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Infect Immun. 2011 Nov; 79(11): 4503–4510.
PMCID: PMC3257912

Protective Role of Naturally Occurring Interleukin-17A-Producing γδ T Cells in the Lung at the Early Stage of Systemic Candidiasis in Mice [down-pointing small open triangle]

G. S. Deepe, Jr, Editor

Abstract

Interleukin-17A (IL-17A)-producing γδ T cells differentiate in the fetal thymus and reside in the peripheral tissues, such as the lungs of naïve adult mice. We show here that naturally occurring γδ T cells play a protective role in the lung at a very early stage after systemic infection with Candida albicans. Selective depletion of neutrophils by in vivo administration of anti-Ly6G monoclonal antibody (MAb) impaired fungal clearance more prominently in the lung than in the kidney 24 h after intravenous infection with C. albicans. Rapid and transient production of IL-23 was detected in the lung at 12 h, preceding IL-17A production and the influx of neutrophils, which reached a peak at 24 h after infection. IL-17A knockout (KO) mice showed reduced infiltration of neutrophils concurrently with impaired fungal clearance in the lung after infection. The major source of IL-17A was the γδ T cell population in the lung, and Cδ KO mice showed little IL-17A production and reduced neutrophil infiltration after infection. Early IL-23 production in a TLR2/MyD88-dependent manner and IL-23-triggered tyrosine kinase 2 (Tyk2) signaling were essential for IL-17A production by γδ T cells. Thus, our study demonstrated a novel role of naturally occurring IL-17A-producing γδ T cells in the first line of host defense against C. albicans infection.

INTRODUCTION

Candida albicans is a dimorphic fungus that causes chronic mucocutaneous candidiasis and, more rarely, multiple-organ failure due to systemic dissemination in immunocompromised hosts (5). Innate immune cells, which recognize components of C. albicans cell walls via pattern recognition receptors (PRRs), are important, not only to clear the microorganism by phagocytosis and killing through oxidative and nonoxidative mechanisms, but also to induce acquired immunity by producing proinflammatory cytokines (18, 20, 29). TLR2, through activation of MyD88, was involved in protection against C. albicans infection by triggering tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and MIP-2 production from macrophages (30, 50). The antifungal activity of neutrophils and the Th1 response induced by dendritic cells (DCs) were impaired in IL-1 receptor (IL-1R) knockout (KO) and MyD88 KO mice after systemic infection with C. albicans (4). More recently, it was revealed that α-mannan, which was exposed on the cell walls of C. albicans, bound to one C-type lectin receptor (CLR), Dectin-2, on DCs and macrophages (39, 40). Dectin-2 KO mice showed impaired Th17 cell differentiation and became susceptible after systemic infection with C. albicans (39). Among innate immune cells, depletion of neutrophils in mice by pretreatment with anti-Gr1 monoclonal antibody (MAb) led to death within 4 days after systemic infection with C. albicans (37), indicating the particular importance of neutrophils for host defense at an early stage when acquired immunity is not completely established, although the detailed mechanism of neutrophil-mediated host defense was not fully understood.

IL-17A is a T cell-derived proinflammatory cytokine that is involved in the mobilization and fungicidal activity of neutrophils (15, 19). The protective roles of IL-17A in a murine model of infection with C. albicans were recently demonstrated. IL-17R KO mice showed markedly decreased neutrophil recruitment to infected tissues and impaired host defense against systemic and oral candidiasis (7, 15). IL-17A KO mice showed increased susceptibility to systemic infection with C. albicans (39). Dectin-2, through activation of caspase-recruiting domain family member 9 (CARD9), induced Th17 cell differentiation (36, 39). In addition to Th17 cells, other cell types were reported to produce IL-17A, including CD8+ T cells, γδ T cells, and NKT cells, although the involvement of these cells in host defense against C. albicans infection was unclear (9).

IL-17A-producing γδ T cells are known as naturally occurring effectors because they are functionally differentiated within the fetal thymus before being exposed to foreign antigens (Ags) (43). In naive mice, IL-17A-producing γδ T cells are widely distributed in various organs but are predominantly found in mucosal tissues, such as the gut, peritoneal cavity, and lung (21, 43). IL-17A-producing γδ T cells play important roles in the first line of host defense against Mycobacterium tuberculosis, Escherichia coli, and Listeria monocytogenes (13, 22, 42, 49). Various exogenous signals were reported to induce IL-17A production by γδ T cells. IL-23 and IL-1β, produced by DCs and macrophages after activation through PRRs, were potent inducers of IL-17A production by γδ T cells (22, 34, 42, 49). γδ T cells expressed TLR2 and Dectin-1, which directly recognized C. albicans and induced IL-17 production and cell proliferation in synergy with IL-23 (23, 26), although the in vivo roles of innate receptors on γδ T cells are still a matter of debate.

