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Published ahead of print on December 18, 2002, doi:10.1164/rccm.200206-589OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 991-998, (2003)
© 2003 American Thoracic Society

Original Article

TA-19, a Novel Protein Antigen of Trichosporon asahii, in Summer-type Hypersensitivity Pneumonitis

Yasujiro Matsunaga, Yutaka Usui and Yasuyuki Yoshizawa

Department of Pulmonary Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku; and Department of Internal Medicine, Tokyo Metropolitan Bokutoh Hospital, Kotohbashi, Sumida-ku, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Yutaka Usui, Department of Pulmonary Medicine, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. E-mail: usuibokutoh{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most common form of hypersensitivity pneumonitis in Japan is summer-type hypersensitivity pneumonitis (SHP), which is caused by the inhalation of Trichosporon asahii or Trichosporon mucoides. To seek protein antigens relevant to the immunopathogenesis of SHP, we constructed a cDNA expression library of T. asahii, a major causative yeast species of SHP. Using the immunoscreening method, we identified and cloned a novel gene encoding a 19-kD protein, named TA-19, which proved to be specifically recognized in the bronchoalveolar lavage (BAL) fluids and sera of patients with SHP. IgG, IgA, and IgM antibodies to the recombinant TA-19 protein were significantly elevated in the sera as well as in the BAL fluids from SHP patients compared with those from non-SHP groups. This protein also induced SHP-specific proliferation of the mononuclear cells from both the peripheral blood and BAL. These results reveal that TA-19 derived from T. asahii may play a relevant role in specific cellular and humoral immune responses in patients with SHP.

Key Words: cDNA expression library • antigen-specific antibody • lymphocyte proliferation assay • bronchoalveolar lavage • G-X-X-X-Q-X-W motif

Hypersensitivity pneumonitis (HP) is an immunologically mediated lung disease that is caused by sensitization to and repeated inhalation of an inhaled antigen (1, 2). The most common HP in Japan is summer-type HP (SHP), which is caused by inhalation of Trichosporon asahii or Trichosporon mucoides (3–8). The clinical and laboratory features of HP have been well defined; however, the precise immune mechanisms involved in the development of the disease have not been fully documented. The immunologic mechanisms involved in the pathogenesis of HP are probably the result of an initial immune complex–mediated lung injury followed by cell-mediated hypersensitivity-induced tissue damage (9).

Antigenic components have been identified for several forms of HP, including mannnose and mannoproteins of T. asahii and T. mucoides in the case of SHP and the carbohydrate moieties of the glycoproteins from Saccharopolyspora rectivirgula in the case of farmer's lung disease (4, 10–15). Although the involvement of protein antigens is suspected, the nature and extent of this involvement remain unknown. Processed extrinsic peptides are presented by antigen-presenting cells in context with major histocompatibility complex (MHC) class II molecules and are recognized by CD4+ T cells, following disease-specific immune reaction (16). Recent studies have revealed that CD4+ T cells play a crucial role in the induction of experimental HP. Schuyler and colleagues established a murine model of experimental HP by an adoptive transfer of S. rectivirgula–sensitized spleen cells (17, 18). In this system, depletion of the CD4+ T cells ablated the ability of recipient animals to express adoptive HP. They also revealed that when CD4+ T cells were polarized into Th1 cells, they were responsible for the successful adoptive transfer in the same murine model system (19). In addition, a few reports have revealed that MHC class II haplotypes might affect the development of pigeon breeder's disease (20). Camarena and colleagues recently proposed that genetic factors located within the MHC class II region contributed to the development of pigeon breeder's disease (21). In case of the SHP, Ando and colleagues reported a significantly higher incidence of human leukocyte antigen (HLA)-DQw3 in SHP patients from Japan (22).

