T cell responses to 53-kDa outer membrane protein of porphyromonas gingivalis in humans with early-onset periodontitis

T Cell Responses to 53-kDa Outer Membrane Protein of Porphyromonas gingivalis in Humans with Early-Onset Periodontitis Hideki Ohyama, Sho Matsushita, Nahoko Kato, Fusanori Nishimura, Kosuke Oyaizu, Susumu Kokeguchi, Hidemi Kurihara, Shogo Takashiba, Yasuharu Nishimura, and Yoji Murayama ABBREVIATIONS Ag53 53-kDa outer membrane protein of P. gingivalis APC antigen presenting cells(s) EOP early-onset periodontitis HLA human leukocyte antigen HPLC high performance liquid chromatography

mAb MBP PBMC PCR-RFLP P. gingivalis TCR

monoclonal antibody maltose binding protein peripheral blood mononuclear cell(s) polymerase chain reaction-restriction fragment length polymorphism Porphyromonas gingivalis T cell receptor

ABSTRACT: Patients with early-onset periodontitis (EOP) are susceptible to infection with periodontopathic bacteria, such as Porphyromonas gingivalis. Ag53, 53-kDa outer membrane protein of P. gingivalis, evokes strong humoral immune responses in EOP patients. In a first step to clarify how host immune cells recognize Ag53, we established Ag53-specific short-term T cell lines from 22 subjects including 6 EOP patients and 16 healthy donors, using overlapping peptides based on Ag53 amino acid sequences. All T cell lines from active EOP patients recognized a common region (p141–181, especially p141–161) on Ag53, while those from healthy donors

showed heterogeneous specificity. p141–181 was not recognized by T cell lines established from EOP patients following therapy. A monoclonal antibody to HLA-DRB1 inhibited Ag53-induced proliferation of most of the T cell lines. Our observations suggest that, although antigenpresenting molecules are common in EOP patients and in healthy individuals, p141–161 includes a major T cell epitope(s) on Ag53 for active EOP patients but not for healthy individuals or inactive EOP patients. Human Immunology, 59, 635– 643 (1998) © American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Science Inc.

INTRODUCTION One of the major causes of tooth loss in adults is periodontal disease which results from loss of periodontal

attachment and resorption of alveolar bone [1]. The disease involves invasion of oral bacteria into periodontal tissue. There are individual differences in onset and progression, which means polymorphism in susceptibility to this disease [2, 3]. Severe destructive early-onset forms of periodontal disease (early-onset periodontitis: EOP) such as juvenile periodontitis, are aggregated in some families, thereby suggesting involvement of genetic factors [4 – 8]. Immune responsiveness to periodontopathic bacteria is likely to be one of these factors [9 –11] since aggregation in families seems to be linked to HLA class II phenotypes, in different races. Among these bacteria, Porphyromonas gingivalis (P. gingivalis), one

From the Department of Periodontology and Endodontology, Okayama University Dental School, Okayama, Japan (H.O., N.K., F.N., K.O., S.K., S.T., Y.M.), the Division of Immunogenetics, Department Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan (S.M., Y.N.), and the Department of Endodontology and Periodontology, Hiroshima University School of Dentistry, Japan (H.K.). Address reprint requests to: Dr. Yoji Murayama, Department of Periodontology and Endodontology, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan; Tel: 181-86-235-6675; Fax: 181-86-235-6679; E-Mail: [email protected]. Received April 7, 1998; revised June 1, 1998; accepted June 2, 1998. Human Immunology 59, 635– 643 (1998) © American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.

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TABLE 1 HLA class II alleles of the subjects used in this study Subjects

Age

Sex

Diag.

DRB1*

DQA1*

DQB1*

DPB1*

H.S. T.K. Y.S S.F T.H H.I N.K M.N H.O M.A T.S K.O E.K M.K H.A H.H T.K S.K C.F K.N Y.A J.K

20 40 24 29 32 22 26 35 29 24 30 28 28 31 38 31 29 40 29 27 29 27

M M F M M M F M M M M M M M M M M M F M M M

EOP EOP EOP EOP EOP EOP Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy

1602/0901 0101/0901 1502/0410 0101/1201 0803/1501 0405/0406 1502/0901 1502/0405 1502/1101 1501/0901 0405/0901 1405/0901 0803/0901 0803/0803 0405/0406 0101/0410 0101/0406 0101/0406 1401/0410 0101/1501 1501/1302 1501/0901