In this study, we found rapid production of IL-17A, which was critical for neutrophil infiltration and host defense in the lung at a very early stage after intravenous infection with C. albicans. Resident γδ T cells in the lung were the major source of early IL-17A production after C. albicans infection. IL-23, which was rapidly produced in the TLR2/MyD88-dependent, but not in the CARD9-dependent, pathway induced IL-17A production by γδ T cells, whereas TLR2 expression on γδ T cells was dispensable for IL-17A production per se. These findings provide new insight into the functions of γδ T cells in the first line of host defense against fungal infection.

MATERIALS AND METHODS

Mice.

C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). IL-17A KO, Cδ KO, TLR2 KO, tyrosine kinase 2 (Tyk2) KO, MyD88 KO, and CARD9 KO mice were generated as previously described (1, 14, 17, 27, 44, 47). These mice were bred under specific-pathogen-free conditions in our institute. Six- to 8-week-old male mice were used for the experiments. This study was approved by the Committee on Ethics in Animal Experiments in the Faculty of Medicine, Kyushu University. Experiments were carried out under the control of the Guideline for Animal Experiments.

Microorganisms.

C. albicans (American Type Culture Collection [ATCC] no. 10261 and SC5314) was cultured in a shaking incubator for 24 h at 37°C in Sabouraud broth (Nihon Pharmaceutical, Tokyo, Japan). C. albicans was washed extensively with phosphate-buffered saline (PBS) before being resuspended in 50% glycerol-containing PBS. Small aliquots were stored at −80°C until use. Mice were intravenously (i.v.) inoculated with 1 × 107 CFU of 10261 or 2 × 105 CFU of SC5314, respectively.

Assessment of fungal growth.

At the indicated time after infection, the lungs were removed and placed in homogenizers containing 3 ml of PBS. The lung homogenates were spread on Sabouraud agar plates (Nihon Pharmaceutical, Tokyo, Japan). After incubation for 24 h at 37°C, colonies were counted.

Lung cell preparation.

The lung was minced to yield 1- to 2-mm pieces and incubated with 1 mg/ml collagenase (Gibco) and 20 μg/ml DNase (DN-25; Sigma) in RPMI 1640 containing 10% fetal calf serum (FCS) for 90 min at 37°C with vigorous vortexing every 15 min. Mononuclear cells were further purified in 33% Percoll by centrifugation at 600 × g for 20 min.

Measurement of IL-17A, IL-23, and IL-1β in the lung and kidney.

After mononuclear cells from the lung were cultured for 24 h, the supernatant was collected and measured for the production of IL-17A and IL-1β with the DuoSet ELISA Development System (R&D Systems) and Mouse IL-23 (p19/p40) ELISA (enzyme-linked immunosorbent assay) Ready-Set-Go (eBioscience), respectively, according to the manufacturers' instructions.

MPO activity.

A Mouse MPO ELISA kit (Hycult Biotech) was used for the measurement of myeloperoxidase (MPO) activity. The protocol followed the manufacturer's instructions. Briefly, after centrifugation of lung homogenates taken from naive or infected mice at 1,500 × g at 4°C for 15 min, MPO activities in the supernatants were analyzed.

Antibodies and flow cytometric analysis.

Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (RM4-5), anti-CD11b (M1/70), and anti-CD45.2 (104) MAbs; allophycocyanin (APC)-conjugated anti-T cell receptor γδ (TCRγδ) (GL3), peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated streptavidin, phycoerythrin (PE)-conjugated anti-CD3ε (145-2C11), anti-Ly6G (1A8), and anti-murine IL-17A (mIL-17A) (TC11-18H10.1) MAbs were purchased from BD Biosciences (San Diego, CA). PE-conjugated anti-Gr1 (RB6-8C5) MAb was purchased from Caltag Laboratories (Burlingame, CA). FITC-conjugated anti-TCRβ (H57-597), APC-conjugated F4/80 (BM8), biotin-conjugated anti-major histocompatibility complex (MHC) class II (M5/114.15.2), anti-F4/80 (BM8), anti-TCRβ (H57-597), and anti-B220 (RA3-6B2) MAbs were purchased from eBioscience (San Diego, CA). Stained cells were run on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining.

