Stem Cells
Vol. 23 No.
7
August 2005, pp.
975
-982
doi:10.1634/stemcells.2004-0227; www.StemCells.com
© 2005 AlphaMed Press
Determination of Thrombopoietin-Derived Peptides Recognized by Both Cellular and Humoral Immunities in Healthy Donors and Patients with Thrombocytopenia
Hiroko Takedatsua,
Kohji Yoshimotob,
Takashi Okamurab,
Hiroshi Miyazakic,
Tomoaki Kuwakic,
Michio Satab,
Kyogo Itoha
a Department of Immunology and
b 2nd Department of Internal Medicine, Kurume University School of Medicine, Kurume, Fukuoka, Japan;
c Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan
Key Words. Human • T cells • Epitope • Thrombopoietin • Thrombocytopenia • Autoimmunity
Correspondence: Hiroko Takedatsu, M.D., Ph.D., Department of Immunology, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan. Telephone: 81-942-31-7551; Fax: 81-942-31-7699; e-mail: takedatu{at}med.kurume-u.ac.jp
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ABSTRACT
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Thrombopoietin (TPO) is a cytokine that promotes megakaryocytopoiesis and thrombopoiesis and is considered a drug suitable for patients with thrombocytopenia. However, unexpected severe thrombocytopenia has developed in some healthy individuals participating in phase I clinical trials with a pegylated recombinant human megakaryocyte growth factor (PEG-rHuMGDF) that contained the first 163 amino acids of endogenous TPO, which resulted in hampering the further development of clinical trials. Autoimmune responses to PEG-rHuMGDF, which cross-reacted with endogenous TPO, were suggested to be involved in this rare but severe adverse event, although the immunogenic epitopes have not yet been determined. To better understand the molecular basis of such autoimmune reactions, we investigated the reactivity of 18 TPO-derived peptides with HLA-A2–binding motifs to plasma and T cells, both from patients with thrombocytopenia (n = 24) and from healthy donors (HDs) (n = 24). Four peptides, including those possessing amino acids in receptor-binding sites, were preferentially reactive to plasma from at least 20% of the patients, whereas one peptide at position 101–109 was equally reactive to those of the patients and the HDs. Each of the five peptides had the ability to induce peptide-specific cytotoxic T lymphocytes (CTLs) in both groups, albeit with less frequency among the patients. More important, each of these five peptides had the ability to induce HLA-A2–restricted and peptide-specific CTL activity reactive to cells that produce TPO. These results may provide new insights to gain a better understanding of autoimmune reactions to TPO.
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INTRODUCTION
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Thrombopoietin (TPO), the c-Mpl ligand, is a key regulator of platelet production [1]. TPO stimulates the growth of committed megakaryocyte progenitors, the progressive maturation of megakaryocytes, and proplatelet formation. TPO is synthesized primarily in the liver as a single 353–amino acid precursor protein. On removal of the 21–amino acid signal peptide, the mature molecule consists of two domains that show considerable homology to erythropoietin and a carbohydrate-rich carboxy-terminus of the protein that is highly glycosylated and important in maintaining protein stability [2]. The production of TPO is elevated in patients with several thrombocytopenic disorders but not in those with immune thrombocytopenic purpura (ITP) [3, 4]. Two recombinant TPOs were provided for use in extensive clinical trials. One of these TPOs was recombinant hTPO (rHuTPO), a glycosylated molecule with an identical amino acid sequence to that of endogenous TPO, whereas the other was pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a nonglycosylated molecule that contained the first 163 amino acids of endogenous TPO, that is, the biologically active domain, which was coupled to polyethylene glycol [1, 2, 5]. Both reagents are potent stimulators of platelet production in humans and thus have the ability to rescue the extent of thrombocytopenia associated with chemotherapy, thus providing the potential advantage of reducing the need for platelet transfusions [6, 7]. However, in clinical studies conducted during the past decade, PEG-rHuMGDF induced antibodies that cross-reacted with endogenous TPO, and severe thrombocytopenia persisted in 4% of healthy volunteers and 0.6% of oncology patients who received intensive chemotherapy [5]. It was of note that immuno-competent individuals were likely to become thrombocytopenic at lower doses, since healthy volunteers became thrombocytopenic after receiving two or three administrations whereas oncology patients became thrombocytopenic after receiving multiple administrations [2]. Such results suggest that autoimmune reactions are responsible for the observed adverse effects. However, the immunogenic epitopes of TPO recognized by host T cells have not yet been determined. We previously reported that antibodies reactive to cytotoxic T lymphocyte (CTL) epitope peptides derived from cancer-associated self-antigens were detected in healthy donors (HDs), patients with atopic disease, and cancer patients [8–10]. Immunoglobulin G (IgG) reactive to certain CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients [9]. In contrast, increases in the levels of IgG to such peptides have been shown to be well-correlated with the overall survival of cancer patients vaccinated with these peptides [10]. These results suggest the positive role of IgG reactive to CTL epitope peptides in patients with atopic diseases and cancer patients. To better understand the immunogenic epitopes of PEG-rHuMGDF, we investigated in the present study the reactivity of 18 TPO-derived peptides with HLA-A2–binding motifs to plasma and T cells, both from patients with thrombocytopenia (n = 24) and from HDs (n = 24), respectively; we then report five such epitope peptides.