This study was undertaken to identify a protein antigen(s) that could evoke both humoral and cell-mediated hypersensitivity in SHP. We prepared a cDNA expression library of T. asahii, the most common yeast in the genus Trichosporon in the environment (23), and performed immunoscreening with bronchoalveolar lavage (BAL) fluids and sera of SHP patients. We cloned the full-length cDNA of one new gene. The corresponding amino acid sequence revealed a protein with a predicted molecular weight (Mr) of 19 kD and presented tandem repeats of a G-X-X-X-Q-X-W motif. This protein, named TA-19, represents a new member of the Ricin superfamily (24). This is the first study to clone a full-length cDNA of a protein antigen potentially involved in pathology of HP.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
SHP was diagnosed as defined by Shimazu and colleagues (4). We obtained informed consent from all subjects and approval of this study from institutional review boards for human study. BAL fluids or sera were collected from 21 patients with SHP (13 men and 8 women with a mean age of 48 years; range, 23–68 years), 12 patients with idiopathic pulmonary fibrosis (IPF; 9 men and 3 women with a mean age of 55 years; range, 19–74 years), 9 patients with interstitial pneumonia with collagen vascular disease (6 men and 3 women with a mean age of 65 years; range, 44–76 years), 6 patients with eosinophilic pneumonia (2 men and 4 women with a mean age of 40 years; range, 21–67 years), 3 patients with pulmonary tuberculosis (2 men and 1 woman with a mean age of 57 years; range, 52–66 years), 7 patients with pulmonary sarcoidosis (Sar; 4 men and 3 women with a mean age of 46 years; range, 26–64 years), and 22 normal volunteers (NVs; 13 men and 9 women with a mean age 28 years; range, 22–47 years) and were stored at -30°C until use.

Fresh BAL cells were obtained from 5 patients with SHP (2 men and 3 women with a mean age of 38 years; range, 24–57 years) and 14 patients with Sar (8 men and 6 women with a mean age of 52 years; range, 44–74 years), and fresh peripheral blood was obtained from 8 patients with SHP (6 men and 2 women with a mean age of 57 years; range, 49–62 years), 13 patients with pulmonary Sar (7 men and 6 women with a mean age of 53 years; range, 44–74 years), 5 patients with IPF (4 men and 1 woman with a mean age of 62 years; range, 54–74 years), and 7 NVs (6 men and 1 woman with a mean age of 35 years; range, 25–52 years). None of the patients had previously taken immunosuppressive drugs such as glucocorticoids.

BAL Fluids and Sera for Library Screening
Pooled BAL fluids and sera from 4 patients (a 59-year-old man, a 60-year-old man, a 64-year-old man, a 68-year-old man) with SHP from our first group of 21 patients were used for screening of the cDNA expression library (discussed later here). The patients had been confirmed to show significantly high titers of T. asahii–specific IgG and IgA antibodies in the BAL fluids, as well as in the sera measured by the enzyme-linked immunosorbent assay described by Yoshizawa and colleagues (25).

Yeast Strains
Dr. Uchida from the Teikyo University Institute of Medical Mycology kindly provided the following yeast strains for use in this study: T. mucoides, TIMM 1573 strain; T. asahii, TIMM 1318 strain; Candida albicans, TIMM 3169 strain; Candida guilliermondii, TIMM 0260 strain; Candida parapsilosis, TIMM 3172 strain; and Cryptococcus neoformans (Crn), TIMM 317 strain. They were grown in Sabouraud liquid medium at 28°C, harvested by centrifugation, washed three times with saline, and stored at -80°C until use.

Preparation of T. asahii cDNA Expression Library
Total RNA was extracted by the method described by Schmitt and colleagues (26), and a recombinant cDNA expression library was prepared with a vector kit ({lambda}ZIPLOX; GIBCO BRL, Gaithersburg, MD) and Gigapack III Gold packaging extracts (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

Screening of Expression Library
The T. asahii cDNA expression library was screened as described elsewhere (27), with the pooled BAL fluids and sera of patients with SHP. In brief, some 60,000 plaques were plated at 3,000 plaque forming units (PFU) per plate, and two replicas were prepared on nitrocellulose membranes; one was treated with pooled patient sera at a dilution of 1:200, and the other was treated with corresponding undiluted BAL fluids. The binding of IgG antibodies specific to the proteins expressed in the library was detected with biotin-labeled goat anti-human IgG (Tago, Burlingame, CA), followed by peroxidase-conjugated streptavidin (Dako, Carpinteria, CA). In the screening steps, we isolated only those plaques that were reacted strongly with both the sera and BAL fluids from the SHP patients but remained undetectable with the sera and BAL fluids from the NV and patients with pulmonary disease other than SHP.