Lung cells were stimulated with or without 10 ng/ml of recombinant IL-23 (rIL-23) (R&D systems) for 6 h at 37°C. Brefeldin A (BFA) (10 μg/ml) was added for the last 3 h of incubation. After incubation, cells were stained with various MAbs for 30 min at 4°C. Intracellular staining was performed according to the manufacturer's instructions (BD Biosciences). Briefly, 100 μl of BD Cytofix/Cytoperm solution (BD Biosciences) was added to the cell suspension with mild mixing and then placed at 4°C for 20 min. The fixed cells were washed twice with 250 μl of BD Perm/Wash solution (BD Biosciences) and stained intracellularly with PE-conjugated anti-mIL-17A MAb for 30 min at 4°C.

In vivo depletion of neutrophils.

One hundred micrograms of anti-Ly6G MAb (1A8) or the isotype-matched MAb was administered intraperitoneally (i.p.) 1 day before infection.

In vitro mixed-cell culture.

Cells from the lungs of TLR2 KO mice (CD45.2) were mixed in a 1:1 ratio with lung cells from wild-type (WT) mice (CD45.1). The mixed cells were stimulated with 100 ng/ml of the TLR2 ligand Pam3CSK4 for 24 h at 37°C, and 10 μg/ml of BFA was added for last 3 h of incubation. IL-17A production by γδ T cells was analyzed by the intracellular-staining method described above.

Statistics.

Statistical significance was calculated by Student's t test using Prism software (GraphPad, San Diego, CA). Differences with P values of <0.05 were considered to be statistically significant.

RESULTS

Neutrophils are required for host defense against candidiasis.

To examine the in vivo significance of the early infiltration of neutrophils in host defense against systemic infection with C. albicans, we pretreated mice with neutrophil-specific anti-Ly6G MAb (10) and then infected them intravenously with either a virulent or an avirulent strain of C. albicans. Infiltration of neutrophils into the peripheral tissues of mice pretreated with anti-Ly6G MAb was decreased by 24 h in a mouse systemic-infection model of C. albicans (see Fig. S1 in the supplemental material). Neutrophil-depleted mice died within 2 days after infection irrespective of the virulence of C. albicans. In contrast, all mice treated with an isotype-matched MAb survived beyond 5 days (Fig. 1 A). To examine the protective role of early-infiltrated neutrophils, fungal clearances in the periphery were analyzed 24 h after infection. We found that fungal clearance of both a virulent and an avirulent strain of C. albicans in the lung, but not in the kidney, spleen, and liver, was significantly impaired 24 h after infection (Fig. 1B). Histological analysis showed that the mycelial form of C. albicans was observed with equal frequency in the kidneys of both neutrophil-depleted and control mice (Fig. 1C), whereas fungal growth of the yeast form of C. albicans was observed in the lungs of neutrophil-depleted mice but was less frequently detected in control mice (Fig. 1C). These results suggest the importance of neutrophils for host defense in the lung at an early stage after systemic C. albicans infection.

Fig. 1.
Susceptibility of mice depleted of neutrophils to systemic C. albicans infection. Mice were pretreated with 100 μg of anti-Ly6G MAb (1A8) or isotype-matched MAb (isotype matched) 1 day before i.v. infection with 1 × 107 CFU of strain 10261 ...

Rapid and transient IL-23 production preceded IL-17A production and infiltration of neutrophils in the lung after C. albicans infection.

Since IL-17A has been shown to be involved in the mobilization of neutrophils (19), we measured IL-17A production in the lung after infection with C. albicans. Both a virulent and an avirulent strain of C. albicans in the lung were grown after systemic infection, and thereafter, both strains were cleared completely within a week (Fig. 2 A and data not shown). In response to rapid fungal growth in the lung (Fig. 2A), infiltration of neutrophils and IL-17A production were detected as early as 12 h after infection with both a virulent and an avirulent strain and rapidly ceased (Fig. 2B and E). We also examined the kinetics of IL-23 and IL-1β, which have been shown to promote IL-17A production by Th17, γδ T, NKT, and intestinal innate lymphoid cells (2, 6, 22, 32, 46). Increased production of IL-23 and IL-1β preceding IL-17A production and the influx of neutrophils was observed in the lung after infection with both a virulent and an avirulent strain of C. albicans (Fig. 2B to E). These results suggest that IL-17A is involved in neutrophil-mediated host defense in the lung at an early stage after systemic infection with C. albicans.