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MATERIALS AND METHODS
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Subjects
Twenty-four thrombocytopenic patients and 24 HDs were enrolled in this study after written informed consent was obtained. None of these participants was infected with human immunodeficiency virus (HIV). Peripheral blood mononuclear cells (PBMCs) and plasma samples were prepared from 20 ml of heparinized blood by Ficoll-Conray density-gradient centrifugation. The expression of HLA-A2 molecules on the PBMCs of patients and HDs was determined by flow cytometry, as previously reported [11]. The characteristics of thrombocytopenic patients and HDs are shown in Table 1
. The patients included 14 with ITP, 6 with aplastic anemia (AA), 2 with myelodysplastic syndrome (MDS), 1 with prostate cancer–related bone metastasis, and 1 primary splenomegaly patient (Table 1). The median age of these patients was 57.4 years, which was higher than that of healthy volunteers. The male-to-female ratio was smaller in the ITP and AA groups, as is generally recognized. Because of the sample limitation, the median age was lower and the male-to-female ratio was higher in HDs compared with patients, causing the comparison to be biased. Although we must consider the results carefully, the influence of these differences in this study might be limited. There was no significant difference in terms of platelet count and disease category. The serum TPO level was high in AA patients compared with that of others with thrombocytopenia and HDs, which would be expected based on previously reported observations [12]. None of the patients had antibody against rhTPO protein (data not shown).
Peptides
Eighteen TPO-derived peptides were prepared based on their capacity for binding to HLA-A0201 molecules according to a computer analysis (Bioinformatics and Molecular Analysis Section, National Institutes of Health, Bethesda, MD, http://thr.cit.nih.gov); these peptides are listed in Table 2. All of these peptides were of >90% purity and were purchased from Biologica (Nagoya, Japan, http://www.biologica.co.jp). HIV-derived peptide with an HLA-A2–binding motif was used as a negative control (SLYNTVATL) [11]. All peptides were dissolved with dimethyl sulfoxide at a dose of 10 mg/ml.
Detection of Peptide-Specific IgG
The plasma levels of IgG reactive to the peptides were measured by enzyme-linked immunosorbent assay (ELISA), as previously reported [8]. Briefly, a peptide (20 µg/well)-immobilized plate was blocked with Block Ace (Yukijirushi, Tokyo, http://www.snowbrand.co.jp) and washed with 0.05% Tween 20–phosphate-buffered saline. One hundred microliters per well of plasma diluted with 0.05% Tween 20-Block Ace was added to the plate. After a 2-hour incubation at 37°C, the plate was washed and further incubated for 2 hours with a 1:1000-diluted rabbit anti-human IgG antibody (
-chain–specific; DAKO, Glostrup, Denmark). The plate was washed again, 100 µl of 1:100-diluted horseradish peroxidase-conjugated goat anti-rabbit Ig antibody (DAKO) was added to each well, and the plate was incubated for 40 minutes. Then the plate was washed once again, 100 µl per well of tetramethyl benzidine substrate solution (KPL, Guildford, U.K., http://www.kpl.com) was added to the plate, and the reaction was stopped by the addition of 1 M phosphoric acid. To estimate peptide-specific IgG levels, the optical density (OD) values of each sample were compared with those of serially diluted standard samples, and the values are shown as OD units (Figs. 1–3). The cut-off value of the peptide-specific IgG was determined using HIV peptide as the solid-phase antigen. To confirm the specificity of IgG to the peptide, we cultured 100 µl of sample in the peptide-coated wells to absorb peptide-specific IgG, and we determined the levels of peptide-specific IgG in the resultant sample.
