The Journal of Immunology

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic of North America

Huafeng Wang,*,1 Vanessa LeBert,*,1 Chiung Yu Hung,† Kevin Galles,* Shinobu Saijo,‡ Xin Lin,x Garry T. Cole,† Bruce S. Klein,*,{,‖ and Marcel Wu¨thrich*

Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent con- tributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, capsulatum, and posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal . Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells. The Journal of Immunology, 2014, 192: 1107–1119.

espite several million new systemic fungal infections still largely unknown and need to be determined to develop ra- annually worldwide, there are no commercial vaccines tionale strategies for antifungal vaccines. D available. We and others have engineered vaccines that Fungi-specific T cell responses are initiated through the rec- protect against experimental infection with the dimorphic fungal ognition of -associated molecular patterns by pattern pathogens Blastomyces dermatitidis (1), recognition receptors (PRRs) on innate immune cells. Among the (2), and Coccidioides posadasii (3). Our studies showed that vaccine- best-characterized PRRs that recognize fungi are the ITAM- induced immunity is chiefly mediated by CD4+ T cells (4). Despite coupled receptors Dectin-1, Dectin-2, and Mincle. They are C- the crucial roles of Th1 cells in protective immunity against fungal type lectin receptors (CLRs), which are predominantly expressed infection (3, 5, 6) and the controversial roles of Th17 cells in some in myeloid cells (15, 16). There is accumulating evidence that other infection models (7–13), in our vaccination model Th1 im- stimulation of the most-studied CLR, Dectin-1, by b-glucans munity is dispensable, whereas -specific Th17 cells are induces Th17 differentiation of naive CD4+ T cells (17). Recently, necessary and sufficient for vaccine-induced protection against Viriyakosol et al. (18) showed that Dectin-12/2 mice infected with these three pathogenic fungi that cause the major endemic mycoses have lower levels of Th17 cytokines in their of North America (14). Thus, engaging Th17 cells could be a prom- lungs. Patients who are homozygous for a single polymorphism of ising strategy to develop effective fungal vaccines. However, the Dectin-1 are susceptible to mucocutaneous infec- mechanisms underlying the vaccine-induced Th17 immunity are tions (19, 20) and invasive (21, 22) owing to defective IL-17 production. We have found, however, that Dectin-1 is un- expectedly dispensable in the development of vaccine-induced *Department of Pediatrics, University of Wisconsin Medical School, University of Th17 cell responses and resistance to B. dermatitidis (14). It is un- Wisconsin Hospital and Clinics, Madison, WI 53792; †Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San known whether Dectin-1 is required for the development of vaccine- Antonio, TX 78249; ‡Center for Experimental Medicine and Systems Biology, Uni- induced Th17 cells and resistance to H. capsulatum and C. posadasii versity of Tokyo, Tokyo 108-8639 Japan; xDepartment of Molecular and Cellular Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX infection. 77030; {Internal Medicine, University of Wisconsin Medical School, University In contrast to Dectin-1, few reports describe the role of Dectin-2 ‖ of Wisconsin Hospital and Clinics, Madison, WI 53792; and Medical in driving Th17 responses. In mice, Dectin-2 is required for the and Immunology, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madison, WI 53792 differentiation of Th17 cells induced by C. albicans infection (23). 1H.W. and V.L. contributed equally to this work. In dendritic cells (DCs), Dectin-2 activation by Candida Received for publication September 3, 2013. Accepted for publication November 29, results in the selective activation of the NF-kB subunit c-Rel and + 2013. the production of IL-1b and IL-23 p19, which skews CD4 T cell This work was supported by National Institutes of Health Grants R01 AI093553 responses toward a Th17 profile (24). Although Mincle has been (to M.W.) and R01 AI071118 (to G.T.C.). reported to induce Th1/Th17 immunity in response to the myco- Address correspondence and reprint requests to Dr. Marcel Wu¨thrich, University bacterial cell wall glycolipid trehalose 6,69-dimycolate (TDM) of Wisconsin, Microbial Sciences Building, 1550 Linden Drive, Madison, WI 53706. E-mail address: [email protected] and its synthetic analog trehalose-6,6-dibehenate (TDB) (25), to The online version of this article contains supplemental material. our knowledge its role in driving anti-fungal Th17 responses has Abbreviations used in this article: BMDC, bone marrow–derived dendritic cell; CLR, not been investigated. C-type lectin receptor; DC, dendritic cell; FKS, formalin-killed spherule; LN, lymph Although Dectin-1 recognizes fungi via b-1,3-glucan exposed on node; PRR, pattern recognition receptor; sdLN, skin-draining lymph node; TDB, the cell wall and recruits Syk directly through its hemITAM motif trehalose-6,6-dibehenate; TDM, trehalose 6,69-dimycolate; Tg, transgenic. (26), Dectin-2 and Mincle recognize mannose-like structures (23, Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 27–29) and need to pair with the ITAM-bearing adaptor FcRg to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302314 1108 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY activate the Syk-Card9 pathway (30–32). In mice, Card9 signaling mice were a generous gift from Dr. Victor L. J. Tybulewicz. All mice were induces DC maturation, the production of proinflammatory cyto- 7–8 wk old at the time of experiments. Mice were housed and cared for kines, and the induction of Th17 responses (17). In , a Card9 according to guidelines of the University of Wisconsin Animal Care Committee, who approved all aspects of this work. mutation results in susceptibility to chronic mucocutaneous can- didiasis (33). Notably, C. albicans–induced Th17 responses are Fungi and growth conditions dependent on Card9 (17), and greatly reduced numbers of Th17 B. dermatitidis strains used were ATCC 26199, a wild type virulent strain, cells are associated with patients carrying homozygous Card9 and the isogenic, attenuated mutant lacking BAD1, designated strain #55 mutations (33), indicating that Card9 is essential for antifungal (39). Isolates of B. dermatitidis were maintained as on Middlebrook Th17 responses. However, it is unclear at what stage the Card9 7H10 agar with oleic acid-albumin complex (Sigma, St. Louis, MO) at 39˚C. H. capsulatum strain G217B (provided by George S. Deepe, Uni- signaling pathway governs Th17 cell development in vivo. To versity of Cincinnati, Cincinnati, OH) was maintained on brain-heart in- dissect whether Card9 affects the priming, expansion, differentiation, fusion slants supplemented with 5% sheep blood at 37˚C. C. posadasii contraction, and migration of vaccine-induced Th17 cells, we isolate C735 is a virulent, human clinical strain that was used to generate exploited an adoptive transfer system that we have previously a live, attenuated vaccine strain (DT) by deleting the chitinases 2, 3, and D-arabinotol-2-dehydrogenase genes as described previously (3). The established using TCR transgenic 1807 cells that recognize a shared saprobic phase of both the parental and mutant strains were grown on Ag from B. dermatitidis, H. capsulatum,andC. posadasii (34). On glucose yeast extract medium (1% glucose, 0.5% yeast extract, 1.5% agar) adoptive transfer into recipient mice, 1807 cells become activated, at 30˚C for 3–4 wk to generate a confluent layer of arthroconidia (spores) proliferate, and expand in the draining lymph node (LN); 1807 cells on the agar surface. Formalin-killed endosporulated spherules of C. pos- differentiate into cytokine-producing effector T cells after traffick- adasii were generated as described (40). ing to the site of vaccination and the lung upon challenge and Vaccinations confer resistance against the three dimorphic fungi (14, 34, 35). Mice were vaccinated s.c. at two sites: dorsally and at the base of the tail as Thus, the autologous adoptive transfer system offers a powerful tool follows. B. dermatitidis virulence attenuated BAD12/2 yeast, designated to dissect normal or defective development of vaccine-induced Ag- strain #55, were injected as live or heat-killed cells using a dose range of 6 7 7 specific T cells responsive to multiple dimorphic fungi. 10 –10 yeast per mouse. For vaccination with H. capsulatum G217B, 10 heat-killed or 106 live yeast per mouse was used. For vaccination with Although Card9 and CLRs have been implicated in mediating 4 C. posadasii, mice were immunized with 5.0 3 10 viable spores of the innate resistance to primary fungal infection and priming of Th17 DT strain (3). Formalin-killed spherules (FKS) of C. posadasii were used cells, their role in vaccine-induced resistance to fungi and the effect at 106 per mouse for vaccination. Resistance experiments included one on the sequential stages of T cell development has not been in- (B. dermatitidis and H. capsulatum) or two (C. posadasii) booster vacci- vestigated. In this study, we demonstrate that the adaptor Card9 nation(s) of the same vaccine dose and site 2 weeks apart. is indispensable for the acquisition of vaccine immunity and the Experimental infection development of Th17 cells against all three systemic dimorphic Mice were infected intratracheally with 2 3 103 or 2 3 104 wild type fungi of North America, but the upstream CLRs play distinctly 26199 B. dermatitidis yeast as described previously (14). At day 4 after different roles for each pathogen. We also pinpoint at what stage of infection, coinciding with the peak of T cell influx (14), the mice were the immune response Card9 controls Th17 cell development and sacrificed and lung T cells were analyzed by FACS analysis. Extent of lung show that this adaptor promotes the differentiation of antifungal infection was examined 2 wk after infection and determined by plating Th17 cells, but does not influence downstream stages of T cell homogenized lung and enumerating yeast CFU on brain-heart infusion (Difco) agar. For H. capsulatum infections, mice were challenged with a sub- expansion, activation, contraction, or migration to the lung upon lethal dose of 1–2 3 105 G217B intratracheally, and cellular analysis was challenge. With B. dermatitidis, the upstream PRR Dectin-2, but performed 4 d later. Lung burden was determined 13 d after infection by neither Mincle nor Dectin-1, recognizes yeast and induces ITAM plating lung homogenates on sheep blood containing Mycosel plates and signaling that initiates the development of Th17 cells and vaccine enumerating CFUs. For C. posadasii infections, mice were challenged by the intranasal route with a lethal dose of 50–80 spores of the virulent, immunity to fungal infection. Conversely, for the related dimor- parental strain 4 wk after completion of the vaccination protocol (34). The phic fungi H. capsulatum and C. posadasii, while Card9 is re- fungal burden determined as CFUs in lungs and spleens of the vaccinated quired for the induction of vaccine immunity, both Dectin-1 and and unvaccinated mice was conducted at day 14 after infection by plating Dectin-2, but not Mincle, participate in the recognition and in- organ homogenates on glucose yeast extract plates containing 50 mg/ml duction of protective Th17 cell responses. Our data offer insight chloramphenicol. into the mechanisms that underpin acquisition of vaccine immu- Adoptive transfer of transgenic 1807 T-cells and surface nity to pathogenic fungi. staining Single-cell suspensions of 106 magnetic bead purified CD4+ cells from Materials and Methods 1807 Tg Thy1.1+ mice were injected i.v. into Thy1.2+ C57BL/6 recipients. Ethics statement When indicated, transgenic 1807 cells were labeled with CFSE (Molecular Probes) before adoptive transfer. After vaccination or infection, single-cell All animal procedures were performed in accordance with the recom- suspensions from draining lymph nodes (brachial and inguinal) and lung mendations in the Guide for the Care and Use of Laboratory Animals of the cells of recipient mice were stained with mAbs directed against the fol- National Institutes of Health. Care was taken to minimize animal suffering. lowing surface markers: CD4, CD8, Thy1.1, CD44, CD62L, and B220 (as The work was done with