Cloning of TA-19 cDNA
Nucleotide sequences in both strands of the insert cDNA that encoded the SHP-specific recombinant protein were determined by using a dRhodamine Terminator Cycle Sequencing kit and an ABI 377 DNA sequencer equipped with ABI Prism Model for data recording and analysis (Perkin-Elmer Applied Biosystems, Forester City, CA). A SMART Rapid Amplification of cDNA Ends kit (Clontech Laboratories Inc., Palo Alto, CA) was used to elongate the 5'-cDNA end. Two successive and specific oligonucleotide primers for TA-19 were used: 5'-CCACTCCTGGTTCTTGTTGCCGGTGC-3'and 5'-AGACCTGGAGGCCGTTGCCGTTGG-3'. Both nucleotide and deduced amino acid sequences were analyzed with known sequences using basic local alignment search tool (BLAST) search (28) and ExPASy proteomics tools (ExPASy, Geneva, Switzerland).

Amplification and Dot-blot Hybridization of Genomic DNA
Genomic DNA was extracted from each yeast strain and applied to polymerase chain reaction amplification with two sets of TA-19–specific primers (F1: 5'-AAGCTACCACGATGCTTTCC-3' and R1: 5'-GTAGCACTGCCAGAGGTGG-3'; F2: 5'-ACCTCTGGCAGTGCTACCC-3' and R2: 5'-ATCTGTTCATTCATAAGCC-3'). Genomic DNA was serially diluted and spotted onto a nylon membrane. TA-19 genomic DNA fragment was amplified with F1 and R1, labeled with digoxigenin-11-dUTP (DIG DNA Labeling and Detection Kit; Boehringer-Mannheim, Indianapolis, IN), and used as a probe. The signals were detected according to the manufacturer's instructions.

Northern Blot Analysis
Northern blotting was performed as described elsewhere (27). Five micrograms of total RNA from T. asahii was reverse transcribed to synthesize cDNA with oligo(dT) primer (GIBCO) and Moloney murine leukemia virus reverse transcriptase (SUPERSCRIPT RT; GIBCO). TA-19 cDNA fragment was amplified with F1 and R1 oligonucleotides, labeled with digoxigenin-11-dUTP, and used as a probe. Twenty micrograms of total RNA extracted from each yeast strain was subjected to electrophoresis. The signals were detected according to the manufacturer's instructions.

Preparation of Recombinant Protein
The TA-19 cDNA was subcloned into the pGEX 6p-2 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) and used to transform BL21 Escherichia coli (Stratagene). Glutathione- S-transferase (GST) and a recombinant protein fused with GST (hereinafter referred to as the "fusion protein") were prepared from BL21 according to the manufacturer's instructions. The recombinant proteins were purified with GST-conjugated sepharose beads (Amersham Pharmacia Biotech) as instructed by the manufacturer.

Western Blot
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the methods of Laemmli (29). Proteins were transferred electrically to a polyvinylidene difluoride membrane with a Multiphor II electrophoresis system (Amersham Pharmacia Biotech). The binding of specific antibodies to the proteins was detected with biotin-conjugated goat anti-human IgG as the second antibody, followed by peroxidase-conjugated streptavidin (Dako).

Inhibition Assay for Cross-reactivity
T. asahii, T. mucoides, and C. neoformans were separately grown in Sabouraud liquid medium. The pelleted yeast was resuspended in saline and sonicated at 4°C. After centrifugation, the soluble fraction was recovered, filtrated, and frozen at -30°C until use. The patient's serum was diluted to a concentration of 1:200, mixed with yeast lysate at concentrations ranging from 10 µg to 1 mg per 10 ml, and applied to the Western immunoblot of the fusion protein.

Antibody Assay
TA-19–specific IgG, IgA, and IgM antibody activities in the BAL fluids and sera were measured by the enzyme-linked immunosorbent assay. Flat-bottomed microplates (Nunc Immunoplate; Nalge Nunc International, Rochester, NY) were coated with 250 ng of fusion protein or 150 ng of GST alone diluted in carbonate-buffer (pH 9.6) overnight at 4°C. The plates were washed with Tris-buffered saline containing 0.05% Tween 20 and blocked with 1% bovine serum albumin for 60 minutes. After washing in Tris-buffered saline containing 0.05% Tween 20, 100 µL of BAL fluid diluted to a concentration of 1:2 or serum diluted to a concentration of 1:200 was added to each well and incubated for 2 hours at room temperature. Before adding the diluted serum to each well, it was preincubated with 10 µg of native BL21 lysate for 2 hours at room temperature just before applying to each well to eliminate antibody binding to the BL21 lysate mingled with the purified recombinant proteins. After washing with Tris-buffered saline containing 0.05% Tween 20, the plates were incubated for 30 minutes with alkaline phosphatase-conjugated goat anti-human IgG, IgA, or IgM (all from Tago and all labeled with biotin) at room temperature. After the plates were washed again, they were developed with o-phenylendiamine (Wako Pharmapheutical, Tokyo, Japan) at room temperature for 15 minutes. The reaction was stopped by 25 µL of 2N HCl. The amount of antibody in each sample was expressed as the optical density at 490 nm in a well coated with fusion protein after subtracting the background absorbance value of a well coated with GST only.