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Figure 1. Detection of plasma levels of IgGs reactive to peptides. The representative enzyme-linked immunosorbent assay results of two reproducible experiments are shown. Serially diluted plasma samples from thrombocytopenic patients and healthy donors were individually examined. The representative IgG reactions, which are considered positive and negative to peptides, and an HIV peptide are shown as OD unit. The background OD value of cross-reactivity of IgGs to the peptide-uncoated wells was subtracted from the value. Abbreviations: IgG, immunoglobulin G; OD, optical density.
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Figure 2. Specificity of IgG reactive to peptide. To confirm the specificity of IgGs to the peptides, 100 µl of sample plasma from patients was incubated in a plate coated with the corresponding peptide, an irrelevant peptide, or an HIV peptide. Thereafter, the level of IgG reactive to the corresponding peptide in the resultant sample was determined. The representative results of two reproducible experiments are shown. A two-tailed Student’s t-test was used for the statistical analysis of the IgG reactivity. *p < .05 compared with the IgG reactivity of samples absorbed by the irrelevant peptide. Abbreviation: IgG, immunoglobulin G.
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Figure 3. IgG reactivity to TPO protein. The reactivity of peptide-specific IgG in the plasma from patients was absorbed by the immobilized corresponding peptide but not by the immobilized rhTPO or human albumin. One hundred microliters of sample plasma from patients was incubated in a plate coated with the corresponding peptide, the rhTPO protein, or human albumin. Thereafter, the level of IgG reactive to the corresponding peptide in the resultant sample was determined. The representative results of two reproducible experiments are shown. A two-tailed Student’s t-test was used for the statistical analysis of the IgG reactivity. *p < .05 compared with the IgG reactivity of samples absorbed by human albumin. Abbreviations: IgG, immunoglobulin G; TPO, thrombopoietin.
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Detection of Peptide-Specific CTL
PBMCs (1 x 105 cells/well) were incubated with 10 µM of each peptide in a U-bottom–type 96-well microculture plate (Nunc, Roskilde, Denmark, http://www.nuncbrand.com) in 200 µl of culture medium. The medium consisted of 45% RPMI-1640, 45% AIM-V (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 10% fetal calf serum (FCS), 100 U/ml interleukin (IL)-2, and 0.1 mM minimal essential medium (MEM) nonessential amino acid solution (Invitrogen). On the third, sixth, and ninth days, half of the medium was removed and replaced with new medium containing the corresponding peptide (20 µg/ml). On the 12th day of culture, the harvested cells were tested for their ability to produce interferon (IFN)- in response to HLA-A2–expressing T2 cells, which were preloaded with either a corresponding peptide or the HIV peptide, taken as a negative control. Four wells were prepared for each peptide, and the assays were performed in duplicate. The background IFN- production in response to the HIV peptide was subtracted from the values given in the data. The peptide-specific CTL was considered to be positive when the mean value of IFN- production by the peptide-stimulated PBMCs in response to a corresponding peptide was significantly (p < .05) higher than that produced in response to the HIV peptide. The production of IFN- was examined by ELISA, as reported previously [11]. For the inhibition assay, 100 ng each of HLA-A*0201 cDNA, which was inserted into the eukaryotic expression vector pCR3.1 (Invitrogen), or 100 ng of HLA-A*0201 cDNA with 100 ng of TPO cDNA, which was inserted into pEAK10CV (Edge BioSystems, Gaithersburg, MD, http://www.edgebio.com), was mixed in 100 µl of Opti-MEM (Invitrogen) with 0.3 or 0.6 µl of Fugene 6 (Invitrogen), respectively, and incubated for 30 minutes. The mixture was then added to the COS-7 cells (1 x 103 cells), which were then incubated for 3 hours. One hundred milliliters of RPMI 1640 containing 20% FCS was added, and COS-7 cells were cultured for 2 days. The peptide-stimulated CTLs were positively purified using a CD8 Isolation Kit (DYNAL A.S., Oslo, Norway, http://www.dynal.no), and their peptide-specific IFN- production in response to target cells was measured in the presence of 20 µg/ml of each monoclonal antibody (mAb).