the approval of the Institutional Animal Care and a dump marker to exclude events that are stained nonspecifically). mAbs Use Committee of the University of Wisconsin–Madison. were obtained from BD Pharmingen (San Diego, CA) and eBioscience (San Diego, CA), and cytometry data were gathered with an LSRII Flow Mouse strains Cytometer (BD Biosciences, San Jose). Data were analyzed with FlowJo + a software (Tree Star, Ashland, OR). The number of 1807 CD4 T cells in Inbred wild type C57BL/6 and congenic B6.PL-Thy1 /Cy (stock #00406) + + mice carrying the Thy 1.1 allele were obtained from Jackson Laboratories a lung was calculated by multiplying the percentage of Thy1.1 CD4 cells (Bar Harbor, ME). B6.129P2-Fcer1gtm1Rav N12 mice (model no. 583) that by the number of viable cells as determined by trypan blue dye exclusion. lack FcRg were purchased from Taconic. Blastomyces-specific TCR trans- Intracellular cytokine staining genic (Tg) 1807 mice were generated in our laboratory and were back- crossed to congenic Thy1.1+ mice as described elsewhere (35). Card92/2 Effector T cells from the site of vaccination and lung were obtained as (36), Dectin-22/2 (23), Mincle2/2 (37), and Syk+/2 (38) were bred at our described previously (14). An aliquot of isolated cells was stained for facility. Radiation chimeras were generated by transferring fetal liver cells surface CD4 and Thy 1.1 to determine the percentage of transferred 1807 from day 15–17 Syk2/2 and Syk+/+ embryos (womb mates that were cells. The numbers of 1807 cells in the lung was derived by multiplying the identified by PCR) into lethally irradiated C57BL6 recipients (38). Syk+/2 percentage of cells by the total number of cells per organ isolated. The rest The Journal of Immunology 1109 of the cells were stimulated with anti-CD3 and anti-CD28 mAb in the Dectin-1-Fc was a gift from Dr. Gordon D. Brown (University of Aber- presence of Golgi-Stop (BD Pharmingen). After 4–6 h of stimulation, cells deen, Aberdeen, U.K.) (42). Human IgG1 Fc recombinant protein was were stained for surface markers, fixed, and permeabilized using the in purchased from Invitrogen. Cytofix/Cytoperm kit (BD Pharmingen) and stained with anticytokine Abs as described (14). FACS assay for the binding of Fc fusion proteins to fungi Generation of bone marrow dendritic cells Fc fragment alone or Fc fusion proteins were added at 10–20 mg/ml to 2 3 106 B. dermatitidis,107 H. capsulatum,or23 105 C. posadasii in 100 ml Bone marrow–derived dendritic cells (BMDCs) were obtained from the binding buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 0.05% femurs and tibias of individual mice. Each bone was flushed with 10 ml Tween-20, pH 7.4) and incubated for 2 h at room temperature or overnight of 1% FBS in RPMI 1640 through a 22-gauge needle. RBCs were lysed at 4˚C. The fungal cells were washed twice with binding buffer and in- followed by wash and resuspension of cells in 10% FBS in RPMI 1640 cubated for an additional 30 min at room temperature with 1:100 diluted medium. In a Petri dish, 2 3 106 bone marrow cells were plated in 10 ml PE-conjugated goat anti-human Fc Ab (Jackson ImmunoResearch Labo- of RPMI 1640 containing 10% FBS plus penicillin-streptomycin (HyClone), ratories) in binding buffer. The cells were then washed three times with 2-ME, and 20 ng/ml of rGM-CSF. The culture media were refreshed every binding buffer before fixed with 1% formaldehyde in binding buffer. 3 d, and BMDCs were harvested after 10 d for in vitro coculture assays. Bound Fc proteins were quantified by flow cytometry. Ex vivo and in vitro coculture for cytokine protein measurement CLR reporter cell construction, stimulation, and binding by Ex vivo cell culture supernatants were generated using the brachial and fungi inguinal draining lymph nodes harvested from mice 10 d after vaccination, B3Z cells carrying an NFAT-lacZ reporter construct have been described washed, and resuspended in complete RPMI 1640 containing 10 mg/ml preciously (43) and were provided by Dr. Nilabh Shastri (University of yeast cell wall membrane Ag (CW/M) (1), and plated in 96-well plates at 3 5 California, Berkeley, CA). B3Z cells expressing Dectin-2 or a wild type or a concentration of 5 10 cells/well. Supernatants were collected from a signaling-defective FcRg-chain that carries two tyrosine-to-phenylalanine ex vivo cocultures after three days of incubation at 37˚C and 5% CO2 (14). mutations within the ITAM motif (31), as well as BWZ cells expressing In vitro cell culture supernatants were generated using BMDCs plated in 5 3 5 Dectin-1–CD3z (the extracellular domain of mouse Dectin-1 fused to CD3z complete RPMI 1640 at a concentration of 10 cells/well with 3 10 intracellular tail) (44) and the parent BWZ cells (45), were a generous gift heat-killed strain #55 yeast overnight. After 24 h, single-cell suspensions from Dr. Caetano Reis e Sousa (London Research Institute, London, U.K.). of magnetic bead purified CD4+ naive 1807 cells from brachial, axillary, To construct Mincle-expressing reporter cells, the full-length cDNA of mouse cervical, and inguinal lymph nodes were added at a concentration of 3 5 Mincle was subcloned from the pEF1HISB-mMincle plasmid into a pFB- 2 10 cells/well. Supernatants were collected from in vitro cocultures IRES-EGFP retroviral vector. B3Z cells were retrovirally transduced with g after three days of incubation at 37˚C and 5% CO2.IFN- and IL-17 pFB-mMincle-IRES-EGFP, selected based on EGFP expression, and ver- (R&D Systems) were measured by ELISA according to the manu- ified by cell surface staining and FACS analysis using an anti-mouse facturer’s specifications (detection limits, 0.05 ng/ml and 0.02 ng/ml, Mincle mAb (MBL). To generate B3Z cells coexpressing Mincle and the respectively). adaptor FcRg, Mincle-expressing reporter cells were retrovirally trans- Isolation of effector cells at the site of vaccination duced with pMX-IP-FcRg-chain (wild type) or pMX-IP-FcRgm(mutant) (31). FcRg expression was verified with Western blotting using an anti- Mice were vaccinated once s.c. with 107 heat-killed strain #55 yeast at FcRg Ab (Upstate). a single dorsal site. On day 10 after vaccination, inflamed s.c. tissue was For B3Z/BWZ cell stimulation, 105 B3Z/BWZ cells/well in a 96-well excised from the site of vaccination, placed in ice-cold collagenase buffer plate were incubated for 18 h with heat-killed fungal cells. lacZ activity and minced into fine pieces. To digest the tissue, 5 ml of dissociation buffer was measured in total cell lysates using CPRG (Roche) as a substrate. OD (0.025 mg/ml Liberase [Roche Diagnostics] and 50 mg/ml DNAse I [Sigma- 560 was measured using OD 620 as a reference. For the B3Z/BWZ cell Aldrich] in collagenase buffer) was added and samples were incubated at binding assay, 2 3 105 B3Z/BWZ cells were prestained with CFSE 6 6 37˚C and 5% CO2 for 30 min. To release single cells further, the tissue was (Molecular Probes) and incubated for 6 h with 10 B. dermatitidis,23 10 mashed with the back of a 10-ml syringe plunger through a 70-mmcell H. capsulatum,or23 105 C. posadasii prestained with LIVE/DEAD Violet strainer with an additional 5 ml of dissociation buffer and incubated for staining (Invitrogen). Bound fungal cells were quantified with flow another 30 min. The dissolved tissue was washed with ice-cold PBS cytometry. containing 5 mM EDTA and 1% BSA and strained again. Filtered cells were spun at 1500 rpm, the supernatant was carefully aspirated, and cells Statistical analysis were resuspended in complete media for stimulation. Differences in the number and percentage of activated, proliferating or RT-PCR analysis for the detection of Card9-associated gene cytokine-producing T cells, and in the number of CFUs were analyzed using transcripts the Wilcoxon rank test for nonparametric data or the t test when data were normally distributed (46). Factorial ANOVA analysis using the SAF BMDCs were raised from 15 individual Card92/2 or wild type mice and software was used to compare the vaccine-induced reduction in lung CFUs 3 3 105 nonadherent DCs from each individual mouse were cocultured between different strains of mice; p , 0.05 is considered statistically with 3 3 105 B. dermatitidis vaccine yeast or medium alone in a 24-well significant. plate for 24 h. Total RNA from the BMDC cultures described above was purified using the Qiagen RNeasy kit (catalog #74106) according to the Results manufacturer’s protocol. Genomic DNA was removed using Turbo DNase (Ambion, catalog no. AM2238). Following DNase treatment, the RNA was Vaccine-induced immunity against systemic dimorphic fungi cleaned up using Qiagen RNeasy columns according to the manufacturer’s requires the adaptor Card9 protocol and by performing additional washes with buffer RPE and 80% ethanol to remove contaminants. The n-fold change of gene expression for We previously reported that vaccine-induced Th17 cells were yeast stimulation versus no stimulation was calculated using the compar- necessary and sufficient to protect mice against the three major ative CT method (41). systemic mycoses in North America (14). Because Card9 has been reported to be required for the development of Th17 cells in Generation of Fc fusion proteins responses to primary C. albicans infection (17), we investigated The Dectin-2-Fc expression vector pSecTag2(C)-mDectin-2-Fc was pro- whether Card92/2 mice can acquire vaccine-induced immunity vided by Dr. Philip R. Taylor (Cardiff University, Cardiff, U.K.) (27). To against infection with the systemic dimorphic fungi. Card92/2 construct the Mincle-Fc expression vector, the extracellular domain of mouse Mincle (amino acids 46–214) was amplified by PCR from a mice were unable to control the live, virulence-attenuated #55 pEF1HISB-mMincle plasmid provided by Dr. Christine Wells (University vaccine strain of B. dermatitidis and succumbed to widespread of Queensland, Brisbane, QLD, Australia) (37) and introduced in-frame into dissemination and infiltration of the lungs by the yeast (Fig. 1A). the KpnIandPstI sites of pSecTag2(C)-mDectin-2-Fc to replace the Dectin-2 To circumvent susceptibility of Card92/2 mice to the vaccine coding sequence. The pSecTag2(C)-mDectin-2-Fc and pSecTag2(C)-mMincle- Fc constructs were separately transfected into HEK293T cells using Lip- strain, we immunized them with heat-killed yeast and tested their ofectamine 2000 (Invitrogen). Soluble Dectin-2-Fc and Mincle-Fc ability to resist a lethal pulmonary infection with wild type yeast. 2 2 were expressed and purified as described previously (27). Soluble Vaccinated Card9 / mice had a burden of lung infection similar 1110 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY to unvaccinated littermates, which was more than 5 logs higher To test whether Card9 is necessary for the acquisition of than in vaccinated wild type controls, indicating the adaptor vaccine-induced immunity and the development of Th17 cell responses Card9 is essential for the acquisition of immunity to B. derma- to other systemic dimorphic fungi, we vaccinated mice with titidis infection (Fig. 1B). Interestingly, unvaccinated Card92/2 H. capsulatum and C. posadasii. Card92/2 mice vaccinated with mice were more susceptible to primary infection than unvacci- live G217B H. capsulatum yeast as described (14) contained the nated wild type controls, suggesting that Card9 also contributes vaccine in the s.c. tissue and did not succumb to dissemination to innate resistance. To determine whether Card9 is required for (data not shown), but they failed to acquire resistance to pulmo- the development of Th17 cells, we studied the recruitment of Ag- nary challenge (Fig. 1B) and recruited reduced numbers and fre- specific Th17 cells to the lung upon challenge. Vaccinated Card92/2 quencies of IL-17– and IFN-g–producing 1807 cells to the lungs mice recruited reduced numbers and frequencies of IL-17– and IFN- (Fig. 1C–D). Card92/2 mice succumbed to dissemination when g–producing Ag-specific 1807 cells to the lungs compared with vaccinated s.c. with live C. posadasii conidia from wild type strain vaccinated wild type controls (Fig. 1C–D). Thus, the adaptor Card9 C735 (C.-Y. Hung, M. del Pilar Jimenez-Alzate, A. Gonzalez, is required for the development of antifungal Th17 responses after M. Wu¨thrich, B.S. Klein, and G.T. Cole, submitted for publication). vaccination with B. dermatitidis. To determine whether Th17 cells develop in the absence of Card9,

