INDEX
Epitope analysis of the Japanese cypress pollen major allergen, Cha o 1, recognized by a human T cell clone.

Shigeru Takagia, Yoshihisa Nakamuraa, Motohiko Suzukia, Hirotaka Itoa, Shingo Murakamia and Nobuo Ohtab.
Department of Otorhinolaryngology, and bDepartment of Medical Zoology, Nagoya City University Medical School, Nagoya, Japan.
Short title: Human T cell epitope of Cha o 1.
Key words: Altered peptide, Cha o 1, Japanese cypress, T cell epitope
Correspondences: Shigeru Takagi, Department of Otorhinolaryngology, Nagoya City University Medical School, 1 Azakawasumi, Mizuhocho, Mizuhoku, Nagoya 467-8601, JAPAN
Tel. +81-52-853-8256, Fax. +81-52-851-5300
e-mail. shigeten@gc5.so-net.ne.jp

FOOTNOTE
Abbreviations use: APC, antigen presenting cell; CAP-RAST, CAP radioallergosorbent
test ; FACS, fluorescent activated cell sorter; ELISA, enzyme-linked
immunosorbent assay; HLA, human leukocyte antigen; IFN, Interferon;
MHC, major histocompatibility complex; MMC, Mitomycin C; PBL,
peripheral blood leukocytes ; PBMC, peripheral blood mononuclear
cells; PBS, phosphate buffered saline; PPD, purified protein delivertive;
and TCR, T cell receptor

SUMMARY
One CD4+ T cell clone with specificity for Cha o 1, a major allergen of Japanese cypress pollen, was established from peripheral blood of a patient with Japanese cypress pollinosis and epitope analysis was done using synthesized overlapping oligopeptides.
We screened MHC-binding regions of Cha o 1 by testing HLA-DR molecules to solid-phase synthetic peptides. Based on the results, we synthesized peptides, and a peptide, Cha o 1:202-217 (GHSDIYSDDKSMKVTV), induced a proliferative response of this clone. However, three altered peptides carrying a single amino acid substitution to alanine or serine, K214A, V215S and T216A, remarkably lost its proliferative activity. This result suggested that these amino acid residues were important for the eptiope activity. In addition, it was also suggested that a residue of 216T was important for binding between the epitope peptide and antigen presenting cells (APC), because T216A did not inhibit binding between APC and a labeled original epitope peptide in an analysis using FACScan (Beckton-Dickinson, Sandiego, CA). Our results suggest a possibility of immunotherapy of Japanese cypress pollinosis through manipulating T cell responses in humans.

INTRODUCTION
Antigens are processed to peptides by antigen presenting cells (APC), and are presented to T cells by appropriate major histocompatibility complex (MHC) class II molecules of the APC. CD4+ T cells become activated to proliferate and exert various biological functions when T cell epitope peptides, together with MHC, are recognized by specific T cell receptor (TCR) molecules. Since it was demonstrated that the administration of T cell epitope peptide from a certain allergenic protein could induce non-responsiveness of specific T cells to a subsequent allergenic protein challenge (1-6), use of allergen peptides containing a T cell epitope for immunotherapy instead of whole allergen proteins, known as "peptide immunotherapy" has been suggested(2,3,7-9). In general, T cell epitopes of allergens are different from B cell epitopes where are IgE-binding sites. Therefore, peptide immunotherapy has received much attention as a possible immunotherapy without anaphylaxis. In such situation, the T cell recognizing epitopes of important major allergens have been studied in the hope of their clinical use.
In Japan, the most common allergic disease is pollinosis due to Cryptomeria japonica (Japanese cedar). Two major allergenic proteins of this pollen, Cry j 1 and Cry j 2, were isolated (10, 11) and the amino acid sequences were deduced (12-15). Furthermore, T cell-recognizing epitopes of Cry j 1 and Cry j 2 were identified (16). Many patients with cedar pollinosis continue to have allergic symptoms even after the cedar pollen season. It is thought that such symptoms are caused by pollen of Chamaecyparis obtusa (Japanese cypress) which is scattered continuously after the cedar pollen season (17). Because a larger area has been reforested with cypress trees than with cedar in recent years in Japan, the number of patients suffering from cypress pollen allergies might increase. Thereforepollinosis due to the Japanese cypress should be given more attention.
Cha o 1 was identified as a major allergenic protein of Japanese cypress pollen, and the amino acid sequences were deduced (18). While there have been many immunological studies on Cry j 1 or Cry j 2, there are only a few on Cha o 1. In this study, a Cha o 1- specific human CD4+ T cell clone was generated, and one of the T cell-recognizing epitopes of the Japanese cypress was identified. Furthermore, amino acid residues contributing to the activity of this peptide were studied using its amino acid substitution peptides (altered peptides). Together with those results, we discuss about the role of Japanese cypress pollen in the Japanese pollinosis, and also about future of altered peptides in immunotherapy of allergic disease.