Cytotoxicity Assay
The peptide-reactive CTLs were further cultured in the culture medium described above without peptides to obtain a sufficient number of cells to carry out a cytotoxicity assay. These cells were tested for cytotoxicity against the targets by the standard 51Cr-release assay, as reported previously [11]. The targets used in this study were T2 cells preloaded with peptides and Hep-G2 TPO-producing hepatocellular adenocarcinoma cell line (HLA-A 0201/2402, B 35/5105, C 0401/1602, DR 1302/1602, DQA1 0102, DQB1 0604/0502) [13].
Statistical Analysis
The two-tailed Student’s t-test was used to compare the levels of IgG, IFN- production, and percent lysis. A p value of less than .05 was considered significant.
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RESULTS
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Detection of IgG Reactive to TPO-Derived Peptide
We first investigated the levels of IgG reactive to 18 kinds of TPO-derived peptides in the patients (n = 24) and HDs (n = 24). Representative results showing positive and negative responses are shown in Figure 1
. A linear inverse correlation was observed between the OD values of peptide-specific ELISA and plasma dilutions. The cut-off value was set as 0.06 of the OD values (mean ± 2 SD) at a plasma dilution of 1:100, based on the fact that the mean ± 2 SD of the HIV peptide-specific IgG of HD was 0.02 ± 0.04. Under this condition, significant levels of IgG reactive to the TPO peptide at position of 101–110 (TPO-101) were detectable in eight (33%) and seven (29%) of the patients and HDs, respectively. IgG reactive to TPO peptides at positions 89–98 (TPO-89), 109–118 (TPO-109), 162–171 (TPO-162), and 164–173 (TPO-164) was detectable in five, six, five, and seven patients, whereas these levels were found in one, zero, two, and two HDs, respectively. IgG reactive to some other peptides was also detectable in a few patients, with much lower frequency in HDs, whereas IgG reactive to TPO-35 was detected only in HDs but not in the patients. The overall summary is given in Table 2. Collectively, the mean number of peptides recognized by TPO peptide–reactive IgG was 2.2 in ITP patients, 1.0 in AA patients, 3.8 in the patients with other thrombocytopenic disorders, and 0.8 in the HDs. Based on these results, six peptides (TPO-35, -89, 101, -109, -162, and -164) were intensively investigated in the following experiments.
Specificity of Peptide-Reactive IgG
The specificity of the peptide-specific IgG was confirmed by an absorption test using immobilized peptides. The representative results are shown in Figure 2. IgG reactive to each of the six peptides (TPO-35, -89, -101, -109, -162, and -164) was absorbed by the corresponding peptide but not by the irrelevant TPO-derived peptides or HIV peptide. IgG reactive to the other peptides (TPO-60, -94, -119,-191, and -201) was also absorbed by the corresponding peptide but not by a negative control (HIV) peptide (data not shown).
Anti-peptide IgG reactive to the CTL epitope usually failed to recognize the parent protein, as reported previously [11, 14]. We then considered whether the anti-TPO peptide–reactive IgG shown above would recognize TPO. As expected, the patients’ plasma containing the anti-TPO peptide IgG failed to react to the native (dot-plot method) and denatured (Western blot method) rhTPO protein (data not shown). Furthermore, the reactivity of anti-TPO peptide–reactive IgG was not absorbed by the rhTPO, whereas it was absorbed by the corresponding peptide. Representative results of IgG reactive to the six peptides (TPO-35, -89, -101, -109, -162, and -164) are shown in Figure 3. The present results did not indicate any reactivity of the anti-TPO peptide IgG to the whole TPO protein.
Induction of Peptide-Reactive CTLs by TPO-Derived Peptide
Next, the six peptides (TPO-35, -89, -101, -109, -162, and -164) were tested for their ability to induce HLA-A2–restricted CTL activity in PBMCs from 6 HLA-A2+ patients and 10 HDs. The PBMCs were repeatedly stimulated in vitro with each of the six TPO-derived peptides or with an HIV peptide as a control for up to 14 days, followed by a test of their ability to produce IFN in response to T2 cells prepulsed with a corresponding peptide or an HIV peptide as a negative control (Tables 3, 4). The TPO-35 peptide failed to induce peptide-reactive CTLs from any of the thrombocytopenic patients and HDs; thereafter, we used this peptide as a negative control. None of the patients or HDs reacted to the HIV peptide. In contrast, TPO-101, -109, -162, and -164 peptides induced peptide-reactive CTLs in one of five to six thrombocytopenic patients. CTL precursors to these peptides were also found in HDs at a higher frequency. Namely, the TPO-89 and -164 peptides induced peptide-reactive CTLs in 3 of 10 HDs, the TPO-101 and -109 peptides induced peptide-reactive CTLs in 2 of 10 HDs, and the TPO-162 peptides induced peptide-reactive CTLs in 1 of 10 HDs.