Lymphoproliferation Assay
Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Paque (Pharmacia) centrifugation of fresh peripheral blood with heparin, washed three times with cold phosphate-buffered saline, and resuspended in RPMI 1640 medium (GIBCO) containing 10% fetal calf serum, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid) buffer, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate at 2 x 105 per well. BAL cells were washed three times with cold phosphate-buffered saline, resuspended in RPMI 1640 medium at 2 x 105 per well, and cultured in 96-well, round-bottomed microtiter plates (Sumitomo Bakelite Co., Tokyo, Japan) for 6 days at 37°C in humidified air with 5% CO2. Triplicate cultures were done for each condition, that is, with medium only, GST (15 µg/ml) only, or 25-µg/ml fusion protein. Six days later, culture cells were pulsed with 1 µCi of 3H-thymidine (DuPont, NEN Research Products, Inc., Boston, MA) for 16 hours and harvested. The uptake of 3H-thymidine was determined with a scintillation counter. The stimulation index was calculated as the mean counts per minute of stimulated cultures divided by the mean counts per minute of unstimulated cultures.

Statistical Analysis
The Mann-Whitney U test was used to compare the levels of antibodies and the lymphoproliferative responses in different groups of subjects; p values of < 0.05 were considered significant in the analysis.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Molecular Characterization of TA-19
The screening of 240,000 plaques from the T. asahii cDNA expression library identified two plaques with insert cDNA of approximately 0.8 kbp. Both plaques reacted with all the BAL fluids and sera from the four SHP patients studied, but neither reacted with BAL fluids or sera from the patients with eosinophilic pneumonia, Sar, tuberculosis, IPF, or interstitial pneumonia with collagen vascular disease or from the NV group. DNA sequencing of the inserts showed that the two clones overlapped and were fragments of an identical cDNA. 5'-Rapid amplification of cDNA ends produced a substantially longer and complete cDNA of 864 bp containing a single open reading frame encoding a protein of 182 amino acids. The predicted Mr was 19 kD (Figure 1) . Northern blot analysis performed to confirm the size of the full-length cDNA demonstrated an approximately 0.9-kb band (Figure 2) . Sequencing of the amplified products of T. asahii genomic DNA with the two sets of specific oligonucleotides F1 and R1 and F2 and R2 revealed the presence of two introns. The nucleotide sequence of the genomic DNA was submitted to DNA data bank of Japan (accession number AB038374).



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  		Figure 1. Nucleotide sequence of the TA-19 cDNA and the deduced amino acid sequence. The CT-rich sequence, implicated as functional components of gene promotor, is underlined. Two intron insertion sites are indicated by downward arrows. The putative signal sequence is underlined with a broken line. Potential sites for O-glycosylation sites are expressed in bold, and potential sites for N-glycosylation are expressed in bold italics. Tandem repeats of G-X-X-X-Q-X-W are expressed in bold and underlined. *TAA stop codon. Nucleotides and amino acid sequences were submitted to DNA data bank of Japan under accession number AB038374.

 


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  		Figure 2. Northern blot analysis of TA-19 mRNA expression in yeasts. (A) Lane 1, RNA molecular marker; lane 2, T. mucoides; lane 3, T. asahii; lane 4, C. albicans; lane 5, C. guilliermondii; lane 6, C. parapsilosis; lane 7, C. neoformans. (B) 28S and 18S rRNA stained with ethidium bromide.

 
The deduced amino acid sequence analysis revealed that the N-terminal amino acids composed a block of hydrophobic residues typical of signal sequences with potential sites for the signal peptidase after residue 17 or 19 (30), followed by a serine/threonine-rich region. Putative O-glycosylation sites (31) and N-glycosylation sites (32) were found. Notably, this new protein contained multiple short conserved motifs, "Gly (G)-X-X-X-Gln (Q)-X-Trp/Phe/Tyr (W/F/Y)," characteristic of a member of the "Ricin superfamily" proposed by Hirabayashi and colleagues (24). We named this new protein TA-19. Figures 3 and 4 showed comparisons of amino acid sequences and domain structures of proteins with the same consensus motif.



