was used in this study. Malassezia in detail, additional experiment of in vivo Malassezia infection should be conducted. However, no such in vivo models Fungi. The fungal strains and Institute of Food Microbiology (IFM) number with pathogenic invasion are available in mice so far, partly used in this study (Table 1) were inoculated on potato dextrose agar (PDA; because Malassezia species are residents with weak pathogenicity Difco Laboratories) slants and incubated for 3 to 14 days at 25 °C. Fungal in healthy animals. Instead, we injected WT and Mincle- spores or mycelia were collected and suspended in 0.85% NaCl or 0.1% deficient mice with M. pachydermatis i.p. and cytokine produc- Tween80 solution. Some Malassezia species were cultured on PDA in the tion in the peritoneal cavitiy was examined. Malassezia induced presence of olive oil or on CHROMagar Malassezia Candida medium detectable amount of inflammatory cytokines, such as IL-6 and (CHROMagar). TNF (data not shown), in WT mice, whereas it was impaired in Glycoarray. Preparation of glycoarray, hybridization of Ig-fusion protein, and Mincle-deficient mice (Fig. 3C). Malassezia-induced neutrophil data analysis were performed as described (17). infiltration into peritoneal cavity was also significantly sup- pressed in Mincle-deficient mice (Fig. 3D). RT-PCR. Gene-specific primer sequences were as follows: MIP-2, 5Ј-GCTTC- In normal skin, Malassezia species can live as commensal. CTCGGGCACTCCAGAC-3Ј (forward) 5Ј-TTAGCCTTGCCTTTGTTCAGTAT-3Ј (re- However, in atopic/eczema dermatitis syndrome and , verse); TNF␣,5Ј-GCGACGTGGAACTGGCAGAAG-3Ј (forward) 5Ј-GGTACAAC- these fungi elicit an inflammatory response in the skin lesions CCATCGGCTGGCA-3Ј (reverse); KC 5Ј-GCCAATGAGCTGCGCTGTCAATGC-3Ј (7). Mincle expression is up-regulated by several stresses (1), and 5Ј-CTTGGGGACACCTTTTAGCATCTT-3Ј (reverse); IL-10, 5Ј-TAGAGCTGCGGACT- indeed we observed that it was slightly induced on dermal GCCTTCA-3Ј (forward) 5Ј-TCATGGCCTTGTAGACACCTTG-3Ј (reverse); ␤-actin, dendritic cells upon stimulation with chemokines or LPS (data 5Ј-TGGAATCCTGTGGCATCCATGAAAC-3Ј (forward) 5Ј-TAAAACGCAGCTCAG- Ј not shown). Therefore, it is likely that Mincle-induced inflam- TAACAGTCCG-3 (reverse). matory cytokine may contribute to the maintenance of the lesions. Reagent. LPS (L4516) and Zymosan (Z4250) were purchased from SIGMA. Candida albicans Cell wall mannan (MG001), Mannan from Yeast (21338–34) Multiple diseases, such as tinea versicolor, atopic dermatitis, and D-Galacto-D-mannan from Ceratonia siliqua (48230) were from takara and lethal sepsis, have been shown to be caused by invasive Bio Inc. and SIGMA and nacalai tesque, respectively. Recombinant M. furfur infection of Malassezia fungus. The identification of Mincle as a peroxisomal membrane protein (Mal f2) and Cyclophilin (Mal f6) were from specific receptor for Malassezia will provide valuable informa- takara Bio Inc. tion for the development of therapy and effective drugs against Malassezia-related diseases. Construct. cDNAs for FcR␥ and Mincle were cloned by PCR and inserted into Very recently, it was reported that Mincle is involved in pMX-IRES-rCD2 and pMX-IRES-hCD8 vectors (25), respectively. response to C. albicans (23). Three strains of C. albicans were not recognized by Mincle in our reporter systems, although these Ig Fusion Protein. The extracellular domain of Mincle (a.a. 46–214) was fused strains differ from that used by Wells, et al. (23). This implies that to the N terminus of hIgG Fc region by PCR and inserted into the XhoI fragment Mincle may distinguish structural difference of the substrain of of pME18S-SLAMsig-hIgG Fc. The preparation of Ig fusion protein was de- C. albicans. Alternatively, Mincle alone may not be sufficient to scribed elsewhere (2). 293T cells were transiently transfected with pME18S- IMMUNOLOGY SLAMsig-hIgG Fc (Ig) or pME18S-SLAMsig-hIgG Fc-Mincle (Ig-Mincle). Cells recognize C. albicans to induce cell activation and some other were cultured in protein-free medium (PFHM-II). Filtered supernatant was receptor(s) expressed in macrophages may cooperate with applied to Protein A-Sepharose column and bound fraction was eluted with 50 Mincle to recognize C. albicans. mM diethylamine and immediately neutralized with Tris-HCl (pH 7.5). The major fraction was dialyzed against PBS and used as purified Ig fusion solution. Materials and Methods Mice. Mincle-deficient mice were established using R1 embryonic stem cells ACKNOWLEDGMENTS. We thank Drs. R. Tanaka, N. Shibata, and H. Hara for following a general procedure as described elsewhere (24), and used as discussion; Drs. A. Mori, S. Seki, and Mr. T. Ishikawa for technical assistance; C57BL/6 and 129 mixed genetic background. All mice were maintained in a and Ms. H. Yamaguchi for secretarial help.

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