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The Journal of Immunology, 1998, 161: 448-457.
Copyright © 1998 by The American Association of Immunologists

T Cell Epitopes in Japanese Cedar (Cryptomeria japonica) Pollen Allergens: Choice of Major T Cell Epitopes in Cry j 1 and Cry j 2 Toward Design of the Peptide-Based Immunotherapeutics for the Management of Japanese Cedar Pollinosis1

Toshio Sone2,3, Keiko Morikubo, Michinori Miyahara, Naoki Komiyama, Kimiko Shimizu, Hajime Tsunoo and Kohsuke Kino

Department of Pharmaceutical Research, Meiji Institute of Health Science, Odawara, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Japanese cedar pollinosis is caused by exposure to Japanese cedar (Cryptomeria japonica) pollen, of which two components, Cry j 1 and Cry j 2, are believed to be the major allergens. T cell lines specific to either Cry j 1 or rCry j 2 were reactive to various portions of each panel of overlapping peptides derived from Cry j 1 or Cry j 2. Two peptides, p211–225 and p108–120, from among six major T cell epitopes identified in Cry j 1 sequence, and three peptides, p182–200, p344–355, and p66–80, from among five in Cry j 2, were chosen to design an artificial polypeptide (named Cry-consensus) based on a difference among the types of the restriction molecules capable of presenting these peptides. After construction of a DNA encoding these peptides in order, Cry-consensus was expressed in Escherichia coli. Five of six T cell epitopes, except for Cry j 2 p344–355, in Cry-consensus were recognized by the T cell clones specific to each peptide. PBMC from allergic patients induced higher proliferation under stimulation from Cry-consensus than individual peptides. Eighty-eight percent of the PBMC (15 of 17) showed proliferation under the Cry-consensus stimulation. Thus, several major T cell epitopes from Cry j 1 and Cry j 2 can be chosen in the design of peptide-based immunotherapeutics for the management of Japanese cedar pollinosis in subjects having various types of HLA class II molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During the determination of the primary structure of several major allergenic proteins derived from various sources, T cell epitopes on the molecules were identified (1). Recently, it has been demonstrated that the injection or oral administration of a peptide containing at least one immunodominant T cell epitope from a certain allergenic protein can induce T cell nonresponsiveness to a subsequent allergenic protein or allergen challenge in experimental murine models in vitro and in vivo (2, 3, 4, 5, 6, 7). A clinical trial of peptide containing major T cell epitopes from Fel d 1 is now under investigation (8, 9).

Studies of the mapping of T cell epitopes in the primary structure of allergenic proteins (10, 11, 12, 13, 14) revealed that two or more major T cell epitopes exist in the sequence of the allergen. A T cell epitope, regarded as immunodominant, of a particular allergenic protein is presumed to be different among individual allergic patients. Usually, IgE in each serum from allergic patients is reactive to at least two or more components of a particular allergen (1). These findings suggest that use of several major T cell epitopes from more than one major component of a certain allergen may be necessary for the development of peptide-based immunotherapeutics for the management of the symptoms of allergic diseases.

Pollen from Japanese cedar (Cryptomeria japonica) is a seasonal aeroallergen in Japan. More than 10% of the population suffer from pollinosis caused by exposure to the pollen (15). Two major allergenic proteins of this pollen, Cry j 1 and Cry j 2, have been isolated (15, 16). IgE specific to Cry j 1 was detected in up to 95% of patients suffering from the pollinosis, while that for Cry j 2 in about 70% (16, 17, 18, 19). A Western immunoblot of a crude extract of Japanese cedar pollen showed that more than 50% of IgE from individual patients bind to 40- to 50-kDa polypeptides, corresponding to Cry j 1 and Cry j 2 (20, 21). Therefore, both Cry j 1 and Cry j 2 are thought to be important in the pathogenesis of Japanese cedar pollinosis. Recently, the amino acid sequences of Cry j 1 and Cry j 2 were deduced from the nucleotide sequences of cDNAs coding for Cry j 1 (22, 23) and Cry j 2 (19, 24).

In the present study, we show the existence of major T cell epitopes in Cry j 1 and Cry j 2 sequences using T cell lines (TCL)4 specific to either Cry j 1 or Cry j 2. A recombined polypeptide, designated Cry-consensus, is prepared. It contains five of six T cell epitopes chosen from major and minor T cell epitopes from Cry j 1 and Cry j 2 sequences, based on a difference among the types of restriction molecules capable of presenting these peptides.5 The proliferative response of PBMC by allergic patients to the stimulation with Cry-consensus is measured. Following this, a need is shown for the concurrent use of the several major T cell epitopes from Cry j 1 and Cry j 2 in the design of peptide-based immunotherapeutics for the management of Japanese cedar pollinosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Donors

The blood donors who participated in this study were recruited from within our own Institute. Eighteen donors (16 male and 2 female aged 21–45 yr) were allergic patients suffering from Japanese cedar pollinosis, a diagnosis made on the bases of their case histories and the presence of allergen-specific IgE, as measured by the radio-allergosorbent test (RAST; Pharmacia Japan, Tokyo, Japan) and Ala-STAT (Diagnostic Products, Los Angeles, CA). All patients and two healthy individuals (aged 28 and 46 yr) gave informed consent. The genotypes of HLA class II loci of the allergic patients (shown in Table IGo) were determined by PCR/sequence-specific-oligonucleotide probe (SSOP) analysis (25) using DNA prepared from EBV-transformed B cell lines (EBV-B cells) established from each PBMC.