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  		Figure 3. Multiple amino acid sequence alignments of TA-19 and the other members with the G-X-X-X-Q-X-W motif. Conserved amino acids between sequences are shaded. The amino acid matches are indicated by asterisks, conservative substitution by double dots, and less conservative substitution by a single dot. Dashes represent gaps where the corresponding amino acid residues in the other sequences are absent.

 


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  		Figure 4. Domain structures of putative superfamily of proteins with the G-X-X-X-Q-X-W motif. The members are classified into a lectin group and a hydrolytic enzyme group. Two consecutive consensus motifs are expressed by a shaded square box in each unit segment. Most members have signal sequences in the N-termini.

 
Genomic polymerase chain reaction with two sets of TA-19–specific oligonucleotides gave a single band only for T. asahii, and genomic DNA dot blot hybridization revealed positive signals for only T. asahii (data not shown).

Expression, Antigenicity, and Purification of Recombinant Fusion Protein
Predicting that the block of N-terminal amino acids are signal peptides, the TA-19 cDNA encoding Glu (E)-20 to Asn (N)-182 was cloned into pGEX 6p-2 plasmid and expressed in a bacterial system. The immunoblots in Figures 5A and 5B were performed with the serum from a pool of patients with SHP. Unpurified bacterial lysate demonstrated recombinant protein at a molecular weight of 43 kD, a value that corresponded to the predicted molecular mass encoded by the clone (Figure 5A, lane 1). Preincubation of the 10 ml of 1:200 diluted serum with 10 mg of native BL21 lysate (lane 2) demonstrated a single 43-kD band equal in size to the purified fusion protein (lane 3). The 29-kD GST protein did not react with the patient's serum (lane 4). We used this purified fusion protein for further examinations. The patient's serum was preincubated with a soluble fraction of each lysate of T. asahii, T. mucoides, and C. neoformans and then applied to the Western immunoblot. As shown in Figure 5B, preincubation with 1 mg of T. asahii lysate per 10 ml of 1:200 diluted serum, but not with T. mucoides or C. neoformans lysate, abolished the antibody binding.



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  		Figure 5. (A) Western immunoblot of the recombinant proteins with a pooled sera of patients with SHP. Lane 1, unpurified fusion protein reacted with a serum of patients with SHP; lane 2, unpurified fusion protein reacted with the patient's serum preincubated with an excessive lysate of native BL21; lane 3, purified fusion protein reacted with the untreated patient's serum; lane 4, purified GST protein reacted with the untreated patient's serum. (B) Western immunoblot of purified fusion protein with a pooled sera of patients with SHP. The serum was preincubated with an excessive lysate of T. mucoides (lane 1), T. asahii (lane 2), or C. neoformans (lane 3).

 
Levels of Antibodies to the Recombinant Protein
Figure 6 shows the levels of TA-19–specific IgG, IgA, and IgM antibodies in BAL fluids from patients with SHP, healthy volunteers, and patients with three other respiratory diseases (IPF, interstitial pneumonia with collagen vascular disease, and eosinophilic pneumonia). Levels of three classes of antibodies in the BAL fluid were significantly higher in the SHP patients. In fact, the absorbance values were negligible in all of the subjects except the SHP patients. Serum levels of IgG, IgA, and IgM antibodies were also significantly higher in the SHP patients than in all of the other groups (Figure 7) , although a few non-SHP subjects showed elevated IgG or IgA titers up to 1.06.



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  		Figure 6. Bronchoalveolar lavage (BAL) fluid levels of IgG, IgA, and IgM antibodies to the TA-19/GST fusion protein. BAL fluids from patients with SHP, NVs, patients with IPF, interstitial pneumonia with collagen vascular disease (IP-CVD), and eosinophilic pneumonia (EP) were examined. Horizontal bars indicate the mean values.

 


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  		Figure 7. Serum levels of IgG, IgA, and IgM antibodies to the fusion protein. Horizontal bars indicate the mean values.