FIGURE 1. Card9 is required for vaccine control and the acquisition of adaptive immunity to dimorphic fungi. (A) Wild type and Card92/2 mice were vaccinated s.c. with 105 or 106 live B. dermatitidis yeast and survival recorded when mice were moribund. n = 10 mice/group and are representative of two experiments. (B) Mice were vaccinated with 106 heat-killed (HK) B. dermatitidis,107 HK or live H. capsulatum yeast, or 106 FKS of C. posadasii or not as a control and challenged with 2 3 103 B. dermatitidis or 2 3 105 H. capsulatum yeast 2 wk or 80 C.p. spores 3 wk after booster vaccination. Lung CFUs were determined when unvaccinated mice were moribund $ 14 d after infection with B. dermatitidis or C. posadasii and at day 13 after infection with H. capsulatum. Data are the mean 6 SEM (n = 8–12 mice/group) and are representative of three to five experiments. *p , 0.05 versus vaccine-induced reduction in lung CFUs in wild type mice. Numbers indicate the n-fold difference in lung CFUs versus unvaccinated controls. (C) Wild type and Card92/2 mice received 106 naive, CD4+ purified 1807 T cells and were vaccinated with HK B. dermatitidis or H. capsulatum yeast, FKS from C. posadasii or not. Five weeks later, mice were challenged with 2 3 104 B. dermatitidis, 2 3 105 H. capsulatum yeast or 106 FKS from C. posadasii and the number of cytokine-producing 1807 cells enumerated by FACS at day 4 after infection. Data are the mean 6 SEM of three independent experiments (n = 16–20 mice/ group). *p , 0.05 versus vaccinated wild type controls. (D) The frequency of cytokine-producing 1807 cells in the lung at day 4 after infection. Data are the mean 6 SEM (n = 16–20 mice/group); representative of three experiments. *p , 0.05 versus vaccinated wild type controls. ND; not detected. The Journal of Immunology 1111 knockout mice were instead vaccinated with either FKS or live time points (days 14 and 35 after vaccination), activated 1807 cells conidia of an attenuated DT vaccine strain. This strain lacks the underwent similar contraction. Upon recall at day 4 after infection, + chitinases 2, 3, and D-arabinotol-2-dehydrogenase genes (3) and the frequency and number of activated CD44 1807 cells were fails to endosporulate and disseminate in s.c. vaccinated Card92/2 comparable in the skin-draining lymph node (sdLN) and in the lung mice. Card92/2 mice vaccinated with FKS failed to acquire re- of Card92/2 and wild type recipient mice (Fig. 2B–C). These data sistance to challenge (Fig. 1B) and recruited lower numbers and indicate that the adaptor Card9 is not required for the activation, frequencies of IL-17– and IFN-g–producing 1807 cells to the proliferation, and contraction of Ag-specific 1807 cells in the sdLN. lungs upon challenge compared with wild type controls (Fig. 1C– Although the expansion of 1807 cells was minimally affected by the D). Vaccination with the attenuated DT vaccine strain yielded absence of Card9, this did not affect the number of 1807 cells in the similar results (data not shown). In contrast to primary infection sdLN and the lung at day 4 after challenge. with B. dermatitidis and H. capsulatum, unvaccinated Card92/2 Th17 cell differentiation requires Card9 mice were not more susceptible than wild type controls to infection 2/2 with C. posadasii. Thus, the Card9 signaling pathway is required for Because Card9 mice failed to recruit IL-17– and IFN-g– the development of vaccine-induced resistance and antifungal Th17 producing 1807 cells to the lung (Fig. 1C–D), we investigated responses to the three major systemic dimorphic fungi. whether Card9 governs the differentiation of effector T cells during the vaccine-priming phase. We analyzed T cell differenti- Expansion and activation of Ag-specific cells does not require ation by three independent approaches. First, we harvested acti- Card9 vated T cells at the peak of expansion from the sdLN and Receptor signaling pathways can affect T cell development at stimulated them ex vivo with cell wall membrane (CW/M) Ag. multiple stages. In the setting of LCMV infection, Tc1 cells require T cells from Card92/2 mice produced .8-fold less IL-17 protein intrinsic TLR/Myd88 signals not for activation, expansion, or in response to fungal Ag than did wild type T cells (Fig. 3A). differentiation, but to survive apoptosis after these events (47). Similarly, IFN-g protein levels were reduced by at least 30-fold Because Card9 is required for the development of vaccine-induced compared with wild type controls. Although Card9 is primarily anti-fungal Th17 cells, we investigated the stage of the immune responsible for receptor signaling in myeloid but not lymphoid response at which Card9 governs T cell priming. To do so, we adop- cells, we sought to exclude formally a T cell–intrinsic role of tively transferred magnetic bead-purified CD4+, CFSE-labeled Card9 for the differentiation of Th17 cells. Thus, in a second naive T cells from 1807 mice into Card92/2 and wild type recipi- approach we cocultured BMDCs from Card92/2 and wild type mice ents prior to vaccination with B. dermatitidis andanalyzedTcell together with vaccine yeast and naive wild type 1807 cells in vitro. development at serial time points thereafter. At day 7 after vacci- 1807 cells cocultured with Card92/2 DCs produced ∼7-fold less IL- nation, mice lacking Card9 showed no deficit in the proliferation 17 than did cocultures with wild type DCs (Fig. 3B). Third, to and activation, but had a small reduction in expansion of Ag-specific investigate Th17 cell differentiation in vivo, we determined the 1807 cells (Fig. 2A–C), indicating that Card9 minimally affects the frequency of cytokine-producing wild type 1807 cells at the site early stages of vaccine-induced T cell development. At subsequent of vaccination. The frequency of IL-17– and IFN-g–producing