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  		Table I. HLA class II genotypes of 18 patients with Japanese cedar pollinosis

 
Preparation of Cry j 1 and rCry j 2

Crude extract of C. japonica pollen was prepared in a well-established manner (23). Cry j 1 was purified by a well-established procedure (15, 23) from C. japonica pollen collected in Atami, Shizuoka Prefecture (Japan).

The existence of two mature forms of Cry j 2 was reported: one is composed of 388 amino acids at positions from 46 to 434, and another is of 379 at positions from 55 to 434, in case that Met, a start codon in the cDNA coded for Cry j 2, is numbered at position 1 (24). Expression of rCry j 2 was performed according to the procedure previously described (19), with minor modifications. Briefly, a 1383-bp fragment of DNA encoding Cry j 2, where it codes from Ala at position 55 to the stop codon, was amplified by PCR using cDNA coded for entire sequence of Cry j 2 (pCCII-1) (19) as a template. The oligonucleotide primers used in this amplification contain a BamHI site upstream from the gene coding for Ala at the N terminus and a PstI site downstream coding for the stop codon. The PCR product was digested with BamHI and PstI and then ligated into the BamHI-PstI site of the expression plasmid, pQE-9 (Qiagen, Catsworth, CA) (named pQECryII). Expression of rCry j 2 in Escherichia coli M15 (pREP4) by the addition of isopropyl-1-thio-ß-D-galactopyranoside was performed according to the manufacturer’s protocol. After solubilization of the inclusion body in the E. coli with 6 M guanidine/20 mM Tris-HCl, pH 8, rCry j 2 was purified by affinity chromatography on nickel-nitrilotriacetic acid (NTA) agarose. The protein bound to the resin was eluted by 20 mM Tris-HCl, pH 4.5, containing 8 M urea. The purified protein was detected as one band (>90% purity) on a SDS-PAGE gel after staining with Coomassie brilliant blue R-250. rCry j 2 was dialyzed against PBS containing 8 M urea and then adjusted to 5 to 10 mg/ml. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL).

Synthesis of peptides

A panel of 69 overlapping peptides was synthesized according to the Cry j 1 sequence deduced from a cDNA coded for Cry j 1 (pCCI2-2) (23), using a solid-phase peptide synthesizer, PSSM-8 (Shimadzu, Kyoto, Japan), which uses the F-moc strategy. In addition, a panel of 74 overlapping peptides was synthesized according to the Cry j 2 sequence (19). The pentadecapeptides were overlapped for 10 amino acids. Each peptide was applied to a reverse-phase HPLC using a protein C4 column (Vydac, Hesperia, CA), and the main peak was collected. Purity of the peptides was estimated to >90%, as judged by the height of a main peak by mass spectrometry (Kompact Maldi I; Shimadzu). After lyophilization, each peptide was dissolved in 8 M urea/PBS to a concentration of 2 mM and stored at -20°C before use. Four hundred-fold dilution of 8 M urea/PBS containing each peptide and rCry j 2 with culture media did not show any cytotoxic effect on PBMC, EBV-B cells, TCLs, and T cell clones (TCC) during culture.

Construction of a DNA (pQEF4.1) and expression of Cry-consensus

DNA fragments encoding p211–225 and p108–120 from Cry j 1, and p181–200, p344–365, and p66–80 from Cry j 2 were amplified by PCR using each set of primers (F and R) shown in Table IIGo and cDNAs coding for Cry j 1 (pCCI2-2) (23) and Cry j 2 (pCCII-1) (19) as templates.


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  		Table II. Oligonucleotides used for PCR to construct pQEF4.1 coding for Cry-consensus

 
After DNA fragments A and B were digested with SalI and PstI, respectively, they were mixed and reacted with a Klenow fragment (Takara Biomedicals, Shiga, Japan) to convert the termini to blunt ends. In this step, the SalI site was changed to code for Arg-Arg residues. The mixture was ligated using DNA ligation kit Ver.1 (Takara Biomedicals). PCR was performed using A:F and B:R primer and an aliquot of ligation mixture as a template. The products were digested with SmaI and SalI and inserted into the pUC19 SmaI-SalI site (pUC19-A-B).