0101/0301 0101/0301 0103/0301 0101/0501 0103/0102 03/03 0103/0301 0103/0301 0103/0501 0102/0301 0301/0301 0101/0301 0103/0301 0103/0103 0301/0301 0101/0301 0101/0301 0101/0301 0101/0301 0101/0102 0102/0102 0102/0301

0502/0303 0501/0303 0601/0402 0501/0301 0601/0602 0401/0302 0601/0303 0601/0401 0601/0301 0602/0303 0401/0303 0503/0303 0601/0301 0601/0601 0401/0302 0501/0402 0501/0302 0501/0302 0503/0402 0501/0602 0602/0604 0602/0303

0501/1401 0201/0501 0901/0201 0201/0301 0201/0202 0501/0201 0201/0501 0501/0501 0901/0201 0501/0301 0501/0501 0901/0201 1301/0501 0501/0201 1401/0201 0402/0501 0402/0201 0201/0202 0301/0501 0201/0301 0401/0202 0201/0201

of the most frequently implicated pathogens in periodontal diseases, has various possible proteins recognized by human T cells [12, 13], including 53-kDa outer membrane protein antigen (Ag53), a major protein reacting with sera from patients with periodontal diseases [14, 15]. The initiation of antigen specific immune responses includes T cell recognition of an antigenic peptide-MHC complex via TCR. In particular, the exogenous antigens presented by MHC class II molecules activate CD41 helper T cells which derive effector functions through various biological activities of secreted lymphokines. To determine if T cell-derived effector functions induced by Ag53 are desirable, it is essential to determine the specific region(s) of this antigen protein recognized through TCR. We noted an association between HLA class II alleles and the susceptibility to EOP [16]. We suggested that the antigen specific immune responsiveness to the major T cell epitope of periodontopathic bacteria is controlled by polymorphism of HLA class II alleles. In the present study, we attempted to clarify how Ag53 is involved in the immunological aspects of periodontal diseases and we established Ag53-specific shortterm polyclonal T cell lines from PBMC to determine T cell epitopes of Ag53 and HLA class II molecules recognized by T cells from EOP patients and from healthy individuals.

DRB3*

DRB4* 0103 0103 0103

DRB5* 02 0102

0101 0101 0103 0103 0103 0202 0202

0202

0103 0103 0103 0103

0102 0102 0101 0102

0103 0103 0103 0103 0103

0301 0103

0101 0101 0101

MATERIALS AND METHODS PBMC Donors Six Japanese EOP patients (5 men and one woman, average age: 27.8 6 7.4 years) and 16 healthy Japanese individuals (14 men and 2 women, average age: 30.1 6 4.3 years) were enrolled in the study. EOP was diagnosed based on the description of Murayama et al. [17]. The healthy individuals were selected by their HLA class II haplotypes which are frequently observed in the Japanese [18]. Results of HLA DNA typing of these 22 subjects are shown in Table 1. HLA class II typing (DRB1, DRB3, DRB5, DQA1, DQB1, and DPB1) was performed using the PCR-RFLP method described elsewhere [19 –22]. The nomenclature of the HLA-DR, DQ, and DP alleles followed the WHO Nomenclature Committee for factors of the HLA system [23]. In no subject was there an abnormal response to PHA. Preparation of MBP-Ag53 Maltose binding protein-Ag53 fusion protein (MBPAg53) was generated using a Protein Fusion and Purification System (New England Biolabs Inc., Beverly, MA). Briefly, cDNA encoding the entire reading frame of Ag53 [24] was amplified by PCR, using the following primers; 59-ATGAAGTTAAACAAAATGTTTTTGGT -39 (forward), 59-CACCTCAAGCTTTTAGAATTCG ATA-39 (reverse) (the Hind III site is underlined). The Hind III-cleaved PCR-amplified fragment was then li-

T-Cell Epitopes of 53-kDa OMP on P. gingivalis

gated to the Hind III site of the pMAL-c2 plasmid vector to generate pMAL-Ag53. E. coli strain TB1 transformed with pMAL-Ag53 was grown overnight in Luria-Bertani medium at 37°C and MBP-Ag53 fusion protein was expressed in these cells, according to the method described by Weller et al [25]. These cells were then sonicated after harvesting by centrifugation and cellular proteins were loaded onto an amylose resin column (New England Biolabs Inc.), as described by Garnier et al. [26] to obtain a fraction containing the fusion protein. MBPAg53 was purified from this fraction by ion-exchanged chromatography followed by gel-filtration chromatography, and identified as a single band on SDS-PAGE.