FIGURE 2. Card9 is dispensable for T cell ex- pansion and activation. (A) Wild type and Card92/2 mice received an adoptive transfer of 106 CD4+-purified, CFSE-labeled, naive 1807 Tg cells and were vaccinated with 106 HK B. darmatttidis yeast or not. Transferred 1807 cells were harvested from the sdLN and pro- liferation was assessed by enumerating the frequency of CFSElow cells (mean 6 SEM; n = 4–6 mice/group; representative of three experiments) at day 7 after vaccination. (B) At serial times after vaccination, the frequency of activated (CD44hi) 1807 Tg cells was determined in the sdLN and in the lung at day four after infection. Data are the mean 6 SEM (n =4–6 mice/group); representative of five experiments. (C) The number of activated (CD44hi) 1807 Tg cells for (B). Data are the mean 6 SEM (n = 4–6 mice/group) and are representative of five experiments. *p , 0.05 versus vaccinated wild type controls. 1112 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY

FIGURE 3. Helper T cells fail to differentiate without Card9 signaling. (A) Primed T cells from sdLN of wild type and Card92/2 mice were cocultured ex vivo with CW/M extract for 3 d, and cytokine production measured by ELISA. Data are the mean 6 SEM (n = 6 mice/group) and are rep- resentative of three experiments. *p , 0.05 versus vacci- nated wild type controls. (B) BMDCs from wild type and Card92/2 mice were cocultured with B. dermatitidis vaccine yeast and CD4+ purified naive 1807 cells for 3 d, and cyto- kine production was measured with ELISA. Data are the mean 6 SEM (n = 3–5 mice/group) and representative of five experiments. *p , 0.05 versus vaccinated wild type controls. (C) CD4+ purified 106 naive wild type 1807 Tg cells were adoptively transferred into wild type and Card92/2 mice prior to vaccination with 107 HK B. dermatitidis yeast. Effector T cells were harvested from the s.c. tissue 10 d after vaccination, and the frequency of cytokine-producing cells was determined by FACS analysis. Data are the mean 6 SEM (n = 6 mice/group) and are representative of three experiments. *p , 0.05 versus vaccinated wild type controls. (D) The changes in cytokine transcript and protein were measured with real-time RT-PCR and ELISA for BMDCs cultured with versus without yeast. Data are the mean 6 SEM (n = 13–15 mice/group). *p , 0.05 versus wild type controls.

1807 cells recruited to the s.c. tissue was reduced in Card92/2 soluble recombinant CLRs by fusing their carbohydrate-recognition versus wild type mice (Fig. 3C). Thus, extrinsic Card9 signaling is domains to the Fc fragment of human IgG1. Soluble Dectin-1-, required for the differentiation of Th17 and Th1 cells during the Dectin-2-, and Mincle-Fc fusion proteins stained B. dermatitidis priming phase of vaccine-induced immunity. vaccine yeast brighter than did Fc-alone (Fig. 4A, left panel). Fluo- rescent microscopy showed punctate staining of Fc fusion proteins Card9-dependent genes induced by vaccine yeast around the cell wall (Fig. 4B). Because the carbohydrate recogni- Upon vaccination, APCs become activated and generate a milieu tion domain of Dectin-2 and Mincle requires calcium for binding, that fosters the generation of Th17 and Th1 cells. To investigate we added 5 mM EDTA to the staining conditions. Staining of the genes that are induced upon yeast exposure, we cocultured the yeast was abrogated by the addition of 5 mM EDTA (data not B. dermatitidis vaccine with BMDCs from 15 individual wild type shown), indicating that the binding of the soluble CLRs to the and Card92/2 mice, harvested their RNA, and analyzed gene yeast occurred in a C-type lectin-specific manner. expression by microarray. Among the upregulated genes, some To investigate whether vaccine yeast are recognized by cell were canonical genes that are known to induce the differentiation surface-bound CLRs and induce downstream receptor signaling, of Th17 cells (data not shown). To verify differential gene ex- we used T hybridoma cells expressing an NFAT-lacZ b-galacto- pression in our microarray, we performed real-time RT-PCR on sidase reporter of ITAM signaling (31, 44). In response to vaccine the individual RNA samples from 15 mice/group (Fig. 3D). IL-6, yeast stimulation, lacZ activity was increased in T hybridoma cells TGF-b, IL-1b, IL-12 p35, and IL-23 p19 were upregulated 33.8-, coexpressing Dectin-2 and FcRg, but not in reporter cells expressing 2.1-, 3.4-, 11.8-, and 10.5-fold in wild type versus Card92/2 DCs. either Dectin-2 or FcRg alone (Fig. 5A). Because surface expression Cytokine transcripts were corroborated with protein measure- of Dectin-2 requires coexpression of FcRg (31), reporter cells ments of the cell culture supernatants by ELISA. IL-6 protein was expressing Dectin-2 and FcRg together bound vaccine yeast (Fig. 4-fold reduced for yeast stimulated wild type versus Card92/2 5B), whereas reporter cells expressing Dectin-2 or FcRg alone DCs. Thus, vaccine yeast–induced expression of these genes by bound yeast poorly (Supplemental Fig. 1A). As expected, reporter DCs is Card9 dependent and likely affects the differentiation cells coexpressing an ITAM signaling-defective mutant of FcRg- phenotype of T cells in Card92/2 mice. chain (31) and Dectin-2 failed to increase reporter activity (Fig. 5A), indicating that ITAM signaling mediates the reporter cell Dectin-2, but not Dectin-1 or Mincle, recognizes activation. However, this reporter cell line bound vaccine yeast as B. dermatitidis vaccine yeast efficiently as T hybridoma cells coexpressing Dectin-2 and wild Because Card9 receives signals from upstream receptors including type FcRg (Supplemental Fig. 1A), indicating that the FcRg the CLRs Dectin-1, Dectin-2, and Mincle, we postulated that these mutations did not affect the ability of Dectin-2 to mediate binding CLRs might recognize B. dermatitidis and the other vaccine yeast of vaccine yeast. In contrast to Dectin-2, reporter cells expressing and elicit vaccine immunity. To test this hypothesis, we generated Dectin-1 or Mincle did not increase lacZ activity (Fig. 5A) and The Journal of Immunology 1113

FIGURE 4. Soluble Dectin-1-Fc, Dectin-2-Fc, and Min- cle-Fc proteins bind three dimorphic fungi. Fungal cells were incubated with Fc fragment alone or Fc fusion pro- teins as indicated by different colors, followed by staining with PE-conjugated anti-human Fc Ab, and analyzed with FACS (A) and microscopy (B). As a negative control, Ab- stained fungal cells without any Fc proteins are indicated by gray filled histograms in (A). Data are representative of two independent experiments. Scale bar, 10 mm.

bound vaccine yeast poorly (Fig. 5B; Supplemental Fig. 1B–C), and the GFP reporter cells confirmed our results with the lacZ although the respective soluble CLRs bound the yeast. Thus, reporting cells (data not shown). Thus, Dectin-1 and Dectin-2, but Dectin-2, but not Dectin-1 or Mincle, recognized B. dermatitidis not Mincle, recognized and responded to both H. capsulatum and vaccine yeast and triggered downstream ITAM signaling through C. posadasii. FcRg. Th17 cell differentiation and vaccine immunity to Dectin-1 and Dectin-2 recognize multiple dimorphic fungi B. dermatitidis requires Dectin2 and FcRg The three CLRs differentially recognize B. dermatitidis; therefore, Because Dectin-2 recognizes and binds B. dermatitidis vaccine we investigated how they recognize H. capsulatum and C. pos- yeast and triggers functional ITAM signaling, we tested to deter- adasii. We incubated the latter two fungi with Fc fusion proteins mine whether Dectin-2 and its downstream signaling components and analyzed them by FACS. All three soluble CLR fusion pro- are necessary to prime Th17 cells and acquire vaccine-induced teins stained H. capsulatum yeast to various degrees (Fig. 4A). resistance. We studied Th17 cell differentiation using several Soluble Dectin-1–Fc stained the yeast strongly, Dectin-2–Fc stained approaches. First, we adoptively transferred naive magnetic intermediately and Mincle-Fc stained the least. All three soluble bead purified CD4+ 1807 cells into Dectin-22/2,FcR-g2/2,and CLR fusion proteins stained C. posadasii efficiently (Fig. 4A). Mincle2/2 mice before vaccination, harvested the primed T cells Fluorescent microscopy showed punctate staining of Fc fusion from the sdLN at the peak of expansion, and stimulated them with proteins on the cell wall of H. capsulatum (Fig. 4B), whereas CW/M Ag in vitro as described above. T cells primed in Dectin-2 a more evenly distributed staining pattern was observed on the and FcRg-deficient mice produced significantly less IL-17 and surface of C. posadasii (Fig. 4B). Dectin-1– and Dectin-2–expressing IFN-g than did T cells from wild type or Mincle2/2 mice (Fig. T hybridoma cells showed increased lacZ reporter activity when 6A). In this assay, both endogenous and adoptively transferred stimulated with H. capsulatum or C. posadasii (Fig. 5A), whereas CD4+ T cells contributed to cytokine production and a T cell in- no increase in reporter activity was detected with Mincle-expressing trinsic contribution cannot be evaluated. Thus, second, to exclude reporter cells. Dectin-1– and Dectin-2–expressing T hybridoma a T cell intrinsic role of either Dectin-2 or FcRg, we studied T cell cells consistently bound H. capsulatum and C. posadasii, whereas differentiation of naive wild type 1807 cells in vitro. When cocul- Mincle-expressing and control reporter cells failed to do so (Fig. tured with BMDCs from Dectin-2 and FcRg deficient mice, 1807 5B; Supplemental Fig. 1). Because all three dimorphic fungi failed cells produced significantly less IL-17 compared with cocultures to increase reporter activity of Mincle-expressing T hybridoma with wild type or Mincle-deficient DCs (Fig. 6B). Third, we cells, we used TDB, a known ligand for Mincle and synthetic studied differentiation of wild type 1807 cells in vivo at the site of analog of the mycobacterial cord factor, as a positive control vaccination. The frequency of 1807 cells producing IL-17 and to stimulate the lacZ reporter cells. In an additional approach, IFN-g was significantly reduced in Dectin-22/2 and FcRg2/2 Dr. Sho Yamasaki’s laboratory tested the recognition of B. derma- compared with wild type recipient mice (Fig. 6C). Fourth, we titidis yeast by surface-bound Mincle using a NFAT-GFP reporter determined the number and frequencies of IL-17– and IFN-g– T cells as described (28). TDB increased the reporter activity of producing 1807 cells that migrated to the lung upon recall. Both the Mincle expressing lacZ reporter cells (Supplemental Fig. 1D) the numbers and frequencies of Ag-specific Th17 and Th1 1807 1114 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY

FIGURE 5. Differential recognition of three dimorphic fungi by surface bound Dectin-1, Dectin-2, and Mincle receptors. (A) BWZ cells and a subline expressing Dectin- 1-CD3z (Dectin-1), as well as B3Z cells expressing Dectin- 2, Mincle, FcRg-chain, Dectin-2 + FcRg, Dectin-2 + FcRgm (a signaling-defective FcRg-chain), Mincle + FcRg, or Mincle + FcRgm were stimulated with the indi- cated numbers of heat-killed B. dermatitidis (left column), H. capsulatum (middle column), or C. posadasii (right column). After 18 h, lacZ activity was measured using a col- orimetric assay and expressed as OD 560/620 values. Data are the mean 6 SD of duplicate wells. (B) Reporter cells expressing Dectin-1-CD3z, Dectin-2 + FcRg, or Mincle + FcRg were prestained with CFSE and incubated for 6 h with heat-killed fungal cells prestained with LIVE/DEAD Violet staining. The numbers indicate the frequencies of B3Z/BWZ cells bound by fungi as analyzed by FACS. Data are representative of two independent experiments.

cells that migrated to the lungs were significantly reduced in mice, compared with wild type controls that were vaccinated vaccinated Dectin-22/2 and FcRg2/2 mice, compared with wild and then challenged with H. capsulatum (Fig. 7A). Fewer IFN-g– type recipient mice (Fig. 6D). Few to no IL-13–expressing Th2 producing T cells also were found in the lungs of vaccinated and cells were recruited to the lungs of any of the mouse strains in- challenged Dectin-22/2 and FcRg2/2 mice compared with wild vestigated (data not shown). Not surprisingly, vaccinated Dec- type mice. Histoplasma vaccinated Dectin-22/2 mice likewise tin-22/2, FcRg2/2, and Syk2/2 mice were unable to acquire reduced lung CFU significantly less after challenge than did resistance to B. dermatitidis infection in contrast to wild type or vaccinated wild type or Mincle2/2 mice (Fig. 7B). Although Mincle2/2 mice (Fig. 6E). These data indicate that the Dectin-2/ Dectin-12/2 mice were nearly 10-fold less resistant than wild type FcRg/Syk/Card9 signaling pathway is indispensable for the dif- mice, this difference did not achieve statistical significance. ferentiation of vaccine-induced Th17 and Th1 cells and ac- In the Coccidioides model, the development of Th17 (and Th1) quired resistance to B. dermatitidis infection. cells were impaired in C. posadasii vaccinated and challenged Dectin-22/2 and FcRg2/2 mice, as compared with wild type and Dectin-2/FcRg/Syk/Card9 pathway is required for Th17 cell Mincle2/2 recipients. The acquisition of vaccine immunity like- differentiation and vaccine resistance to multiple dimorphic wise was blunted in these mice, as determined by the reduction of fungi lung and spleen CFU (Fig. 7C+D). Thus, the Dectin-2/FcRg/Syk/ To investigate which CLRs upstream of Card9 are required for Card9 signaling pathway is required for the optimal development the development of Th17 cells and vaccine-induced resistance to of vaccine-induced Th17 cells and acquired resistance to multiple infection with H. capsulatum and C. posadasii, we studied the re- systemic dimorphic fungi. In addition, whereas Dectin-1 is re- cruitment of primed T cells to the lung and acquired resistance to quired for the acquisition of vaccine-induced Th17 cells and re- infection upon recall in the respective vaccine models. For H. cap- sistance to H. capsulatum, this CLR is dispensable for eliciting sulatum, fewer IL-17–producing 1807 cells migrated to the lungs of these responses to C. posadasii (data not shown and paper in Dectin-12/2,Dectin-22/2,andFcRg2/2 mice, but not Mincle2/2 preparation). The Journal of Immunology 1115

FIGURE 6. FcRg and Dectin-2 are required for T cell differentiation and vaccine immunity to B. dermatitidis infection. (A) At day 7 after vaccination cells, harvested from the sdLN of vaccinated wild type, Dectin-22/2, FcRg2/2, and Mincle2/2 mice were cocultured ex vivo with CW/M extract and cytokine production measured with ELISA. Data are the mean 6 SEM (n = 6 mice/group) and are representative of three experiments. *p , 0.05 versus wild type controls. (B) Wild type, Dectin-22/2, FcRg2/2, and Mincle2/2 BMDCs were cocultured with B.dermatitidis vaccine yeast and CD4+-purified, naive 1807 cells for 3 d, and cytokine production measured by ELISA. Data are the mean 6 SEM (n = 3–5 mice/group) and are representative of three experiments. *p , 0.05 versus wild type controls. (C) Wild type, Dectin-22/2, FcRg2/2, and Mincle2/2 mice received 106 naive, CD4+-purified, wild type 1807 T cells and were vaccinated with 107 HK B. dermatitidis yeast. Ten days after vaccination, the frequency of cytokine-producing 1807 T cells harvested from the site of vaccination was determined with FACS analysis. Data are the mean 6 SEM (n = 6 mice/group) and are representative of three experiments. *p , 0.05 versus wild type controls. (D) Mice described in (C) were challenged with 2 3 104 strain 26199 yeast 5 wk after vaccination, and the numbers and frequencies of cytokine-producing 1807 cells were enumerated with FACS at day 4 after infection. Data are the mean 6 SEM (n = 4–6 mice/group) and are representative of four experiments. *p , 0.05 versus vaccinated wild type controls. (E) Wild type, Dectin-22/2, FcRg2/2, Mincle2/2, and chimeric Syk+/+ or Syk2/2 mice were vaccinated with 106 HK B. dermatitidis yeast or not as a control and challenged with 2 3 103 strain 26199 yeast 2 wk after booster vaccination. Lung CFUs were determined when unvaccinated mice were moribund (.14 d after infection). Data are the mean 6 SEM (n = 8–12 mice/group) and are representative of four experiments. Numbers indicate the n-fold change in lung CFU versus unvaccinated controls. *p , 0.05 versus vaccine-induced reduction in lung CFUs in wild type or Syk+/+ mice.

Discussion systemic mycosis of North America by differentiating naive The development of effective vaccines requires a fundamental fungus-specific T cells into protective Th17 cells. Although Card9 understanding of how protective immune responses are induced. has been implicated in host protection to primary infection and the In this study, we identify the signaling adaptors and PRRs that development of Th17 cells (17), we establish that it has a requisite govern the acquisition of vaccine immunity to the three major role in the induction of vaccine immunity, and we uncover the 1116 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY

FIGURE 7. FcRg and Dectin-2 are required for T cell differentiation and adaptive immunity to C. 1 posadasii and H. capsulatum. (A)106 naı¨ve CD4 purified, wild type 1807 Tg cells were transferred into wild type, Dectin-22/2,FcRg2/2,andMincle2/2 mice prior to vaccination with 107 HK H. capsulatum G217B yeast and challenged with 105 H. capsulatum yeast. At day four postinfection, the numbers and frequencies of cytokine-producing 1807 cells in vac- cinated mice were determined by FACS analysis. Data are the mean 6 SEM (n 5 4–6 mice/group); representative of two experiments. *p , 0.05 versus vaccinated wild type controls. (B) Wild type, Dectin-22/2, FcRg2/2, and Mincle2/2 mice were vaccinated with 107 HK H. capsulatum G217B yeast (or not as a control) and challenged with 105 H. capsulatum yeast 2 wk after booster vaccination. Lung CFU were determined 13 d postinfection. Data are the mean 6 SEM (n 5 8–12 mice/group); representative of two experiments. Numbers indi- cate the n-fold change in lung CFU versus unvac- cinated controls. *p , 0.05 versus vaccine-induced reduction in CFU in wild type mice. (C) Same as (A), but mice were vaccinated with 106 FKS of C. posadasii. Two wk after the boost, the mice were challenged with 106 FKS. Data are the mean 6 SEM (n 5 4–6 mice/group); representative of two experiments. *p , 0.05 versus vaccinated wild type controls. (D) Wild type, Dectin-22/2, FcRg2/2, and Mincle2/2 mice were vaccinated with 106 FKS or 5 3 104 viable spores of the ΔT strain (or not as a control) and challenged with 50–80 C. posadasii spores 4 wk later. Lung and spleen CFU were de- termined 14 d postinfection. Data are the averages 6 SEM of two independent experiments (n 5 8–12 mice/group/experiment); representative of two experi- ments. Numbers indicate the n-fold change in lung CFU versus unvaccinated controls. *p , 0.05 versus vaccine-induced reduction in lung CFU in wild type mice.