After DNA fragments C and D were digested with EcoRV and SalI, respectively, they were mixed and reacted with a Klenow fragment and then ligated. Subsequently, PCR was performed using C:FF and D:RR primers (construction of C*-D*) and an aliquot of ligation mixture as a template. After DNA fragments C*-D* and E were digested with SalI and PstI, respectively, they were mixed and reacted with a Klenow fragment, and then ligated. PCR was conducted using C:FF and E:R primer pairs and an aliquot of ligated mixture as the template (construction of C*-D*-E). C*-D*-E was digested with EcoRV and HindIII. The fragment was mixed with pUC19-A-B after digestion with SalI and HindIII and ligated. Competent cells of E. coli strain JM109 were transformed by a reaction mixture. A plasmid (pUC19F4.1) containing the expected DNA sequence (A-B-C*-D*-E) was selected. After digestion of pUC19F4.1 with SmaI and HindIII, the resulting DNA insert was subcloned into the BamHI-HindIII sites of pQE-11 (Qiagen). Competent cells of the E. coli strain JM109 were transformed by this plasmid. Finally, a plasmid named pQEF4.1 was obtained. Competent cells of E. coli strain M15 (pREP4) were transformed with pQEF4.1 and then grown. Expression, purification, and estimation of the purity (>90%) of Cry-consensus having a m.w. of 10,800 were performed by the same procedure used for the preparation of rCry j 2, as described above. Purified Cry-consensus (first version) was dialyzed against distilled water and then lyophilized. The protein was dissolved in 8 M urea/PBS to a concentration of 10 mg/ml and stored at -20°C before use.

APCs

PBMC were isolated from heparinized blood by density centrifugation on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden). EBV-B cells were established by cultivation of PBMC after in vitro infection of EBV, obtained from the culture of marmoset cell line B95-8 (kindly provided by Dr. Y. Nishimura, Kumamoto University, Kumamoto, Japan) in the presence of 1 µg/ml cyclosporin A (Sandoz, Basel, Switzerland) (26). EBV-B cells were cultured in RPMI 1640 (Nissui Pharmaceutical, Osaka, Japan) supplemented with 10 to 12% FCS (Life Technologies, Grand Island, NY). They were treated with 50 µg/ml mitomycin C (MMC) (Kyowa Hakko, Tokyo, Japan) for 30 min and then washed four times with RPMI 1640, after which they were used as APC.

Generation of Cry j 1- and rCry j 2-specific TCL and TCC

PBMC (4 x 106) were cultured with either 50 µg/ml Cry j 1 or 10 µg/ml rCry j 2 in 2 ml of medium in a 24-well plate for 8 days. The medium used was RPMI 1640 supplemented with 15% human AB serum, antibiotics (streptomycin and penicillin; Life Technologies), and L-glutamine (complete medium). On day 8, culture medium was replaced with freshly prepared complete medium containing 20 U/ml rIL-2 (Boehringer Mannheim, Mannheim, Germany). The culture was continued for an additional 10 to 11 days in the presence of 20 U/ml rIL-2, and the medium was changed once per day. The TCLs generated were stored in liquid nitrogen. These TCLs and MMC-treated autologous EBV-B cells were mixed, and proliferation experiments were performed in the presence, and also the absence, of either 25 µg/ml Cry j 1 or 5 µg/ml rCry j 2. The TCLs that induced three times more proliferation than the control culture (stimulation index (SI) >3) were used for additional experiments. The phenotypes of the TCL were determined by flow cytometry using a FACStar (Becton Dickinson, Mountain View, CA) after staining them with FITC-conjugated anti-CD3 (Leu4), anti-CD4 (Leu3a), anti-CD8 (Leu2a), anti-T{alpha}ß (TCR-{alpha}/ß), and anti-T{gamma}{delta} (TCR-{gamma}{delta}1) mAbs (all from Becton Dickinson). Generation of the TCCs specific to either Cry j 1 or rCry j 2 is described elsewhere.5

T cell proliferative response

The proliferation of TCLs and TCCs was assayed by coculturing the cells (2 x 104) with MMC-treated autologous EBV-B cells (5 x 104) as APC in 0.2 ml of complete medium in a 96-well flat-bottom culture plate for 3 days. Synthetic overlapping peptides at a final concentration of either 1 µM, corresponding to a 38 µg/ml Cry j 1, or 0.5 µM, corresponding to a 21 µg/ml rCry j 2, were added to each well and the cells were incubated for 72 h. Some TCLs induced high backgrounds of up to 5000 cpm, probably due to the use of autologous EBV-B cells as APC. Therefore, the responses of TCLs to the peptides were considered positive when the value of SI was >2. PBMC (4 x 105) were cultured in 0.2 ml complete medium with indicated concentrations of Cry j 1, rCry j 2, Cry-consensus, and peptides in a 96-well flat-bottom plate for 7 days. For the final 16-h incubation, 0.5 µCi [3H]thymidine (NEN, Boston, MA) was added to each culture. After the cells were harvested, incorporation of [3H]thymidine was measured using a BETAmatic II liquid scintillation counter (Kontron, Basel, Germany). All cultures were set up in triplicate.