To test the reactivity of these peptide-reactive CTLs to TPO peptides processed by the intrinsic machinery of the major histocompatibility complex class I molecule, their IFN- production against COS-7 cells, which were transfected with HLA-A2, with or without TPO was examined. The peptide-reactive CTLs exhibited higher levels of IFN- production against the COS-7 cells transfected with HLA-A2 and TPO than those against the COS-7 cells transfected with HLA-A2 but not TPO cells (Fig. 4). The expression of both HLA-A2 and TPO in COS-7–transfected cells was determined by flow cytometry and Western blot analysis, respectively (data not shown). The blocking assay was also performed to confirm the specificity of the response. As a result, the reactivity against COS-7 cells transfected with HLA-A2 and TPO was significantly blocked by the addition of anti-CD8 and anti-HLA class I mAb but not by the other mAb tested. Representative results of TPO-89, -101, -109, and -164 peptide–reactive CTLs are shown in Figure 4. We could not examine the specificity of TPO-162 peptide–reactive CTLs because of the sample limitation.
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Figure 4. The specificity of peptide-stimulated CTLs. Peptide-stimulated CTLs were tested for their IFN- production by recognition of either TPO+ HLA-A2+ COS-7 cells or TPO– HLA-A2+ COS-7 cells at an E:T ratio of 20:1. IFN- production by peptide-specific CTLs in response to HLA-A2+ TPO+ COS-7 cells was also tested in the presence of 20 µg/ml of anti-CD4, anti-CD8, anti-HLA class I, anti-HLA class II, anti-CD14 mAbs. Representative data from HD1, HD5, and HD6 are shown. *p < .05 compared with the IFN- production in response to TPO+ HLA-A2+ COS-7 cells without mAbs by a two-tailed Student’s t-test. Abbreviations: CTL, cytotoxic T lymphocyte; IFN, interferon; mAb, monoclonal antibody; TPO, thrombopoietin.
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These peptide-reactive CTLs from both the patients and HDs were further cultured for 14 days with IL-2 alone, and the cultures were examined for cytotoxicity. The peptide-reactive CTLs exhibited a higher level of cytotoxicity against the T2 cells pulsed with corresponding peptide than against T2 cells pulsed with TPO-35 or HIV peptide. The representative results are shown in Figure 5A. Furthermore, the cold inhibition test showed that the cytotoxicity against TPO+ Hep-G2 cells was significantly blocked by unlabeled T2 cells loaded with the corresponding peptide but not by unlabeled T2 cells loaded with TPO-35 or HIV peptide, used as a negative control (Fig. 5B). These results indicate that the reactivity of peptide-stimulated CTLs against TPO producing HLA-A2+ cells was largely mediated by CD8+ T cells in a peptide-specific and an HLA-A2–restricted manner.
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Figure 5. Cytotoxicity. (A): Peptide-stimulated PBMCs were tested for their cytotoxicity against T2 cells pulsed with the corresponding peptide, TPO-35 peptide, or an HIV peptide by the standard 6-hour 51Cr-release assay. Values represent the means of triplicate assays. A two-tailed Student’s t-test was used for the statistical analysis of the percentage lysis of T2 cells pulsed with the corresponding peptide and that of T2 cells pulsed with TPO-35 peptide. *p < .05. (B): Peptide-stimulated CTLs were tested for the peptide-specificity of their cytotoxicity. Unlabeled T2 cells loaded with the corresponding peptide, TPO-35 peptide, or an HIV peptide were added at a cold-to-hot target cell ratio of 10:1 to ascertain the inhibition of the recognition of TPO+ HepG2 cells. *p < .05 compared with the percentage lysis of HepG2 cells in the presence of unlabelled T2 cells loaded with an HIV peptide by a two-tailed Student’s t-test. Abbreviations: CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cell; TPO, thrombopoietin.