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TABLE 2 Characterization of T cell lines established in this study T cell lines HS TK YS SF TH HI NK MN HO MA TS KO EK MK HA HH TK SK CF KN YA JK

Synthesis of Ag53-Derived Peptides Peptides were synthesized based on the amino acid sequence of Ag53 reported by Hongyo et al. [24], using a solid-phase simultaneous multiple peptide synthesizer PSSM-8 (Shimadzu, Kyoto, Japan), based on F-moc strategy. All peptides were purified using C18 reversephase HPLC (Waters, Milford, MA). Generation of Antigen-Specific Short-Term T Cell Lines Ag53-specific T cell lines were established from PBMC. T cell lines were generated by stimulating PBMC (1 x 105/well) with 5 mg/ml of MBP-Ag53 in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine, 100 units/ml of penicillin, 100 mg/ml of streptomycin, and 10% pooled, heat-inactivated autologous plasma in 96-well flat-bottomed culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ). After 7–9 days, irradiated (3,000 cGy) autologous PBMC (1.5 x 105/well) pulsed with mixed-overlapping peptides of Ag53 (0.5 mM each), human rIL-2 (50 U/ml), and human rIL-4 (10 U/ml) were added to the culture wells carrying T cell blasts and these cells were maintained for an additional 7 days. After specificity assay against the mixed-overlapping peptides of Ag53, aliquots of growing cultures with specificity to Ag53, were combined as a bulk T cell line. Antigen-Induced Proliferative Responses of T Cell Lines Antigen-induced proliferation of the T cell lines was assayed by culturing the T cells (3 x 104/well) in 96-well flat-bottomed culture plates in the presence of the mixed-overlapping peptides of Ag53 (0.1 mM each), and 3,000 cGy-irradiated autologous PBMC (1.5 x 105/well). Cells were cultured for 72 h in the presence of 1 mCi/well of [3H]TdR during the final 16 h period, and following cell harvest, the incorporated radioactivity was measured using Microbeta (Pharmacia Biotech, Uppsala, Sweden). To determine restriction molecules for antigen presenta-

Number of positive linesa 6 4 3 4 4 5 3 4 5 4 4 3 4 2 3 4 2 6 3 2 2 2

a

Number of positive lines was the number of cultures responsive to a mixture of Ag53 peptides out of 60 cultures. Each lines were composed by positive lines pooled together.

tion, the T cell lines were cultured with irradiated autologous PBMC, with or without saturating amounts of anti-HLA class II monoclonal antibodies (mAbs) HU-4 (anti-HLA-DRB11DRB5 monomorphic) [27, 28], HU-11 (anti-HLA-DQ11DQ4) [29], HU-18 (antiHLA-DQ3) [30], HU-46 (anti-HLA-DQ4) [31], or B7/21 (anti-HLA-DP monomorphic) [27]. RESULTS T Cell Epitopes of Ag53 Recognized by Polyclonal T Cell Lines T cell lines specific to Ag53 from PBMC of 22 subjects, including 6 EOP patients and 16 healthy subjects, were established (Table 2). Two to six positive lines were combined for each subject, however the frequency of precursor cells in EOP patients and healthy controls was practically the same. T cell lines from 6 EOP patients (Fig. 1A) showed marked proliferative responses to 12 overlapping peptides of Ag53 (Ag53 p1-21, 141-161, 151-171, 162181, 229-249, 239-259, 268-287, 287-307, 307-327, 333-353, 343-363, 363-383), and those from healthy subjects (Fig. 1B) did so to 20 peptides (Ag53 p1-21, 71-91, 81-101, 91-111, 101-121, 111-131, 141-161, 151-171, 162-181, 181-201, 191-211, 229-249, 249-

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FIGURE 1 Proliferative responses of short-term T cell lines established from EOP patients (A) and healthy donors (B) to overlapping peptides of Ag53. Results are expressed as the mean cpm of duplicate determinations 6 standard error. “Backgraund” indicates proliferative responses of T cell lines without peptides (a). “Mix” indicates proliferative responses of T cell lines to the mixture of 45 peptides of Ag53 (b).