Fluorometric assay for the detection of IgE bound to Cry-consensus

A total of 1 mg of crude extract of pollen and 10 µg of Cry-consensus was coated on a 96-well black plate (Dainippon Pharmaceutical, Osaka, Japan) at 4°C for 16 h. After blocking the plate with Block Ace (Dainippon Pharmaceutical), 100 µl of threefold diluted serum with PBS from 18 patients and 4 healthy individuals was transferred to individual wells. The amounts of IgE bound to the solid-phase allergen were evaluated by the fluorescence-based detection method, as described elsewhere (19). The average value of duplicate measurement was indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping of T cell epitopes

In a preliminary experiment, we tried to determine the T cell epitopes in response to PBMC following stimulation by 2 µM of a panel of 69 overlapping peptides derived from the Cry j 1 sequence in primary culture. A detectable proliferative response of PBMC from the four allergic donors was occasionally induced. Therefore, TCLs specific to either Cry j 1 or rCry j 2 were generated. The phenotypes of all TCLs were CD3+, CD4+, and CD8-. Expression of TCR on the cells was all TCR-{alpha}ß+ and TCR-{gamma}{delta}-.

The proliferative response of Cry j 1-specific TCLs from 18 allergic patients to the stimulation with Cry j 1 and a panel of overlapping peptides from the Cry j 1 sequence was examined. The same experiment was performed in Cry j 2-specific TCLs under the stimulations with rCry j 2 and a panel of overlapping peptides from Cry j 2. As shown in Figure 1Go, all TCLs induced a proliferative response to either Cry j 1 or rCry j 2 stimulation (SI > 3). TCLs specific to Cry j 1 responded to stimulation with a panel of overlapping peptides (SI > 2) in the range of 3 (patient:PP) to 17 (PE) (mean 10.4 ± 4.1). Similarly, those reactive to Cry j 2 were in the range of 2 (PJ) to 15 (PK) (mean 9 ± 3.9). Two neighboring peptides that induced proliferation were counted as one T cell epitope. Therefore, TCLs from allergic patients could recognize multiple T cell epitopes in both the Cry j 1 and the Cry j 2 sequences.



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  		FIGURE 1. Schematic representation of the reactivity of TCLs, generated from 18 patients, to a panel of synthetic peptides derived from Cry j 1 and Cry j 2 sequences. TCLs were cocultured with MMC-treated autologous EBV-B cells in the presence of Cry j 1 (25 µg/ml), rCry j 2 (5 µg/ml), or an overlapping peptide (1 µM from Cry j 1 sequence and 0.5 µM from Cry j 2 sequence), as described under Materials and Methods. Proliferative response of TCLs is evaluated by SI. The number of TCL reactive to a peptide is indicated numerically. An overlapping peptide containing a C terminus in an end in Cry j 1 sequence corresponds at positions from 339 to 353. A small letter at position 380 in an end peptide of Cry j 2 sequence means the addition of Ala existed in a C-terminal extension.

 
As for the Cry j 1 sequence, 55 of 69 (80%) overlapping peptides from Cry j 1 sequence contained T cell epitopes, suggesting that the ratio of antigenic peptides capable of binding to at least one of HLA class II molecules and being recognized by Th cells in the Cry j 1 sequence is calculated to be 58%, in which the antigenic peptide is postulated to be composed of 12 amino acids (27). Several limited regions of the peptides were recognized frequently as T cell epitopes by TCLs. Six peptides, p16–30 (11 of 18 TCLs were reactive to the peptide, 61% recognition), p81–95 (11 of 18, 61%), p106–120 (13 of 18, 72%), p111–125 (10 of 18, 56%), p211–225 (13 of 18, 72%), and p301–315 (10 of 18, 56%), were identified as major T cell epitopes since >50% of the TCLs recognized the peptides as T cell epitopes. Furthermore, three peptides, p66–80, p91–105, and p316–330, were recognized as T cell epitopes by one-half of the TCLs (9 of 18, 50%).

Of 74 overlapping peptides from the Cry j 2 sequence, 53 (72%) were recognized as T cell epitopes by TCLs, suggesting that the ratio of antigenic peptides in the Cry j 2 sequence is calculated to be 53%. Five of the overlapping peptides, p66–80 (11 of 18, 61%), p141–155 (10 of 18, 56%), p186–200 (12 of 18, 67%), p346–360 (12 of 18, 67%), and p351–365 (11 of 18, 61%), were identified as major T cell epitopes. Similarly, three peptides, p81–95, p236–250, and p336–350, were recognized by one-half of the TCLs (9 of 18, 50%). Therefore, the existence of several major T cell epitopes in the Cry j 1 and Cry j 2 sequences was clearly demonstrated.

HLA class II molecules capable of presenting T cell epitopes from Cry j 1 and Cry j 2

We have identified the restriction molecules for the presentation of the several T cell epitopes from the Cry j 1 and Cry j 2 sequences using either TCCs specific to Cry j 1 generated from the patients PB and PJ, or TCCs specific to Cry j 2 from PB, PC, and PR.5 Using these data, we tried to predict the binding property of the antigenic peptides toward HLA class II molecules, comparing the recognition of the T cell epitopes from Cry j 1 and Cry j 2 sequences (Fig. 1Go) with the HLA class II genotypes of 17 allergic patients (except for PF) (Table IGo), knowing that the antigenic peptides can bind to one or more types of HLA class II molecules from a locus (28, 29). As shown in Table IIIGo, Cry j 1 p16–30, one of the peptides containing a major T cell epitope, is presented by the subtype of DQ6. Cry j 1 p106–120 seems to be restricted by DR51 as well as by other types of DR. Cry j 1 p211–225 is mainly presented by DP5. As for the antigenic peptides from Cry j 2, four peptides, p66–80, p81–95, p186–200, and p346–360, are mainly presented by DR51, DP5, DR53, and DQ6, respectively. Cry j 2 p236–250 and p336–350 seem to be restricted by DR15 (as well as other types of DR) and DP5 (as well as other types of DP), respectively.