MATERIALS & METHODS
Cha o 1-specific human T cell clone
We used peripheral blood from a Japanese patient with Japanese cypress pollinosis diagnosed by the clinical symptoms, and also by the presence of Japanese cypress-specific IgE detected by CAP-RAST (Pharmacia, Uppsala, Sweden). A Cha o 1-specific T cell clone was established from the peripheral blood using the method described by Sone et al. (13) with minor modifications. Briefly, peripheral blood mononuclear cells (PBMC) were cultured in RPMI 1640 (Sigma, MO, USA) supplemented with 10% heat-inactivated human male AB sera (Nabi, FL, USA), 100 g/ml of streptomycin, 100 U/ml of penicillin and 50 mM of L-glutamine (GIBCO, NY, USA) with 50 g/ml semi-purified Cha o 1 for 5 days. Semi-purified Cha o 1 was prepared using the method described by Ide and coworkers (19 ). On day 5, human rIL-2 (Behringer Mannheim, Germany) was added to the medium at a concentration of 20 U/ml. On day 10, cloning was done using the micromanipulation method, in which a single cell was microscopically sucked from the cell suspension using a microcapillary tube (20). A single cell culture was done in 96 well U-bottom plates (Corning, NY, USA) in the presence of 5 104 of mitomycin C (MMC, Kyowa Hakko, Tokyo, Japan)-treated autologous PBMC as APC, 20 U/ml of rIL-2 and 1 g/ml of semi-purified Cha o 1 in the RPMI 1640 conditioned medium. When we microscopically observed clonal cell expansion, the cells were transferred to 24 well plates. Autologous APC and 0.04% phytohemagglutinin (Sigma, MO, USA) were added every two weeks. The antigen specificity of the T cell clone was confirmed by its proliferative response to recombinant Cha o 1 (r-Cha o 1) , which was a kind gift from Dr. Kino and colleagues (Meiji Institute of Health Science, Japan). Purified protein delivertive (PPD) of tuberculin was used as a negative control antigen. Out of 18 cells of clonal expansion, only one clonal cell showed Cha o 1-specific proliferative response in vitro. At the same time, we measured cytokines in the supernatants with ELISA kits for interleukin(IL)-4 and interferon (IFN) (Cytoscreen, CA, USA) according to the manufacturer's instructions. Fifty days later, cloned cells were cryopreserved at -80. T cell surface markers were analyzed with anti-CD3, CD4, CD8, TCR / (Becton Dickinson, CA, USA), and anti-TCR / (T cell Sciences, MA, USA) antibodies using FACScan (Becton Dickinson, CA, USA). The HLA class II genotypes were determined by PCR/sequence-specific oligonucleotide probe analysis (21) .

Synthetic oligopeptides
We prepared synthetic oligopeptides to determine an epitope recognized by a Cha o 1-specific T cell clone. Based on screening data of bindings between oligopeptides and purified HLA-class II molecules on filter papers, as described by Fu and coworkers (22), we synthesized peptides from candidate regions of Cha o 1 using the F-moc method. Each synthesized peptide was applied to a reverse-phase HPLC using a protein C4 column sourse, and the main peak was collected (data not shown). After lyophilization, each peptide was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/ml, and stored at -80 before use. We also synthesized a series of altered peptides with a single amino acid substitution on the epitope peptide by the same methods.