Fig. 2.
Kinetics of fungal clearance, neutrophil infiltration, and cytokine production in the lung after systemic infection with C. albicans. After mice were injected intravenously with 1 × 107 CFU of strain 10261 or 2 × 105 CFU of SC5314, fungal ...

IL-17A produced by γδ T cells plays important roles in neutrophil infiltration and fungal clearance at an early stage of candidiasis.

To directly examine the in vivo significance of early IL-17A production for the infiltration of neutrophils and host defense against C. albicans, IL-17A KO mice were inoculated with C. albicans. Although fungal clearance in the lung was significantly impaired In IL-17A KO mice, a reduced but appreciable level of neutrophil infiltration was detected 24 h after infection (Fig. 3 A). It was previously shown that IL-17R-mediated signaling is also involved in cytotoxic activation of neutrophils by enhancing their MPO activity after systemic infection with C. albicans. In agreement with this, MPO activity in the lungs of IL-17A KO mice was significantly reduced 24 h after infection (Fig. 3B). In order to identify the cell subsets responsible for early IL-17A production in the lung after C. albicans infection, lung cells were harvested 24 h after infection, cultured with BFA for 4 h in vitro, and examined by intracellular staining for IL-17A. Most of the IL-17A-producing cells were found in γδ TCR-positive (γδ TCR+) cells, but not in CD4+ cells (Fig. 3C). We next examined the involvement of γδ T cells in IL-17A production and neutrophil infiltration after C. albicans infection using mice genetically lacking γδ T cells. IL-17A production was significantly reduced in Cδ KO mice compared with WT mice at 24 h after intravenous infection with C. albicans (Fig. 3D). Neutrophil infiltration and fungal clearance were impaired in Cδ KO mice 24 h after infection (Fig. 3D), suggesting that γδ TCR+ cells predominantly participate in early IL-17A production, which induces infiltration of neutrophils to clear C. albicans after infection.

Fig. 3.
Protective role of IL-17A-producing γδ T cells after systemic infection with C. albicans. (A) IL-17A KO mice showed impaired neutrophil infiltration and fungal clearance in the lung after infection. WT or IL-17A KO mice were i.v. infected ...

Tyk2-mediated signaling is critical for IL-23-induced IL-17A production by γδ T cells.

Tyk2, a member of the JAK signal transducer family, is involved in intracellular signaling triggered by IL-23 (28, 41, 44). To determine whether IL-23 was involved in IL-17A production by γδ T cells after C. albicans infection, we inoculated C. albicans i.v. into Tyk2 KO mice. IL-17A production in the lung was severely impaired in Tyk2 KO mice, although IL-23 production in Tyk2 KO mice was comparable to that in WT mice (Fig. 4 A). These results demonstrated that IL-17A production by γδ T cells is dependent on the IL-23-Tyk2 pathway after C. albicans infection.

Fig. 4.
IL-17A production by γδ T cells is induced by IL-23 in a TLR2/MyD88-dependent but CARD9-independent manner. (A) Tyk2 signaling was indispensable for IL-17A production by γδ T cells. After i.v. challenge with 1 × ...

The rapid IL-17A production after C. albicans infection is TLR2/MyD88 dependent but CARD9 independent.

TLR2/MyD88 signaling and Dectin-2/CARD9 signaling are important in the production of proinflammatory cytokines in a murine model of candidiasis and human candidiasis (12, 36, 39, 50, 51). We next examined which type of signaling is involved in IL-23 production at an early stage after systemic infection with C. albicans. IL-23 production was detected in the lungs of WT mice at 12 h after infection with C. albicans, but such production was completely abolished in MyD88 KO, as well as TLR2 KO, mice, but not in CARD9 KO mice (Fig. 4B). Consistent with IL-23 production, IL-17A production was detected in the lungs of WT and CARD9 KO mice, but not in MyD88 KO and TLR2 KO mice, at 24 h after systemic infection with C. albicans (Fig. 4B). The infiltration of neutrophils and fungal clearance were strikingly diminished in TLR2 KO mice (Fig. 4C). Thus, TLR2/MyD88-mediated signaling is indispensable for early IL-23 and IL-17A production, which was important for neutrophil-mediated fungal clearance in the lung at 24 h after systemic C. albicans infection.