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  		Table III. Prediction of the type of restriction molecules capable of presenting T cell epitopes from the Cry j 1 and Cry j 2 sequences

 
Properties of Cry-consensus

To use several T cell epitopes together as an immunotherapeutic, we attempted to construct a recombined polypeptide containing the major T cell epitopes of p106–120 and p211–225 from Cry j 1, and p66–80, p182–200, and p346–360 from Cry j 2. To delete Cys residues at position 107 in Cry j 1 and at position 356 in Cry j 2, Cry j 1 p108–120 in place of p106–120 and Cry j 2 p344–355 in place of p346–360 were used. Cry j 1 p16–30 and Cry j 2 p351–365 were eliminated, since a Cys residue at position 24 on Cry j 1 and a residue at position 356 on Cry j 2 are located nearly at the center of the peptide.

PCR primers used for the construction of a pQEF4.1 are shown in Table IIGo. Each DNA fragment was conjugated in the order shown in Figure 2Go. The Arg-Arg residues that are the cleavage site for cathepsin B were inserted into the junction between the two peptides because the enzyme may partially participate in the processing of foreign Ags (30, 31). Finally, a recombined polypeptide, Cry-consensus (first version), expressed in E. coli, was obtained. This protein is readily soluble in distilled water and saline in excess of 10 mg/ml.



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  		FIGURE 2. Nucleotide sequence of pQEF4.1 and amino acid sequence of Cry-consensus. Position of the peptides from Cry j 1 and Cry j 2 sequences is indicated under the amino acid sequence of Cry-consensus. To indicate conjugated position, horizontal and perpendicular bars are inserted in the nucleotide sequence and the amino acid sequence, respectively.

 
The proliferative response of TCCs reactive to individual T cell epitopes was examined (shown in Fig. 3Go). Five of the six TCCs induced proliferation to Cry-consensus stimulation, while two TCCs, PB14-19 and PB12-8 (the value of PB14-19 alone was shown in Fig. 3Go), which is reactive to both Cry j 2 p341–355 and p346–360 peptides, showed a detectable, but very low response. We further examined the proliferative response of two TCCs to the stimulation with Cry j 2 p344–355, pArg-Arg (RR)-344–355, and p344–355-RR, in which the peptides are located in the Cry-consensus (Fig. 2Go). Stimulations by p344–355 and pRR-344–355 induced proliferation in two TCCs, while that by p344–355-RR did not. Consequently, the addition of the RR residues to the downstream of the p344–355 in the Cry-consensus may result in induction of a very low proliferative response of two TCCs. These observations indicate that five peptides in Cry-consensus, not including p344–355, can be recognized by Th cells as T cell epitopes.



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  		FIGURE 3. Recognition of Cry-consensus by TCCs specific to individual T cell epitopes. TCCs reactive to each T cell epitope were cocultured for 3 days with MMC-treated autologous EBV-B cells in the presence of Cry j 1, rCry j 2, each peptide indicated in the columns or originated from Cry-consensus sequence (a lower column), and Cry-consensus. For the final 16-h incubation, [3H]thymidine was added to each culture. Cultures are set up in triplicate, and the mean value is indicated (SEM < 10%).

 
The proliferative response of PBMC to the Cry-consensus in both allergic patients and healthy individuals was also examined. PBMC from the four patients induced proliferation in a Cry-consensus dose-dependent manner, while those from the two healthy donors did not (Fig. 4GoA). An almost equal level of proliferation of PBMC from the allergic patients under stimulation by Cry-consensus and a mixture of six peptides was observed, while occasionally a low, but detectable level of the six peptides was noted (Fig. 4GoB). PBMC from 15 of 17 allergic patients (88%) induced proliferative response (SI > 2) to the stimulation of the Cry-consensus (Fig. 5Go). The mean values of SI under the stimulation by Cry j 1, rCry j 2, and Cry-consensus were 19.6 ± 16, 16.5 ± 13.9, and 8.3 ± 7.8, respectively. Several major T cell epitopes from Cry j 1 and Cry j 2 are appropriate for the design of a peptide-based immunotherapeutic for the management of Japanese cedar pillinosis in subjects having various types of the HLA class II molecules.



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  		FIGURE 4. Proliferative response of PBMC stimulated by Cry-consensus (A), one of the peptides, and mixture of six peptides (B). PBMC from four patients, PJ, PB, PR, and PC, and two healthy individuals, HT and HM, were cultured for 7 days in the presence of Cry j 1, rCry j 2, 0.001 to 25 µg/ml Cry-consensus, each peptide, and the mixture of six peptides. A total of 10 µg/ml of Cry-consensus is equivalent to 1 µM. For the final 16-h incubation, [3H]thymidine was added to each culture. Cultures are set up in triplicate, and the mean value is indicated (SEM < 15%).