Antigen-induced proliferative responses of cloned T cells.
Proliferative responses of cloned T cells were tested by uptake of 3[H]-TdR, as described elsewhere (20). Briefly, T cell clones (1 104 /well) were incubated with MMC-treated autologous PBMC (1 105 /well) as APC and antigens in 200l of medium in a 96-well U-bottom culture plate for 3 days. For the final 16 hours incubation, 1Ci 3H-thymidine (ICN Biomedicals Inc, CA, USA) was added to each culture. After the cells were harvested, incorporation of 3H-thymidine was measured using liquid scintillation counter (Aloka Co., Tokyo, Japan). All cultures were set up in triplicate.

Binding affinity of altered peptide to APC
The binding affinity of altered peptides to MHC was assessed by the inhibitory activity of each altered peptide against binding of the original epitope peptide to APC, as described by Ikagawa et al. (23) with some modifications. Briefly, PBMC (6 105 /well) was incubated with 200M of each altered peptide in 96 well U-bottom plates for four hours. Two micromoler of biotinylated original epitope peptide and the same quantity of FITC-conjugated anti human HLA-DQ antibody (Leinco Technologies, St.Louis, MO, USA) were added to PBL, and the cells were further incubated for four hours. T- and non T-cells in PBL were distinguish by positive or negative binding of anti human HLA-DQ antibody. Then, they were harvested and stained with B-Phycoerythrin-conjugated Streptavidin (Immunotech, Marseille, France). The fluorescence intensity of two colours was measured by FACScan.

RESULTS
Cha o 1-specific T cell clone, ST12
A T cell clone, designated as ST12, was CD3+, CD4+, CD8- and TCR /+ (data not shown), and showed Cha o 1-specific proliferative response (Table 1). ST12 produced IL-4, but no detectable IFN-, in response to semi-purified Cha o 1 and r-Cha o 1 in vitro, indicating that ST12 was a Th2 clone. The HLA class II genotype of this donor was DRB1*0405/0409, DQA1*0302, DQB1*0301 (data not shown).

T cell epitope of ST12
Epitope analysis was done for ST12 cells, and we observed fine specificity ST12 cells to Cha o 1:202-217 at testing synthetic peptide-driven proliferation in vitro (Table 2). Therefore Cha o 1:202-217(GHSDIYSDDKSMKVTV) was used as a T cell of this clone for subsequent experiments. The sequence does not contain reported motif of HLA-DRB1*0405 or HLA-DRB1*0409-binding peptides. This clone also proliferated at stimulation with a homologue region peptide of Cry j 1, Cry j 1:202-217 (Data not shown).

Proliferative responses of ST12 with Cha o 1:202-217-derived altered peptides.
To determine important amino acid residues on the epitope peptide for its activity, we synthesized a series of altered peptides with nonconservative single amino acid substitution at every amino acid residue on the Cha o 1:202-217. In these substitutions, each hydrophilic residue was substituted with hydrophobic alanine, while each hydrophobic residue was substituted with hydrophilic serine. Because alanine and serine have only very small side chains, these substitutions effectively assess the role of the original amino acid side chain. The proliferative responses of ST12 with 0.1M each altered peptide were assessed. Compared with the original epitope peptide, the peptide-induced proliferative response was markedly decreased in D205A (an altered peptide of Cha o 1:202-217 with a substitution to Alanine at aspartic acid), D210A, S212A, M213S, K214A, V215S, T216A and V217S . In particular in K214A, V215S and T216A, the response almost disappeared (Fig. 1).