Exogenous IL-23, induced through TLR2, is involved in IL-17A production by γδ T cells.

A significant fraction of γδ T cells are reported to express TLR2 involved in IL-17A production (23). To further determine whether TLR2 expression on γδ T cells is required for IL-17A production, we set up the following experiment. Lung cells from TLR2 KO mice were stimulated in vitro with a TLR2 ligand, Pam3CSK4, in the presence of lung cells from WT mice, and then IL-17A production by TLR2-deficient γδ T cells was analyzed by flow cytometry. The percentage of IL-17A-producing γδ T cells from TLR2 KO mice increased significantly in the presence of TLR2-sufficient lung cells (Fig. 5 A and B). Therefore, it was revealed that TLR2-mediated signaling in γδ T cells was not required for C. albicans-induced IL-17A production. The above results suggest that IL-17A production by γδ T cells requires IL-23 provided by C. albicans-stimulated lung cells. Therefore, we next examined the ability of exogenous IL-23 to stimulate naive γδ T cells and found that in vitro culture with IL-23 induced the production of IL-17A by resident γδ T cells (Fig. 5C).

Fig. 5.
Exogenous IL-23 induces IL-17A production by γδ T cells. (A and B) TLR2 expression on γδ T cells was not required for IL-17A production. TLR2-deficient lung cells, mixed with (+WT) or without (−WT) TLR2-suffcient ...

DISCUSSION

Local infiltration of neutrophils is one of the earliest events induced by microbial infection and is critical for host defense against various pathogens, including C. albicans. In the present study, we found the involvement of γδ T cells in the infiltration of neutrophils in the lung at an early stage following systemic infection with C. albicans through IL-17A production. IL-23, which was produced in the lungs as early as 12 h after systemic infection with C. albicans in a TLR2/MyD88-dependent but not in a CARD9-dependent manner, stimulated preexisting γδ T cells in the lungs to produce IL-17A. Thus, naturally occurring IL-17A-producing γδ T cells play an important role in the first line of host defense by controlling neutrophil infiltration at an early stage after systemic infection with C. albicans.

We found in this study that neutrophil-depleted mice showed impaired fungal clearance in the lung 24 h after infection. We also found that CARD9 KO mice could survive beyond 3 days, suggesting that CARD9 was dispensable in neutrophil-mediated host defense at an early stage (data not shown). In contrast, CARD9, which was activated through Dectin-2, was important for host defense by inducing Th17 cell differentiation after systemic C. albicans infection (39). Indeed, Dectin-2 KO mice began to die around 12 days after C. albicans infection (39). These results indicate different mechanisms to protect against C. albicans at early and late stages after infection. Interestingly, we found that the yeast form of C. albicans was observed in the lung 1 day after systemic infection whereas another group and our present study showed that the mycelial form of C. albicans was observed in the kidney (39). Interestingly, it was shown that dendritic cells internalize the yeast and mycelial forms of C. albicans with different PRRs and subsequently induce different helper T cell subsets in vivo (11, 38). As such, it was speculated that the PRRs involved in the recognition are different for the yeast and mycelial forms of C. albicans, and consequently, the mechanisms responsible for controlling fungal growth at the early stage differ from those at the late stage after systemic infection with C. albicans. However, to prove this hypothesis, further studies to elucidate the interaction between C. albicans and immune cells should be performed.