 


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  		FIGURE 5. Proliferative response of PBMC by the stimulation with Cry-consensus. PBMC from 17 allergic patients, except for PG and 2 healthy individuals, HT and HM, were cultured for 7 days in the presence of 50 µg/ml Cry j 1, 2 µg/ml rCry j 2, and 10 µg/ml Cry-consensus. For the final 16-h incubation, [3H]thymidine was added to each culture. Cultures are set up in triplicate, and the mean value in terms of SI is indicated (SEM < 15%). The basal responses of individual PBMC to no-allergen stimulation were from 942 cpm to 2185 cpm. The dotted line indicates a value of SI = 2.

 
Determination of IgE bound to Cry-consensus

IgE in sera from 17 of 18 patients with Japanese cedar pollinosis bound to the crude extract of pollen in the range of 50 to 1781 (arbitrary fluorescence units), and to Cry-consensus in the range of 3 to 5 (Table IVGo). Sera from two healthy individuals (HT and HM) showed binding to the crude extract of pollen and Cry-consensus in the range of 3 to 5. After that, no binding of IgE in sera from the allergic patients to Cry-consensus was observed.


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  		Table IV. IgE binding to Cry-consensus determined by fluorometric assay

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunotherapy utilizing extracts from various sources of allergen was introduced early this century and used to ameliorate allergic symptoms for many years (32, 33). Control of pollinosis by immunotherapy that utilizes multiple injections of the extract of a particular pollen remains controversial (32). This is also true for Japanese cedar pollinosis (34). Recently, it was demonstrated that the injection of a peptide containing major T cell epitopes induced peripheral tolerance and moderated the symptoms of autoimmune disease (35) and of allergy in experimental murine models (4, 5, 6) and in humans in vivo (8, 9). In the present study, we selected several major T cell epitopes identified in Cry j 1 and Cry j 2 sequences for the design of a peptide-based immunotherapeutic that would help to manage Japanese cedar pollinosis.

TCLs specific to either Cry j 1 or Cry j 2 recognized multiple T cell epitopes on the Cry j 1 and Cry j 2 sequences. This observation is consistent with the previous report on Der p 1 (36), Der p 2 (10, 11), Lol p 1 (12), Bet v 1 (13, 37), and Fel d 1 (38). We estimate that approximately 58% of Cry j 1 and 53% of Cry j 2 sequences can bind to HLA class II molecules and be presented to Th cells. The fact that various regions of the allergen sequences are recognized as T cell epitopes may be a characteristic feature of the allergenic proteins.

Identification of major T cell epitopes in the allergen sequence is the first step toward the design of peptide-based immunotherapeutics. Fortunately, six and five major T cell epitopes in the Cry j 1 and Cry j 2 sequences, respectively, have been identified. Similarly, three peptides in the Cry j 1 and three in Cry j 2 have been recognized by one-half of the TCLs (Fig. 1Go). Finally, nine peptides from Cry j 1 and eight from Cry j 2 are available for the design of peptide-based immunotherapeutics.

Why were so many major T cell epitopes in Cry j 1 and Cry j 2 sequences identified? Three peptides in Cry j 1, p16–30, p106–120, and p211–225, were presented by DQ6, DR51, and DP5, respectively. Three peptides in Cry j 2, p66–80, p186–200, and p341–360, were presented by DR51, DR53, and DQ6, respectively. The Ag frequencies of DR51 (DRB5*0101), DR53 (DRB4*01), DQ6 (DQB1*0602), and DP5 (DPB1*0501) in the Japanese population are 0.11, 0.50, 0.12, and 0.65, respectively (25, 39). Furthermore, Cry j 1 p16–30 is restricted by subtypes of DQ6, of which the Ag frequency is 0.48. In addition, Cry j 2 p66–80 is thought to be presented by subtypes of DR51. The Ag frequency of DR51 is 0.28. Cry j 1 p106–120 may be restricted mainly by DR51 as well as by other undetermined types of DR. Therefore, major T cell epitopes that have been identified are presented by the particular HLA class II molecules, types of which are found frequently in the Japanese population.

T cell epitopes from Cry j 1 are presented by DR9, DR51, DQ6, and DP5, and those from Cry j 2 are restricted by DR9, DR15, DR51, DR53, DQ6, DP2, and DP5 (Table IIIGo). Other types derived from HLA class II loci may also function as restriction molecules, since various genotypes of the HLA class II molecules have been found in allergic patients (Table IGo); and in addition, various T cell epitopes presented by undetermined types of HLA class II molecules were found to exist (Fig. 1Go). Furthermore, it was reported that DR52 (DRB3*0301) could present Cry j 1 p335–346 to Th cells (40). Up to 90% of the Japanese population is thought to possess one of the types of HLA class II molecules that can present T cell epitopes from Cry j 1 and/or Cry j 2 to Th cells. Clinical studies have shown that more than 10% of the Japanese population suffers from Japanese cedar pollinosis (15). Still, unknown factors may contribute to the pathogenesis of this disease. Therefore, further genetic (41, 42, 43, 44, 45), immunologic, and environmental analyses will be necessary.