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DISCUSSION
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We demonstrated in this study the presence of TPO peptide–specific IgGs in the plasma of both thrombocytopenic patients and HDs. There is a clear trend of a higher frequency of peptide-specific IgG in the thrombocytopenic patients. It was of note that the mean number of peptides recognized by ITP patients was 2.8 times higher than that recognized by the HDs. We also demonstrated the presence of CTL precursors reactive to TPO-derived peptides in the PBMCs of both the patients and HDs, with a trend toward a higher frequency in the HDs. The mechanism responsible for this discrepancy between the frequencies of peptide-reactive IgGs and CTL precursors is currently unknown. Further study of a relatively large number of patients and HDs will be needed to confirm this observation.
TPO possesses both high- and low-affinity cytokine receptor homology (CRH) binding sites, forming a 2:1 complex with these two different CRH domains of c-Mpl. This interaction is consistent with current models of hematopoietic receptor activation. TPO-109 peptide shown in this study contained the sequence from low-affinity receptor binding sites, such as G112, Q113, S115, and G116 [15]. Both the TPO-162 and -164 peptides contained the sequence from high-affinity receptor binding sites, such as F162 and L165 [15]. Although neither the TPO-89 nor the -101 peptide contained these direct interaction sites, both of these peptides contained the residues of the neutralizing epitope, and thus they were both considered to be important for the conformational structure [16]. In addition, it was of note that all five of these immunogenic TPO peptides were found to be located in the 
-helix of the secondary structure of the protein. The stabilization of a helical structure is known to enhance peptide antigenicity [17]. It is possible that the secondary structure is critical in determining the immunogenicity to TPO peptides.
Autoantibodies against cell-surface antigens and nuclear components such as DNA and histone have been detected in the serum of patients with autoimmune diseases and also in that of HDs, albeit at a lower frequency. In addition, we and others previously reported that IgGs reactive to a large number of self-antigens were often detected in the serum samples of cancer patients, atopic dermatitis patients, and HDs [11, 14, 18, 19]. However, the biological roles played by antipeptide IgGs reactive to CTL epitopes are still currently unknown.
It has been reported that peptide-reactive helper T cells can promote the production of other peptide-specific IgGs from B cells by the mechanism of antigen spreading [20, 21]. The immune response of CD8+ T cells is facilitated by the aid of cytokines from helper-CD4+ T cells [22, 23]. CTL and helper T-cell epitopes are sometimes, although not necessarily, in close proximity [24], and they appear to exert a positive influence on each other [25] under circumstances involving DNA or peptide immunization. However, these immune responses are rarely induced under unimmunized circumstances, primarily due to the weaker immunogenicity of nonmutated self-antigens, to which there is little or no T-cell response. We report here that 5 of 18 TPO-derived peptides had the ability to induce both cellular and humoral responses. These immunogenic TPO-derived peptides might be directly or indirectly responsible for eliciting autoreactive immune responses in certain cases, that is, in those cases involving the in vivo administration of recombinant PEG-rHuMGDF, which in turn might be responsible for the unexpected severe thrombocytopenia that has been shown to develop in some healthy individuals participating in phase I clinical trials. TPO is primarily produced by liver cells, and our results demonstrated that the peptide-stimulated CTLs recognized TPO-producing cells. TPO is also produced by vascular endothelial cells and stromal cells in bone marrow [26, 27]. These TPO-producing cells might be destroyed by CTLs reactive to TPO peptides in HLA-A2+ subjects, resulting in the enhancement of autoimmune responses in individuals who received in vivo the recombinant PEG-rHuMGDF. This study failed to show direct evidence of the biological role of anti-TPO peptide IgGs, and future studies shall be undertaken to determine the biological significance of this new type of IgG. Regardless of this limitation, the results of the present study may facilitate a better understanding of the immunological and pathological significance of autoreactivity to TPO.
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ACKNOWLEDGMENTS
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This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12213134 to K.I.) and Research Center of Innovative Cancer Therapy of 21st Century COE Program for Medical Science (to T.O., M.S., and K.I.) and from the Ministry of Health and Welfare, Japan (No. H14-trans-002, 11–16 to K.I.).
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Received September 5, 2004;
accepted for publication March 31, 2005.
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Abstract
Introduction