 
Cell-mediated Hypersensitivity Response to the Recombinant Protein
In separate experiments, we collected fresh BAL cells from patients with Sar as a disease control group. We did so for several reasons. First, Sar is a relatively common disease in Japan. Second, patients with active pulmonary Sar show bronchoalveolar lymphocytosis. Finally, the mechanism of T cell accumulation into the lesions is thought to be antigen driven (38). BAL cell differentials revealed that the mean value of lymphocyte was 69.6% in patients with SHP and 50.0% in patients with pulmonary Sar, showing bronchoalveolar lymphocytosis. Antigen-specific proliferative responses of BAL cells to TA-19 were significantly higher in the patients with SHP than in the patients with Sar (Figure 8) . TA-19 induced marked proliferative responses of BAL cells in all five patients with SHP, and the stimulation index exceeded 2.57. In 14 patients with Sar, the stimulation index was always less than 1.48. TA-19 also induced significant proliferative responses of PBMCs in patients with SHP compared with the Sar, IPF, and NV groups. TA-19–induced proliferative responses of PBMCs were negligible in non-SHP individuals. GST induced a proliferative response in only one sample, a collection of BAL cells from a patient with SHP. The stimulation index in this sample was 2.63 for GST versus 8.81 for the fusion protein.



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  		Figure 8. Proliferative response by BAL cells and PBMCs to the fusion protein and GST only. Horizontal bars indicate the mean values.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mannose and mannoproteins of T. asahii or T. mucoides have been identified as antigenic components of SHP. Antigenic components from culture filtrate antigen of T. asahii purified by Yamasaki and coworkers (11) consisted mainly of polysaccharide. Mizobe and associates (12, 13) further characterized the responsible antigenic component as glucronoxymannan. The antigenic epitope was shown to involve the terminal glucuronic acid residues, and a monoclonal antibody named D-8 was produced. The presence of high titers of antigen-specific antibodies is useful for confirming a diagnosis of HP when characteristic, clinical, radiologic, and functional features are present. However, the presence of antibodies is less useful for the diagnosis of HP, as exposed persons with no apparent lung disease also have antibodies to the causative antigen (34, 35). On the other hand, the presence of specific sensitized T cells reflects a cell-mediated hypersensitivity to the presumptive relevant antigens, a condition more important in distinguishing patients with HP from their asymptomatic counterparts (36, 37). The persistence of cell-mediated hypersensitivity as assessed by the antigen-induced proliferation of sensitized lymphocytes is required to sustain the disease (38).

Several studies have revealed that CD4+ T cells play a crucial role in the induction of HP. Schuyler and colleagues established a murine model of experimental HP through an adoptive transfer of S. rectivirgula–sensitized spleen cells. In their murine model, the depletion of CD4+ T cells ablated the ability of recipient mice to express adoptive HP (16, 17). They further revealed that adoptive HP was transferred by CD4+ Th1 cells but not by CD4+ Th2 cells (18). With the technique of targeted disruption, Gudmundsson and coworkers showed an essential role of T cell–related cytokines in murine models of experimental HP induced by S. rectivirgula (39, 40). When compared with wild-type mice, mice undergoing a disruption of the interferon-{gamma} gene did not develop significant amounts of granuloma formation in the lungs after exposure to the S. rectivirgula antigen. Inversely, interleukin-10 knockout mice showed greater inflammation after antigen exposure. Interferon-{gamma} is a key cytokine for Th1 cells, and interleukin-10 is a key cytokine for Th2 cells (41). Gudmundsson's group also demonstrated that injection of interleukin-12, one of the potential cytokines polarizing naive T cells into Th1 phenotype (42), resulted the development of pulmonary granulomatous inflammation in a resistant strain of mice for experimental HP (43). Finally, Hisauchi-Kojima and colleagues purified a 21-kD protein from pigeon dropping extracts that could induce specific lymphoproliferative responses of PBMCs in patients with pigeon breeder's disease (44). The partially sequenced N-terminal peptides of this 21-kD protein were also confirmed to induce in vitro proliferation of PBMCs from patients with pigeon breeder's disease. Taken together, these observations suggest that cell-mediated hypersensitivity plays a major role in the pathogenesis of HP and that protein antigens might have some roles in the immunopathogenesis of HP.

We have cloned the full-length cDNA of a novel protein antigen TA-19 responsible for both cellular and humoral immune reactions in patients with SHP. This is the first time that the full-length cDNA of a potentially immunogenic protein antigen relevant to HP has been cloned.