stage of the immune response at which it governs priming of Th17 we determined the number of IL-17–producing CD4+ T cells in cells in vivo. Finally, we report distinctly different requirements the lungs of vaccinated mice upon recall after challenge. We also for PRRs upstream of Card9 to induce the development of Th17 enumerated the number of IFN-g–producing CD4+ T cells be- cells and vaccine immunity to the three systemic dimorphic fungi cause they can contribute to vaccine-induced resistance (4), al- of the United States. though Th1 cells are dispensable, unlike Th17 cells (14). We We found that Card9 is essential for the acquisition of vaccine- observed that the numbers of Th17 and Th1 cells in all three induced resistance to infection with the dimorphic fungi B. derma- vaccine models were significantly reduced in vaccinated Card92/2 titidis, H. capsulatum,andC. posadasii.Card92/2 mice failed to versus wild type mice. Using our recently developed adoptive control s.c. delivery of live attenuated vaccine yeast of B. dermatitidis transfer system with TCR transgenic 1807 cells, which recognize and wild type conidia of C. posadasii, but survived vaccination with a shared and protective Ag among the systemic dimorphic fungi live H. capsulatum yeast. In response to vaccination with heat killed (34), we demonstrated that although Card9 is required for the B. dermatitidis yeast, live H. capsulatum yeast or formalin-fixed differentiation of naive T cells into Th17, the adaptor is largely spherules of C. posadasii, Card92/2 mice failed to acquire vaccine dispensable for the activation, expansion, proliferation, and sur- resistance. These results indicate that the Card9 signaling pathway is vival during the contraction phase, and for recruitment to the lung essential and can be harnessed for the induction of vaccine-induced upon recall. Moreover, Card9 not only affected the differentiation protection against dimorphic fungal infection. of Th17 cells; it likewise affected Th1 cells. Although the role of Because the generation of Th17 cells directly correlates with the CLR-Card9 signaling pathway on the development of effector vaccine-induced resistance to the systemic dimorphic fungi (14), Th17 and Th1 cells has not been studied in vivo, the TLR-Myd88 The Journal of Immunology 1117 pathway has been shown to exert an incremental impact on the a severe defect in differentiation of fungus-specific Th17 cells and differentiation of A. fumigatus–specific CD4+ T cells during pul- vaccine immunity, indicating that Dectin-2 is indispensable for the monary infection (48). Myd88-mediated signals were dispensable acquisition of adaptive immunity to B. dermatitidis infection. The for the recruitment and expansion of Ag-specific T cells to the inability of T hybridoma cell–expressed Mincle to trigger NFAT- mediastinal lymph nodes (MLN) and trafficking to the lung after reporter activity or bind to the three dimorphic fungi correlated priming. However, Myd88 signaling was required to enhance T- with the dispensability of this CLR in the vaccine models. bet induction and differentiation of Th1 cells in the MLN. Further Similar to Card9, the upstream receptor Dectin-2 affected the maturation of MLN-primed T cells into fully differentiated IFN- development of both B. dermatitidis–specific Th17 and Th1 but g–producing Th1 cells occurred upon arrival of the cells in the not Th2 cells during vaccination. Therefore, Dectin-2 and Card9 airways and was TLR and Myd88 independent. deficiency did not shift the balance of Th cell differentiation as has To discern how Card9 induces T helper differentiation, we per- been reported for Th17 responses that depended on Dectin-1 formed a microarray analysis and verified differential gene ex- during a pulmonary infection of mice with A. fumigatus (50). pression by real-time RT-PCR. Among those differentially expressed Dectin-1 deficiency disproportionally increased Th1 responses and were genes known to induce Th17 and Th1 cells, such as IL-6, IL- decreased Th17 differentiation. The authors showed that Dectin-1 23, TGF-b, IL-1b, and IL-12 p35. Thus, Card9 governs the ex- mediated signals alter CD4 T cell responses to fungal infection by pression of cytokines that are instrumental in driving differentia- reducing the production of IFN-g and IL-12p40 in innate cells, tion of Th17 and Th1 cells. Using the microarray data, we also thereby decreasing T-bet expression in responding CD4 T cells found noncanonical genes that have not been linked previously to and enhancing Th17 responses (50). Th17 differentiation (V. LeBert, H. Wang and M. Wu¨thrich, un- Despite the fact that cell surface–expressed Dectin-1 and Dectin- published data). We intend to study the role of novel candidates in 2 recognized and bound H. capsulatum yeast and C. posadasii elucidating how Card9 governs the expression of the Th17 and spherules, the effect on Th17 cell priming and the acquisition of Th1 priming cytokines. vaccine immunity was modest, albeit statistically significant. Our data To investigate which PRRs upstream of Card9 are required to are compatible with the interpretation that these two CLRs are re- induce the differentiation of Th17 cells and vaccine-induced im- dundant or cooperate in the innate recognition of these two dimorphic munity, we first tested whether soluble fusion proteins consisting fungi. Receptor collaboration has been reported for Dectin-1 and of the extracellular domain of Dectin-1, Dectin-2, and Mincle and multiple TLRs (51), and for the recognition of by Dectin- human Fc can stain fungal yeast and spherules. Soluble Dectin-1 2 and Mincle through distinct ligands (29). The fact, that H. capsu- and Dectin-2 have been used previously to stain A. fumigatus (42), latum and C. posadasii vaccinated FcRg2/2 mice showed stronger (49), C. albicans (27, 30), and C. immitis (18). phenotypes in acquiring vaccine-induced immunity and Th17 cells Here, all three soluble Fc chimeras tested were able to bind the compared with Dectin-22/2 mice is compatible with the notion of three dimorphic fungi; however, the binding efficiency between receptor cooperation. Alternatively, other FcRg coupled CLR alone soluble receptors and fungal species varied presumably based on or together with Dectin-2 could play a prominent role in innate fungal respective ligand expression and size of the organisms. Second, to recognition as well. A recently identified CLR, MCL, is a potential explore whether cell surface–bound CLRs can recognize and bind candidate, and mediates TDM-induced acquired immune responses the fungal vaccine strains we cocultured T cell hybridoma cells (52); however, its role in vaccine immunity to fungi has not been that were engineered to express a b-galactosidase NFAT-lacZ re- studied. porter of ITAM signaling (43). By using this nonmyeloid reporter A prior study showed that b-glucan on H. capsulatum yeast was system, we were able to distinguish between signaling events shielded by a-glucan and not recognized by Dectin-1 (53), whereas triggered by putative CLR ligands versus other ligands. To our we observed that Dectin-1 expressing reporter cells bound to H. surprise, some of the hybridoma cell–expressed CLRs showed a rec- capsulatum, which triggered lacZ reporter activity in vitro. Conse- ognition pattern different from soluble CLR chimera. For example, quently, in vivo, the induction of Th17 cells, the acquisition of soluble Dectin-1 stained B. dermatitidis vaccine yeast, but surface vaccine-induced resistance to H. capsulatum infection, and resis- expressed receptor did not recognize the vaccine yeast. Similarly, tance to primary infection were all blunted in Dectin-12/2 versus soluble Mincle stained all three dimorphic fungi, but Mincle wild type control mice. The differing results between the two expressing reporter cells did not become activated and failed to studies may be due to strain specific differences in the cell wall bind the three fungi. On the other hand, soluble and surface- composition. We used the North American, RFLP class 2 and expressed Dectin-2 yielded consistent results for each of the fungi chemotype I strain G217B (ATCC 26032) that mostly lacks a-1,3- tested. Although soluble CLR chimeras gave valid binding results, glucan on the yeast surface. In contrast, Rappleye et al. (53) used they were less reliable than surface expressed CLRs in forecasting the Central and South American, RFLP class 3 and chemotype II the in vivo role of CLRs in vaccine resistance. yeast strain G186AR (ATCC 26029), which expresses surface One potential explanation for the discrepant findings between a-1,3-glucan that shields b-glucan from detection by Dectin-1. In the two assays could be that only surface-exposed fungal ligands a recent report (54), liquid grown G217B yeast bound to Dectin- can be recognized by cell surface–expressed receptors, but not 1–expressing 3T3 fibroblasts when yeast were harvested during ligands that are embedded deeper in the cell wall; however, the the exponential phase, but not during the stationary phase (54). In latter might still be accessible by soluble CLRs. Thus, cell sur- our study, we grew G217B yeast for 3 to 7 d on slants and heat- face–expressed receptors on the T hybridoma reporter cells helped killed them to perform overnight incubation in the lacZ reporter to predict the importance of the receptor for the priming of T assay. Surface staining of yeast with Dectin-1 fusion protein was helper responses in vivo. Indeed, the failure of cell surface– comparable in live versus heat-killed cells, indicating that heat in- expressed Dectin-1 to recognize B. dermatitidis is consistent with activation did not increase exposure of b-glucan. However, we have our previous data showing that Dectin-1 is dispensable for the seen large differences in the gross appearance of the yeast cell wall induction of Th17 and Th1 cells and vaccine immunity (14). by electron microscopy (H. Wang and M. Wu¨thrich, unpublished data) Similarly, the phenotypes of Dectin-2 and Mincle in the vacci- when comparing liquid versus agar grown yeast, which could nation model with B. dermatitidis showed good fidelity with the account for the different Dectin-1 binding phenotypes. Nonethe- results of the NFAT reporter cells. Dectin-22/2 mice showed less, our data suggest that b-glucan is not shielded in H. capsu- 1118 DECTIN-2 INDUCES ANTIFUNGAL T CELL IMMUNITY latum strain G217B yeast in vivo, and it is recognized by Dectin-1 5. Chai, L. Y., F. van de Veerdonk, R. J. Marijnissen, S. C. Cheng, A. L. Khoo, during primary and adaptive immunity to recruit and prime Th17 M. Hectors, K. Lagrou, A. G. Vonk, J. Maertens, L. A. Joosten, et al. 2010. Anti- human host defence relies on type 1 T helper (Th1), rather than type cells, respectively. 