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269, 268-287, 277-297, 307-327, 333-353, 343-363, 353-373, 397-416). T cell lines from all EOP patients recognized a limited number of peptides in the locus of amino acid residues from 141 to 181 of Ag53; in particular 4 out of 6 T cell lines showed proliferative response to Ag53 p141-161. There was no peptide species

T-Cell Epitopes of 53-kDa OMP on P. gingivalis

639

responses were measured in the presence of anti-HLA class II mAbs. As shown in Fig. 4, proliferative responses of 16 of 22 T cell lines, including 4 EOP patients (TK, YS, SF and TH) and 12 healthy subjects (MN, MA, TS, EK, MK, HA, HH, TK, SK, CF, KN and JK), were inhibited by HU-4 [27, 28], by over 50%. Peptidedriven responses of YS, SF and TH lines from EOP patients, and EK, MK, HA and HH lines from healthy donors were almost completely inhibited by anti-HLADRB11B5 antibody. There was no T cell line the proliferative response of which was inhibited by over 50%, by the addition of HU-11 [29], HU-18 [30], HU-46 [31] and B7/21 [27] mAbs, even though these amounts of mAbs were sufficient to inhibit antigen presentation by APC [27, 29 –31]. The isotype-matched control had no inhibitory effects in these conditions (data not shown).

FIGURE 2 Peptides recognized by T cell lines from EOP patients (1) and healthy donors (s). Horizontal axis means the percentage of numbers of T cell lines showing the proliferative response to each peptide, in respective groups. Because proliferative responses of medium alone were highly diverse among individuals, “positive response” was judged as follows: (cpm with a peptide - cpm with medium alone) / cpm with medium alone . 5.

recognized in common by most of the T cell lines from healthy subjects (Fig. 2). To evaluate the effect of clinical intervention on T cell epitopes, T cell lines from EOP patients, SF and YS, were established following 6 months of periodontal treatment (chemotherapy and debridement of the lesion). As shown in Fig. 3, the peptides to which T cell lines from patients (SF and YS lines) showed the most intensive responses, changed 6 months after the treatment, whereas those peptides of healthy donors (NK and MA lines) were preserved even after 6 months. T cell lines from patients had no proliferative response to peptides covering amino acid residues from 141 to 181, following periodontal therapy. In striking contrast, the T cell line from a healthy subject (MA line) showed proliferative response to p141–161 on Ag53, as it did 6 months earlier. Antigen-Presenting HLA Class II Molecules To determine major antigen-presenting HLA class II molecules in T cell response to Ag53, T cell proliferative

DISCUSSION T cell proliferation assays were done using synthetic peptides. These T cell lines could also recognize processed protein (data not shown), since these lines were generated by stimulating PBMC with MBP-Ag53 fusion protein. Ag53 p141–161 was commonly recognized by 4 out of 6 T cell lines established from EOP subjects. However, a common HLA class II allele was not found in the 4 EOP donors. This suggests that Ag53 p141–161 is not presented by a common HLA class II allele among patients, and/or this region contains a cluster of many T cell epitopes. The 3 T cell lines established from healthy subjects recognized peptide Ag53 p141–161. These T cell lines were established from donors carrying the HLADRB1*1501-DQB1*0602 or HLA-DRB1*1401DQB1*0503 haplotype, which we earlier found to be associated with Japanese EOP patients [16]. T cell responses to this peptide were restricted by these haplotypes in these 3 T cell lines, in a manner similar to findings in EOP patients, since these lines showed proliferative responses when allogenic PBMC carrying HLADRB1*1501-DQB1*0602 (MA and JK lines) or HLADRB1*1401-DQB1*0503 (CF line) haplotype were used as APC (data not shown). This suggests that (a) helper T cells carrying HLA-DRB1*1501-DQB1*0602 and/or DRB1*1401-DQB1*0503 haplotypes are likely to respond to Ag53 p141–161; and (b) other genetic or environmental factors are involved in the development of EOP. We then did a homology search for p141–161, p151–171 and p161–181, but found no homologous peptide fragments in other antigens of microorganismorigin. Allele-specific peptide-binding motifs have been an-

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FIGURE 3 Changes of the region of Ag53 recognized by T cell lines as periodontal therapy was continued. The loci recognized by T cells established in the different terms were detected. In EOP patients, T cell lines were established prior to initiation of periodontal therapy (a), and after termination of therapy (6 months after first visit) (b). In healthy donors, T cell lines were established first (c), and 6 months after the first establishment (d).

alyzed [32, 33] and we speculated that peptide ligands could possibly bind with individual HLA molecules. Indeed, Ag53 p141–161 includes motifs which may explain binding to heterogeneous HLA class II allelic products, as shown in Table 3 [34]. Nevertheless, among 10 T cell lines established from healthy donors carrying these haplotypes, the lines established from DRB1*1501-bearing individuals alone (MA and JK) showed proliferative responses to Ag53 p141–161. We consider that (a) Ag53 p141–161 binds strongly to