TA-19 is a member of the "Ricin superfamily," which was recently proposed by Hirabayashi and colleagues (24). When Hirabayashi's group cloned the cDNA of a novel galactose-binding lectin from annelida, they found that this protein, named EW29, had multiple short consensus amino acid motif G-X-X-Q-X-W. Many of the proteins listed in this superfamily have carbohydrate recognition domains. Proteins with the G-X-X-Q-X-W motif are largely divided into a lectin group and an enzyme group (Figures 3 and 4). The nature of TA-19 and its biologic implications have yet to be determined. The other characteristic feature of the superfamily is hydrophobic signal peptides in the N-terminus. Generally, signal peptides of the protein are located at the N-terminus and are cleaved under a secretory pathway through the endoplasmic reticulum and Golgi apparatus (45). In addition, Prosite analysis (46) strongly suggests that the localization of TA-19 is extracellular (data not shown). Accordingly, we generated a TA-19 recombinant protein after the predicted signal sequence. T. asahii and T. mucoides, formerly denominated as Trichosporon cutaneum (47), are arthrospore-forming yeasts from the same family of Cryptococcaceae as C. neoformans and C. albicans. Serologic cross-reactions among T. cutaneum, Cryptococcus species, and Candida species are well known (4, 48). Antibody absorption of our patient's serum with various yeast lysates suggested that the epitopes of TA-19 recognized by the serum of patients with SHP did not cross-react with C. neoformans or T. mucoides (Figure 5B). Thus, we considered it worthwhile to examine the relevance of TA-19 to immune reactions in SHP.

Levels of IgG, IgA, and IgM antibodies to TA-19 in BAL fluids were preferentially elevated in patients with SHP (Figure 6). None of the non-SHP individuals studied exhibited elevated levels of IgG, IgA, or IgM in BAL fluids. Thus, measurement of antibodies to TA-19 in BAL fluid was confirmed to be both specific and highly sensitive in the identification of individuals exposed to T. asahii. Serum levels of IgG, IgA, and IgM were also significantly higher in patients with SHP than in all of the other groups (Figure 7). A few non-SHP subjects exceptionally exhibited slightly elevated IgG or IgA titers. The precise reason for the elevation was unknown at present. One possible explanation is that they were really exposed to T. asahii, and another is that they had cross-reacting antibodies to T. asahii. The former seems to be less likely because that non-SHP subjects studied did not show elevated levels of IgG, IgA, or IgM in the BAL fluids without exception. The latter possibility remains to be determined. BAL fluid is usually superior to serum as a source of antibodies in terms of monitoring local pulmonary inflammation. The local inflammation is remarkably different from systemic immune response (25, 49). For this reason, we conclude that the measurement of specific antibodies to TA-19 in BAL fluid is the most reliable method to identify individuals exposed to T. asahii at present.

Cell-mediated hypersensitivity response to TA-19 by BAL cells was found in all five SHP patients studied, but not in the patients with Sar. Stimulation indices of PBMCs were also significantly higher in the SHP patients than in the Sar, IPF, and NV groups. PBMCs from non-SHP individuals did not respond to TA-19. In a study on a series of SHP patients by Ando and colleagues (10), BAL cells from 6 out of 14 patients and PBMC from 3 out of 13 patients responded to culture-filtrate antigen of T. cutaneum. Thus, the purified antigen, TA-19, was confirmed to be not only specific, but highly sensitive for the evaluation of cell-mediated hypersensitivity in patients with SHP. The higher sensitivity of BAL cells to TA-19 compared with the PBMCs may indicate that antigen-specific T cells are scarce in the peripheral circulation but abundant at the site of disease.

In conclusion, we have cloned the full-length cDNA of TA-19, a novel protein confirmed to be relevant to the cellular and humoral immunities in SHP. This 19-kD protein may be an important antigenic component to elicit SHP. Although the exact biologic implications of TA-19 in the development of SHP await further studies, these results define a new protein antigen that may participate in the immunopathogenesis of SHP and prove useful for the diagnosis of the disease. TA-19 will also be useful for further studies on the epidemiology and pathogenesis of SHP.


   Acknowledgments
 
The authors are indebted to Dr. Ucida, Teikyo Institute of Medical Mycology, Tokyo, Japan, for providing yeast strains used in the study.

Received in original form June 21, 2002; accepted in final form December 16, 2002


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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