17 T helper (Th17), cellular immunity. Immunology 130: 46–54. Dectin-1 induces two independent signaling pathways—one 6. Allendo¨rfer, R., G. D. Brunner, and G. S. Deepe, Jr. 1999. Complex requirements for nascent and memory immunity in pulmonary . J. Immunol. through Syk and one through Raf-1—to control adaptive immu- 162: 7389–7396. nity (55, 56), but we have not determined whether the FcRg/Card9 7. Lin, L., A. S. Ibrahim, X. Xu, J. M. Farber, V. Avanesian, B. Baquir, Y. Fu, and Dectin-1 phenotypes in the vaccination model with H. cap- S. W. French, J. E. Edwards, Jr., and B. Spellberg. 2009. Th1-Th17 cells mediate protective adaptive immunity against Staphylococcus aureus and Candida sulatum are functionally linked or unrelated/Raf-1 dependent. albicans infection in mice. PLoS Pathog. 5: e1000703. The fungal ligands of most CLRs remain largely unclear. It is 8. Zelante, T., A. De Luca, C. D’Angelo, S. Moretti, and L. Romani. 2009. IL-17/ likely that differences in the cell wall composition and structure Th17 in anti-fungal immunity: what’s new? Eur. J. Immunol. 39: 645–648. 9. Zelante, T., A. De Luca, P. Bonifazi, C. Montagnoli, S. Bozza, S. Moretti, between the endemic fungi account for the differential recognition M. L. Belladonna, C. Vacca, C. Conte, P. Mosci, et al. 2007. IL-23 and the Th17 by Dectin-1, Dectin-2, and Mincle. In addition, individual CLR can pathway promote inflammation and impair antifungal immune resistance. Eur. recognize several distinct ligands within one fungal species and J. Immunol. 37: 2695–2706. 10. Hernandez-Santos, N., A. R. Huppler, A. C.Peterson,S.A.Khader,K.C.McKenna, among different fungi and microorganisms. For example, Mincle and S. L. Gaffen. 2013. Th17 cells confer long-term adaptive immunity to oral recognizes TDM from mycobacteria, corynebacteria, and Nocardia mucosal Candida albicans infections. Mucosal Immunol. 6: 900–910. (57), its synthetic analog TDB (25) and two structurally different 11. Pandiyan, P., H. R. Conti, L. Zheng, A. C. Peterson, D. R. Mathern, N. Herna´ndez-Santos, M. Edgerton, S. L. Gaffen, and M. J. Lenardo. 2011. CD4 glycolipids 44-1 and 44-2 from Malassezia (29). Similarly, Dectin-2 (+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance recognizes the terminal mannose of N-linked glycan (27) and O- host resistance in mouse Candida albicans Th17 cell infection model. Immunity linked a-1,2 mannosyl residues from Malassezia (29). Thus, it is 34: 422–434. 12. Rudner, X. L., K. I. Happel, E. A. Young, and J. E. Shellito. 2007. Interleukin-23 conceivable that Dectin-2 recognizes ligands in the systemic en- (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection. Infect. demic fungi that are structurally distinct from those that have been Immun. 75: 3055–3061. described above. 13. Deepe, G. S., Jr., and R. S. Gibbons. 2009. Interleukins 17 and 23 influence the host response to Histoplasma capsulatum. J. Infect. Dis. 200: 142–151. CLRs have been of burgeoning interest as targets to enhance 14. Wu¨thrich, M., B. Gern, C. Y. Hung, K. Ersland, N. Rocco, J. Pick-Jacobs, vaccine efficacy (56, 58). A promising strategy is to use carbo- K. Galles, H. Filutowicz, T. Warner, M. Evans, et al. 2011. Vaccine-induced hydrate ligands of CLRs to drive vaccine responses (59, 60). For protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice. J. Clin. Invest. 121: 554–568. example, the particulate b-glucan curdlan, a well-defined ligand 15. Vautier, S., D. M. MacCallum, and G. D. Brown. 2012. C-type lectin receptors for Dectin-1, has been reported to be a potent adjuvant that primes and cytokines in fungal immunity. Cytokine 58: 89–99. 16. Osorio, F., and C. Reis e Sousa. 2011. Myeloid C-type lectin receptors in anti-Candida Th17 responses (61). Moreover, porous b-glucan pathogen recognition and host defense. Immunity 34: 651–664. particles that consist primarily of b-1,3-D-glucans and allow for 17. LeibundGut-Landmann, S., O. Gross, M. J. Robinson, F. Osorio, E. C. Slack, high Ag loading have been exploited as a receptor-targeted vac- S. V. Tsoni, E. Schweighoffer, V. Tybulewicz, G. D. Brown, J. Ruland, and C. Reis e Sousa. 2007. Syk- and CARD9-dependent coupling of innate immunity cine delivery system (62). Thus, the glucan particle-based vaccine to the induction of T helper cells that produce interleukin 17. Nat. Immunol. 8: platform combines adjuvanticity and Ag delivery to induce strong 630–638. Th17- and Th1-biased CD4+ T cell responses. Because mannans 18. Viriyakosol, S., Mdel. P. Jimenez, M. A. Gurney, M. E. Ashbaugh, and J. Fierer. 2013. Dectin-1 is required for resistance to in mice. MBio. are recognized by Dectin-2 (23, 27, 29), we postulate that they can 4: e00597–e12. be harnessed as adjuvants for vaccination to combat infection with 19. Ferwerda, B., G. Ferwerda, T. S. Plantinga, J. A. Willment, A. B. van Spriel, the systemic dimorphic fungi of North America that collectively H. Venselaar, C. C. Elbers, M. D. Johnson, A. Cambi, C. Huysamen, et al. 2009. Human dectin-1 deficiency and mucocutaneous fungal infections. N. Engl. account for nearly a million new infections annually. J. Med. 361: 1760–1767. 20. Plantinga, T. S., W. J. van der Velden, B. Ferwerda, A. B. van Spriel, G. Adema, T. Feuth, J. P. Donnelly, G. D. Brown, B. J. Kullberg, N. M. Blijlevens, and Acknowledgments M. G. Netea. 2009. Early stop polymorphism in human DECTIN-1 is associated We thank the laboratory of Dr. Sho Yamasaki for validating the recogni- with increased candida colonization in hematopoietic stem cell transplant tion of B. dermatitidis by Mincle expressing NFAT-GFP reporter cells; recipients. Clin. Infect. Dis. 49: 724–732. Dr. Victor L. J. Tybulewicz for Syk2/2 breeder mice; Dr. George S. Deepe 21. Chai, L. Y., M. G. de Boer, W. J. van der Velden, T. S. Plantinga, A. B. van Spriel, C. Jacobs, C. J. Halkes, A. G. Vonk, N. M. Blijlevens, J. T. van Dissel, for H. capsulatum G217B yeast; Dr. Philip R. Taylor for the Dectin-2-Fc et al. 2011. The Y238X stop codon polymorphism in the human b-glucan re- expression vector pSecTag2(C)-mDectin-2-Fc; Dr. Christine A. Wells and ceptor dectin-1 and susceptibility to invasive aspergillosis. J. Infect. Dis. 203: Robert B. Ashman for the pEF1HISB-mMincle plasmid; Dr. Gordon 736–743. D. Brown for soluble Dectin-1-Fc fusion protein; Dr. Nilabh Shastri for 22. Cunha, C., M. Di Ianni, S. Bozza, G. Giovannini, S. Zagarella, T. Zelante, B3Z parent cells; and Dr. Caetano Reis e Sousa for the pFB-IRES-EGFP ret- C. D’Angelo, A. Pierini, L. Pitzurra, F. Falzetti, et al. 2010. Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hema- roviral vector, the B3Z reporter cells expressing Dectin-2 and negative topoietic transplantation through impairment of both recipient- and donor- control constructs, and BWZ cells expressing Dectin-1. dependent mechanisms of antifungal immunity. Blood 116: 5394–5402. 23. Saijo, S., S. Ikeda, K. Yamabe, S. Kakuta, H. Ishigame, A. Akitsu, N. Fujikado, T. Kusaka, S. Kubo, S. H. Chung, et al. 2010. Dectin-2 recognition of alpha- Disclosures mannans and induction of Th17 cell differentiation is essential for host defense The authors have no financial conflicts of interest. against Candida albicans. Immunity 32: 681–691. 24. Gringhuis, S. I., B. A. Wevers, T. M. Kaptein, T. M. van Capel, B. Theelen, T. Boekhout, E. C. de Jong, and T. B. Geijtenbeek. 2011. Selective C-Rel ac- tivation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin- References 2. PLoS Pathog. 7: e1001259. 1. Wu¨thrich, M., H. I. Filutowicz, and B. S. Klein. 2000. Mutation of the WI-1 gene 25. Schoenen, H., B. Bodendorfer, K. Hitchens, S. Manzanero, K. Werninghaus, yields an attenuated blastomyces dermatitidis strain that induces host resistance. F. Nimmerjahn, E. M. Agger, S. Stenger, P. Andersen, J. Ruland, et al. 2010. J. Clin. Invest. 106: 1381–1389. Cutting edge: Mincle is essential for recognition and adjuvanticity of the my- 2. Wu¨thrich, M., H. I. Filutowicz, T. Warner, G. S. Deepe, Jr., and B. S. Klein. cobacterial cord factor and its synthetic analog trehalose-dibehenate. J. Immunol. 2003. Vaccine immunity to pathogenic fungi overcomes the requirement for 184: 2756–2760. CD4 help in exogenous antigen presentation to CD8+ T cells: implications for 26. Reid, D. M., N. A. Gow, and G. D. Brown. 2009. Pattern recognition: recent vaccine development in immune-deficient hosts. J. Exp. Med. 197: 1405–1416. insights from Dectin-1. Curr. Opin. Immunol. 21: 30–37. 3. Xue, J., X. Chen, D. Selby, C. Y. Hung, J. J. Yu, and G. T. Cole. 2009. A ge- 27. McGreal, E. P., M. Rosas, G. D. Brown, S. Zamze, S. Y. Wong, S. Gordon, netically engineered live attenuated vaccine of Coccidioides posadasii protects L. Martinez-Pomares, and P. R. Taylor. 2006. The carbohydrate-recognition BALB/c mice against coccidioidomycosis. Infect. Immun. 77: 3196–3208. domain of Dectin-2 is a C-type lectin with specificity for high mannose. 4. Wu¨thrich, M., H. I. Filutowicz, T. Warner, and B. S. Klein. 2002. Requisite Glycobiology 16: 422–430. elements in vaccine immunity to Blastomyces dermatitidis: plasticity uncovers 28. Yamasaki, S., M. Matsumoto, O. Takeuchi, T. Matsuzawa, E. Ishikawa, vaccine potential in immune-deficient hosts. J. Immunol. 169: 6969–6976. M. Sakuma, H. Tateno, J. Uno, J. Hirabayashi, Y. Mikami, et al. 2009. C-type The Journal of Immunology 1119