The use of one or two major T cell epitopes as peptide-based immunotherapeutics has been proposed by others (4, 5, 6, 7). In our observations, stimulation by a 15-mer peptide containing a T cell epitope has occasionally induced a low, but detectable proliferative response in PBMC from allergic patients (Fig. 4GoB). Individual TCLs specific to either Cry j 1 or rCry j 2 strongly responded to the stimulation of several different peptides, which are considered to contain an immunodominant T cell epitope. Such peptides appear to localize in several limited regions in which major T cell epitopes exist (Fig. 1Go), leading to the conclusion that the concurrent use of the several major T cell epitopes might be more effective than only one or two.

The choice of the T cell epitopes from Cry j 1 and Cry j 2 was made based on the following reasons: 1) The peptides containing a major T cell epitope should be chosen, as mentioned above. 2) The peptides from Cry j 1 rather than Cry j 2 are preferentially chosen, since IgE in sera from the allergic patients compared with the Cry j 1 is higher than that in Cry j 2. Consequently, Cry j 1 is thought to be somewhat more important than Cry j 2 in the pathogenesis of Japanese cedar pollinosis (16, 17, 18). 3) The peptides restricted by the different types of HLA class II molecules should be chosen, since these may be efficiently presented by them to Th cells. 4) The influences of the Cys residue(s) and the formation of the intra- and intermolecular disulfide bond in an artificial polypeptide on release of the antigenic peptides capable of binding to restriction molecules in APC remain unknown; therefore, the peptides containing the Cys residue(s) may at present be eliminated.

Five major T cell epitopes, p16–30, p81–95, p106–120, p211–225, and p301–315, in Cry j 1 could be used for the design of a peptide-based immunotherapeutic. Two peptides were chosen: one was p108–120 restricted by DR51 in place of p106–120, in which the Cys residue at position 107 exists, and another was p211–225 presented by DP5. Three peptides, p16–30, p81–95, and p301–315, were eliminated since p16–30 contains a Cys residue at position 24, located at the center of the peptide, and p81–95 and p301–315 are presented by undetermined restriction molecules.

Similarly, four peptides, p66–80, p186–200, p346–360, and p351–365, in Cry j 2 could be used. First, p186–180 was chosen since the peptide was restricted by DR53. Additionally, elongation of the four amino acids at positions 182–185 toward the peptide resulted in the insertion of another T cell epitope restricted by DP2. Then p182–200 was chosen in place of p186–200. After this, p66–80 was chosen, since two peptides, Cry j 1 p106–120 and Cry j 2 p66–80, are not only restricted by DR51 (DRB5*0101), but also by the subtypes of DR51 and other types of DR molecule (Table IIIGo). Cry j 2 p336–365 contained at least three T cell epitopes (recognized by 15 of 18 allergic patients) and is a very important region for the design of a peptide-based immunotherapeutic. But two Cys residues at positions 336 and 356 existed in the peptide. A Cys residue at position 356 existed nearly in the center of p351–365; therefore, the peptide was eliminated. Two peptides, Cry j 2 p336–350 (recognized by 9 of 18 allergic patients) and Cry j 1 p211–225 (13 of 18), were restricted by DP5; Cry j 1 p211–225, rather than Cry j 2 p336–350, was preferentially chosen. The difference in the recognition between the two peptides among the patients may be caused by the antigenicity of the two peptides; Cry j 1 p 211–225 was recognized by all patients possessing DP5 (11 of 11), while Cry j 2 p336–350 was recognized by almost one-half of the patients (5 of 11), suggesting that the affinity of Cry j 1 p211–225 for binding toward DP5 might be higher than that of Cry j 2 p336–350. Two TCCs generated from PB, and two from PR could induce almost equal levels of proliferation under the stimulation by p341–355 and p346–360,5 suggesting that p346–355 was the core sequence for its recognition by the Th cells. The crystal structure of HLA-DR1 and DR3 complexed with particular peptides (46, 47) revealed that the amino acid residues of dodecamer participate in its binding to them. Two amino acids were then added to the upstream of the peptide. No peptide restricted by DQ6 was chosen. Finally, p344–355 was chosen in place of p346–355. Then in the 18 patients used in this study, at least one of the six major and minor T cell epitopes identified to date was recognized (Fig. 1Go).

The mechanism of processing the foreign Ags in APC is not yet well understood. The addition of the inhibitors for cathepsins prevents release of the antigenic peptides from the Ags in APC, suggesting that cathepsin B (30, 31) and cathepsin D (48) may participate in part in the processing of the foreign Ags. Cathepsin D cleaves hydrophobic-hydrophobic residues. Addition of two hydrophobic amino acids to the junction of the two peptides could result in the formation of some new antigenic peptides, because the hydrophobic side chains function as the binder for the pockets 1, 4, 6, and 9 situated in the cleft of HLA class II molecules (46, 47). It is rare that Arg residue can function as an anchor residue for the binding to HLA class II molecules, except DR51 (49). Therefore, Arg-Arg residues were inserted into the junction of two peptides.