H. Ohyama et al.

DRB1*1501; or (b) healthy donors have an immunological tolerance to Ag53 p141–161. Ag53 p141–161, where T cell lines from EOP patients showed a marked proliferative response, could not be recognized by T cell lines established from treated EOP patients, which means suggesting that the region probably includes a major T cell epitope(s) with which one could assess disease activity. This specific question will be addressed, using a larger number of patients and controls before and after required treatments. These phenomena could be explained either by (a) the reduction of Ag53 antigen protein in the lesion following treatment, which included chemical and mechanical bacterial elimination using antibiotics and debridement; or (b) “epitope spreading” as reported by McRae et al. [35]. It is conceivable that elimination of bacteria by treatment could reduce the clonal size of T cells specific to Ag53 p141–181, especially p141–161. The effector functions of these T cells specific to this region, e.g. lymphokine

T-Cell Epitopes of 53-kDa OMP on P. gingivalis

FIGURE 4 Inhibition of proliferative responses of T cell lines to overlapping peptides of Ag53 from EOP patients (A) and healthy donors (B), by using anti-HLA class II mAbs. HU-4 (anti-HLA-DRB11DRB5 monomorphic), HU-11 (anti-HLA-DQ11DQ4), HU-18 (anti-HLA-DQ3), HU-46 (antiHLA-DQ4), or B7/21 (anti-HLA-DP monomorphic) were cocultured with T cells, irradiated autologous PBMC and Ag53 peptides mixture. All mAbs in ascites forms were tested at a final dilution of 1:200, which corresponds to 15–25 mg/ml of mAbs.

production, might play a role in the pathogenesis of EOP. If such is indeed the case, it may be that donors carrying HLA-DRB1*1501-DQB1*0602, whose T cell line exhibited marked proliferative response to p141– 161, (e.g. healthy subject M.A) are more susceptible to EOP. Interestingly, the region from 141 to 155 of Ag53 p141–161 was recognized by IgG in many EOP patients’ sera [36], which corroborates observations by others [37] that B cell epitopes locate near T cell epitopes. TABLE 3 Motifs which could bind to HLA class II molecules on Ag53 p141–161 HLA class II alleles DRB1*0101 DRB1*0405 DRB1*1201 DRB1*1501

Peptide sequence NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ NKVPIIVKRAAIRASMTITQQ

Alignment was on the anchor at position 1 of the motifs. Residues indicated in underlined bold type correspond to the report by the Rammensee et al. [24}.

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In our study, many T cell lines specific to Ag53 were restricted by HLA-DR molecules. However, none of anti-HLA class II mAbs markedly inhibited proliferative responses of some T cell lines (EOP patients of HS and HI, and healthy donors of HO and KO). These results suggest that: (a) there is no difference between EOP patients and healthy donors in HLA restriction patterns; and/or (b) HLA-DRB3 and DRB4 gene products, whose antigen-presenting function cannot be blocked by the mAbs used in our study, may be involved in presentation for this protein. Although the results in this study suggest that DRB1 may be major antigen presenting molecule in the response to Ag53, our earlier work indicated that the HLA-DQB1 molecule might primarily confer the susceptibility to EOP [16]. DQB1 molecules may be related to the immune suppression [38 – 40]. Indeed, an increase in Dcpm was observed in T cell lines from EOP patient SF, and healthy donors NK, MA, MK, HH and TK in the presence of anti-HLA-DQ antibodies. However, it is not clear at this stage how T cell responses induced by the antigen complexed with HLA-DQ molecules participate in the host defense against P. gingivalis. Further study is under way, for further detailed characterization of T-cell responses to this antigenic peptide, including cytokine production, cytotoxic activity, helper activity and expression of cell surface effector molecules. ACKNOWLEDGMENTS

We thank Drs. S. Ohkuma and Y. Hirata for HLA DNA typing. This work was supported in part by Grant-in-Aid for

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Scientific Research (B: No. 09470425), Grant-in-Aid for Encouragement of Young Scientists (No. 09771668) from the Ministry of Education, Science, Sports and Culture, Grant for Cooperative Research of Hansen’s Disease from the Ministry of Health and Welfare of Japan, and Grant for US-Japan Cooperative Medical Science Program Leprosy Panel.

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15.

16.

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