In this study, we demonstrated that TLR2 was indispensable for IL-17A production by γδ T cells at an early stage after systemic infection with C. albicans. Although γδ T cells expressed TLR2 involved in IL-17A production and proliferation (23, 33), we could not observe such evidence resulting from direct recognition by γδ T cells. IL-17A production by γδ T cells was induced in response to IL-23, but TLR2 expression on γδ T cells was dispensable for the effector function. Consistent with this, IL-17A production was not induced by purified γδ T cells after stimulation with the TLR2 ligand Pam3CSK4 alone (33, 34). We also found that the number of γδ T cells in the lung did not increase within 24 h after systemic infection with C. albicans (data not shown), suggesting that TLR2 induced IL-17A production but not proliferation of γδ T cells in the lung at this stage. Treg cells express TLR2, which is involved in maintaining suppressive function and survival (45). TLR2-dependent IL-10 production by C. albicans-infected splenocytes was induced after stimulation with heat-killed C. albicans (31). In fact, we found that IL-10 production in the lung was observed 24 h after systemic infection with C. albicans (data not shown). Although the involvement of Treg cells in host defense in the lung after systemic C. albicans infection was unclear, IL-17A production by γδ T cells was induced in the presence of IL-10 after infection. Taken together, the data show that TLR2, but not other PRRs, is important for protection in the lung at an early stage after systemic C. albicans infection by regulating IL-17A production by γδ T cells. Our data also suggest the involvement of factors other than IL-17A for neutrophil-mediated host defense against C. albicans, because IL-17-independent neutrophil infiltration was observed in the lung 24 h after infection. It was previously shown that neutrophil infiltration after systemic infection with C. albicans was partly induced via Dectin-1-mediated signaling, which augments the production of IL-6 and the chemokines MCP-1 and MIP-1α, granulocyte colony-stimulating factor (G-CSF), and granulocyte-monocyte colony-stimulating factor (GM-CSF) (48). Among these chemoattractants, increased expression of G-CSF in the lung was observed independently of IL-17A in a mouse intratracheal infection model with Mycobacterium bovis BCG (49). Indeed, G-CSF-deficient mice had reduced numbers of neutrophils in the blood 7 days after systemic infection with C. albicans (3). Moreover, candidacidal activity was slightly impaired in G-CSF-deficient neutrophils. These results suggest that G-CSF may act synergistically with IL-17A for infiltration of neutrophils and fungal clearance in the lung after systemic infection with C. albicans.

IL-23 is an inflammatory cytokine that induces IL-17A production, not only by γδ T cells, but also by Th17, NKT, and intestinal innate lymphoid cells (2, 6, 22, 32). Consistent with previous findings, IL-17A production by γδ T cells was induced in response to IL-23 at an early stage after C. albicans infection. Intriguingly, other T cell subsets did not produce IL-17A in the lung, indicating that resident IL-17A-producing γδ T cells are a unique subset able to respond to IL-23 immediately after systemic infection with C. albicans. Supporting the rapid response, it was shown that more than 50% of γδ T cells in mucosal tissues, such as peritoneal exudate cells and lamina propria lymphocytes, constitutively expressed IL-23R in naive mice (35). Similar to γδ T cells, Th17 cells express IL-23R, which induces IL-17A production and cell proliferation (2, 24). In addition to its protective roles, IL-23 potentially induces autoimmune disorders, such as experimental allergic encephalomyelitis, inflammatory bowel disease (IBD), and rheumatoid arthritis (RA), in which Th17 cells, as well as IL-17A-producing γδ T cells, are involved (8, 16, 25). In this regard, since responsiveness to IL-23 should be tightly regulated, it is interesting to elucidate the mechanism for maintaining IL-23R expression on IL-17A-producing γδ T cells involved in host defense against systemic C. albicans infection.

In conclusion, we demonstrated the involvement of IL-17A-producing γδ T cells at an early stage of host defense in the lung against fungal infection. Although Th17 cells are functionally differentiated in the periphery after systemic infection with C. albicans (39), IL-17A-producing γδ T cells, which arise within the thymus, are abundantly present in the peripheral tissues of naive mice independent of pathogens (43). This unique developmental pathway and tissue localization of naturally occurring IL-17A-producing γδ T cells may be important for protection in a first line of host defense against pathogens, including C. albicans.

Supplementary Material

Supplemental Material:

ACKNOWLEDGMENTS

We thank Kiyomi Akasaki, Akiko Yano, Mihoko Ookubo, and Miki Kijima for secretarial assistance and laboratory members for their helpful discussions. We thank Hiroji Chibana for providing a virulent strain of C. albicans, SC5314.

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (K.S. and Y.Y.), by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (K.S.), and by the program of Founding Research Centers for Emerging and Reemerging Infectious Diseases launched as a project commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Y.Y.).

We have no financial conflict of interest.

Footnotes

Supplemental material for this article can be found at http://iai.asm.org.

[down-pointing small open triangle]Published ahead of print on 29 August 2011.

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