We have constructed a DNA, pQEF4.1, encoding Cry j 1 p211–225 and p108–120, and Cry j 2 p182–200, p344–355, and p66–80 in that order (Fig. 2Go). Cry j 2 p66–80 was placed at the last position to prevent the addition of the Arg-Arg residue, since Tyr at position 73 and additional Arg in the sequence just fitted to two anchor amino acids, P1 and P9, respectively, of the peptide motifs for DR51 ligands (49). After addition of the Arg-Arg residues to the downstream of the peptides, the other four peptides did not fit into the peptide ligand motifs for DR53 (DRB4*0101) (50, 51), DP2 (DPB1*0201), DR15 (DRB1*1501), and DR4 (DRB1*0405) (49) frequently found in the allergic patients (more than 4 patients possessing of the types among 17), although the motifs for DP5 (DPB1*0501) and DQ6 (DQB1*0601 and DQB1*0602) have not been well defined. Therefore, other four peptides were randomly arranged in order (Fig. 2Go).

Five of six T cell epitopes, not including Cry j 2 p344–355, in the Cry-consensus can function as T cell epitopes by the estimation of reactivity of the individual TCCs under stimulation by each peptide, while a T cell epitope (Cry j 2 p344–355) in it induced a very low proliferative response in two TCCs, PB14-19 and PB12-8 (Fig. 3Go). Subsequent examination using three synthetic peptides, p344–355, pRR-344–355, and p344–355-RR, revealed that the addition of Arg-Arg residue to the downstream of p344–355 in Cry-consensus may interfere with the binding of p344–355 to the DQ6 molecule.

A higher level of proliferative response of PBMC from allergic patients was induced by stimulation using Cry-consensus and a mixture of the peptide, rather than one of the peptides (Fig. 4Go). The stimulation by Cry-consensus resulted in the induction of proliferation (SI > 2) in PBMC from 15 of the 17 patients (88%). The level of proliferation under stimulation by Cry-consensus was one-half compared with those by Cry j 1 or rCry j 2 (Fig. 5Go). Furthermore, no binding of the IgE in sera from the allergic patients to the Cry-consensus is apparent (Table IVGo). Therefore, these observations reveal that an artificial polypeptide, arranging the several T cell epitopes in tandem such as Cry-consensus, can be practically available for its use as a peptide-based immunotherapeutic. It can be used in subjects having various types of HLA class II molecules for the management of Japanese cedar pollinosis.

Based on our observations, we considered the design of the second version of Cry-consensus with slight modifications. The main alterations of the Cry-consensus are as follows: 1) The removal of a His-tag by the use of other expression systems will be necessary. 2) The use of Cry j 2 p344–365 or the altered peptide of which a Cys residue at position 356 is changed to Ser or Ala residue in place of p344–355 may be available in case this T cell epitope would be chosen. In this case, the effect of altered peptide on proliferative response of the TCCs specific to the epitope would be evaluated. 3) It should be most effective to design a recombined polypeptide containing more T cell epitopes such as Cry j 1 p81–95 and p301–315 that contain no Cys residue, although restriction molecules for the peptides are not known at present. In these cases, the artificial polypeptide reactive to IgE detected by ELISA, as well as capable of releasing histamine from PBMC, should be eliminated.

In the present study, we have demonstrated that several major T cell epitopes exist in the Cry j 1 and Cry j 2 sequences. Proliferative response of PBMC from allergic patients by stimulation with Cry-consensus revealed that concurrent use of several major T cell epitopes in Cry j 1 and Cry j 2 sequences is available for the design of peptide-based immunotherapeutics. These treatments may be useful in the management of Japanese cedar pollinosis in subjects having various types of HLA class II molecules.


   Acknowledgments
 
We thank Dr. T. Tana, Dr. N. Kamikawaji, and Dr. T. Sasazuki (Kyushu University, Fukuoka, Japan), and Dr. K. Hirayama and Ms. M. Kikuchi (Saitama Medical School, Saitama, Japan) for supporting our special HLA class II DNA typing technique. We also thank Dr. R. Walton (this Institute) for proofreading our manuscript.


   Footnotes
 
1 This work was supported by Meiji Milk Products. Back

2 Address correspondence and reprint requests to Dr. Toshio Sone, Department of Medical Zoology, Saitama Medical School, 38 Morohongo, Moroyama, Iruma, Saitama 350-0495, Japan. Back

3 Present address: Department of Medical Zoology, Saitama Medical School, Saitama 350-0495, Japan. Back

4 Abbreviations used in this paper: TCL, T cell line; EBV-B cell, Epstein-Barr virus-transformed B cell; MMC, mitomycin C; SI, stimulation index; TCC, T cell clone. Back

5 T. Sone, K. Morikubo, N. Komiyama, K. Shimizu, H. Tsunoo, and K. Kino. 1998. Peptide specificity, HLA class II restriction, and T cell subsets of T cell clones specific to either Cry j 1 or Cry j 2, the major allergens of Japanese cedar (Cryptomeria japonica) pollen. Submitted for publication. Back

Received for publication August 11, 1997. Accepted for publication March 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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