Binding affinity of altered peptides to APC
To assess in which site these residues played an important role, anchoring to the T cell and/or APC site, we tested the binding affinity of altered peptides to APC, which are HLA class II-positive population in PBMC. A cell, of which fluorescence intensity was more than 100 in FACScan analysis, was assigned as positive binding to the biotinylated
epitope peptide. The pre-incubation with peptide, except T216A, inhibited the binding of biotinylated original peptide (Table 3). This results suggested that the 216T of the Cha o 1:202-217 was an important residue for binding to MHC.

DISCUSSION
In the present study, we identified one of the T cell-recognizing epitopes of Cha o 1, the major allergen of Japanese cypress in humans. We used a simple screening method of peptide-MHC class binding for estimating T cell-recognizing epitopes. The identified epitope was located in the bind-positive regions. Recently, Cha o 1:209-228 was reported as a T cell epitope of Cha o 1 in B10.S mice (24). In case of T cell-recognizing epitopes of Cry j 1 of which amino acid sequence is highly similar to Cha o 1 (79-80%), Cry j 1:211-225 was shown to be one of major T cell epitopes because this peptide stimulated T cells from 72% of pollinosis patients. The determined epitope, Cha o1:202-217, recognized by the human Th2 cells overlapped with the Cry j 1 epitope. Although cross-allergenicity between Japanese cedar and cypress has been already suggested at the antibody level (10), the present report shows that this cross-allergenicity exists even on a T cell level in humans. In addition, our altered peptide study showed that the three residues of Cha o 1:202-217 (214K, 215V and 216T) were important for inducing proliferative responses of ST12 cells. Therefore the neighboring residues seem to be responsible for the cross reactivity of Cry j 1 and Cha o 1, although T cell epitopes depend on MHC alleles. In the present study, we tested only one T cell clone, and it is needed to study the case of other MHC alleles.
There are two functional sites of amino acid residues on epitope peptides, although these are not clearly separated. One is important site for binding to TCR (T cell epitope), and the other is site for binding to MHC (MHC anchor). Since it has been reported that altered peptides result in T cell anergy (25) and increase IFN- production from T cells (23), altered peptides have been well studied, because these phenomena indicate the possibility that peptide immunotherapy could become more effective by using altered peptides. To develop a useful altered peptide for immunotherapy, it will be important to find amino acid residues which effectively alter effects of T cells. Binding motif for various HLA-DR alleles has been reported. In our present study, the sequence was not fit for the reported motif for HLA-DRB1*0405 or HLA-DRB1*0409, this might suggest a possibility of exceptional peptide for such HLA-DR alleles, or a possibility of HLA-DQ-binding is not ruled out. In our study, the 216T residue was shown to be one of MHC anchors, because T216A did not bind to APC. The residues of 214K and 215V were mainly positioned in the TCR site, because K214A and V215S did not stimulate T cells but partially bound to APC. In this sense, these TCR-site residues might be the targets of molecules modification leading to their partial agonist. More detail studies on the possible TCR site residues may lead to the development of useful peptides for immunotherapy.

ACKNOWLEDGEMENTS
The authors wish to thank Dr. K. Kino, Meiji Institute of Health Science, for his kind gift of recombinant Cha o 1. We also thank Dr. Y. Yabu, Dr. J. Fu and Ms. M. Hato for their technical assistance. This study was supported in part by grant from Japan Society for the Promotion of Science (No.13877039) .

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TABLES
Table 1. Cha o 1 specificity of ST12.
Table 2. Epitope analysis of ST12.
Table 3. Inhibitory activity of each altered peptide against binding of biotinylated original epitope peptide (Cha o 1:202-217) to APC

FIGURE LEGEND
Fig.1. Proliferative responses of ST12 with Cha o 1:202-217-derived altered peptides. ST12 (1 104 /well) were incubated with MMC-treated APC (1 105 /well) and 0.1M of each altered peptide for 3 days. For the final 16 hours incubation, 1mCi 3H-thymidine was added to each culture. After the cells were harvested, incorporation of 3H-thymidine was measured. All cultures were set up in triplicate.

Nagoya City University
Medical School