lectin Mincle is an activating receptor for , Malassezia. Proc. 44. Sancho, D., O. P. Joffre, A. M. Keller, N. C. Rogers, D. Martı´nez, P. Hernanz- Natl. Acad. Sci. USA 106: 1897–1902. Falco´n, I. Rosewell, and C. Reis e Sousa. 2009. Identification of a dendritic cell 29. Ishikawa, T., F. Itoh, S. Yoshida, S. Saijo, T. Matsuzawa, T. Gonoi, T. Saito, receptor that couples sensing of necrosis to immunity. Nature 458: 899–903. Y. Okawa, N. Shibata, T. Miyamoto, and S. Yamasaki. 2013. Identification of 45. Sanderson, S., and N. Shastri. 1994. LacZ inducible, antigen/MHC-specific distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the T cell hybrids. Int. Immunol. 6: 369–376. pathogenic fungus Malassezia. Cell Host Microbe 13: 477–488. 46. Fisher, L. D., and G. van Belle. 1993. Biostatistics: A Methodology for the 30. Sato, K., X. L. Yang, T. Yudate, J. S. Chung, J. Wu, K. Luby-Phelps, Health Sciences. John Wiley & Sons, New York, p. 611–613. R. P. Kimberly, D. Underhill, P. D. Cruz, Jr., and K. Ariizumi. 2006. Dectin-2 is 47. Quigley, M., J. Martinez, X. Huang, and Y. Yang. 2009. A critical role for direct a pattern recognition receptor for fungi that couples with the Fc receptor gamma TLR2-MyD88 signaling in CD8 T-cell clonal expansion and memory formation chain to induce innate immune responses. J. Biol. Chem. 281: 38854–38866. following vaccinia viral infection. Blood 113: 2256–2264. 31. Robinson, M. J., F. Osorio, M. Rosas, R. P. Freitas, E. Schweighoffer, O. Gross, 48. Rivera, A., G. Ro, H. L. Van Epps, T. Simpson, I. Leiner, D. B. Sant’Angelo, and J. S. Verbeek, J. Ruland, V. Tybulewicz, G. D. Brown, et al. 2009. Dectin-2 is E. G. Pamer. 2006. Innate immune activation and CD4+ T cell priming during respiratory fungal infection. Immunity 25: 665–675. a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal 49. Sousa, Mda. G., D. M. Reid, E. Schweighoffer, V. Tybulewicz, J. Ruland, infection. J. Exp. Med. 206: 2037–2051. J. Langhorne, S. Yamasaki, P. R. Taylor, S. R. Almeida, and G. D. Brown. 2011. 32. Yamasaki, S., E. Ishikawa, M. Sakuma, H. Hara, K. Ogata, and T. Saito. 2008. Restoration of pattern recognition receptor costimulation to treat chromo- Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nat. , a chronic fungal infection of the skin. Cell Host Microbe 9: 436–443. Immunol. 9: 1179–1188. 50. Rivera, A., T. M. Hohl, N. Collins, I. Leiner, A. Gallegos, S. Saijo, J. W. Coward, 33. Glocker, E. O., A. Hennigs, M. Nabavi, A. A. Scha¨ffer, C. Woellner, U. Salzer, Y. Iwakura, and E. G. Pamer. 2011. Dectin-1 diversifies Aspergillus fumigatus- D. Pfeifer, H. Veelken, K. Warnatz, F. Tahami, et al. 2009. A homozygous specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation. CARD9 mutation in a family with susceptibility to fungal infections. N. Engl. J. Exp. Med. 208: 369–381. J. Med. 361: 1727–1735. 51. Gantner, B. N., R. M. Simmons, S. J. Canavera, S. Akira, and D. M. Underhill. 34. Wu¨thrich, M., C. Y. Hung, B. H. Gern, J. C. Pick-Jacobs, K. J. Galles, 2003. Collaborative induction of inflammatory responses by dectin-1 and Toll- H. I. Filutowicz, G. T. Cole, and B. S. Klein. 2011. A TCR transgenic mouse like receptor 2. J. Exp. Med. 197: 1107–1117. reactive with multiple systemic dimorphic fungi. J. Immunol. 187: 1421–1431. 52. Miyake, Y., K. Toyonaga, D. Mori, S. Kakuta, Y. Hoshino, A. Oyamada, 35. Wu¨thrich, M., K. Ersland, T. Sullivan, K. Galles, and B. S. Klein. 2012. Fungi H. Yamada, K. I. Ono, M. Suyama, Y. Iwakura, Y. Yoshikai, and S. Yamasaki. subvert vaccine T cell priming at the respiratory mucosa by preventing 2013. C-type lectin MCL is an FcRgamma-coupled receptor that mediates the chemokine-induced influx of inflammatory monocytes. Immunity 36: 680–692. adjuvanticity of mycobacterial cord factor. Immunity. 38: 1050–1062. 36. Hsu, Y. M., Y. Zhang, Y. You, D. Wang, H. Li, O. Duramad, X. F. Qin, C. Dong, 53. Rappleye, C. A., L. G. Eissenberg, and W. E. Goldman. 2007. Histoplasma and X. Lin. 2007. The adaptor protein CARD9 is required for innate immune capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta- responses to intracellular pathogens. Nat. Immunol. 8: 198–205. glucan receptor. Proc. Natl. Acad. Sci. USA 104: 1366–1370. 37. Wells, C. A., J. A. Salvage-Jones, X. Li, K. Hitchens, S. Butcher, R. Z. Murray, 54. Edwards, J. A., E. A. Alore, and C. A. Rappleye. 2011. The yeast-phase viru- A. G. Beckhouse, Y. L. Lo, S. Manzanero, C. Cobbold, et al. 2008. The lence requirement for a-glucan synthase differs among Histoplasma capsulatum macrophage-inducible C-type lectin, mincle, is an essential component of the chemotypes. Eukaryot. Cell 10: 87–97. innate immune response to Candida albicans. J. Immunol. 180: 7404–7413. 55. Geijtenbeek, T. B., and S. I. Gringhuis. 2009. Signalling through C-type lectin 38. Turner, M., P. J. Mee, P. S. Costello, O. Williams, A. A. Price, L. P. Duddy, receptors: shaping immune responses. Nat. Rev. Immunol. 9: 465–479. M. T. Furlong, R. L. Geahlen, and V. L. Tybulewicz. 1995. Perinatal lethality and 56. Wu¨thrich, M., G. S. Deepe, Jr., and B. Klein. 2012. Adaptive immunity to fungi. blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378: Annu. Rev. Immunol. 30: 115–148. 57. Ishikawa, E., T. Ishikawa, Y. S. Morita, K. Toyonaga, H. Yamada, O. Takeuchi, 298–302. T. Kinoshita, S. Akira, Y. Yoshikai, and S. Yamasaki. 2009. Direct recognition of 39. Brandhorst, T. T., M. Wu¨thrich, T. Warner, and B. Klein. 1999. Targeted gene the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. disruption reveals an adhesin indispensable for pathogenicity of Blastomyces J. Exp. Med. 206: 2879–2888. dermatitidis. J. Exp. Med. 189: 1207–1216. 58. Hardison, S. E., and G. D. Brown. 2012. C-type lectin receptors orchestrate 40. Levine, H. B., Y. C. Kong, and C. Smith. 1965. Immunization of Mice to antifungal immunity. Nat. Immunol. 13: 817–822. Coccidioides Immitis: Dose, Regimen and Spherulation Stage of Killed Spherule 59. Cutler, J. E., G. S. Deepe, Jr., and B. S. Klein. 2007. Advances in combating Vaccines. J. Immunol. 94: 132–142. fungal diseases: vaccines on the threshold. Nat. Rev. Microbiol. 5: 13–28. 41. Johnson, M. R., K. Wang, J. B. Smith, M. J. Heslin, and R. B. Diasio. 2000. 60. Lang, R., H. Schoenen, and C. Desel. 2011. Targeting Syk-Card9-activating Quantitation of dihydropyrimidine dehydrogenase expression by real-time re- C-type lectin receptors by vaccine adjuvants: findings, implications and open verse transcription polymerase chain reaction. Anal. Biochem. 278: 175–184. questions. Immunobiology 216: 1184–1191. 42. Steele, C., R. R. Rapaka, A. Metz, S. M. Pop, D. L. Williams, S. Gordon, 61. Ba¨r, E., A. Gladiator, S. Bastidas, B. Roschitzki, H. Acha-Orbea, A. Oxenius, and J. K. Kolls, and G. D. Brown. 2005. The beta-glucan receptor dectin-1 recog- S. LeibundGut-Landmann. 2012. A novel Th cell epitope of Candida albicans nizes specific morphologies of Aspergillus fumigatus. PLoS Pathog. 1: e42. mediates protection from fungal infection. J. Immunol. 188: 5636–5643. 43. Karttunen, J., S. Sanderson, and N. Shastri. 1992. Detection of rare antigen- 62. Huang, H., G. R. Ostroff, C. K. Lee, C. A. Specht, and S. M. Levitz. 2010. presenting cells by the lacZ T-cell activation assay suggests an expression Robust stimulation of humoral and cellular immune responses following vac- cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. USA 89: 6020–6024. cination with antigen-loaded beta-glucan particles. MBio. 1: e00164–e10.