Determination of Thrombopoietin-Derived Peptides Recognized by Both Cellular and Humoral Immunities in Healthy Donors and Patients with Thro
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《干细胞学杂志》
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@med.kurume-u.ac.jp
ABSTRACT
Thrombopoietin (TPO), the c-Mpl ligand, is a key regulator of platelet production . 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 . The production of TPO is elevated in patients with several thrombocytopenic disorders but not in those with immune thrombocytopenic purpura (ITP) . 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 . 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 . 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 . 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 . 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 . Immunoglobulin G (IgG) reactive to certain CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients . 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 . 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.
MATERIALS AND METHODS
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 . 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.
Table 3. Induction of TPO peptide–reactive PBMCs in thrombocytopenic patients
Table 4. Induction of TPO peptide–reactive PBMCs in healthy donors
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.
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.
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.
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.
DISCUSSION
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.).
REFERENCES
Kaushansky K. Thrombopoietin. N Engl J Med 1998;339:746–754.
Kuter DJ, Begley CG. Recombinant human thrombopoietin: basic biology and evaluation of clinical studies. Blood 2002;100:3457–3469.
Yang C, Li YC, Kuter DJ. The physiological response of thrombopoietin (c-Mpl ligand) to thrombocytopenia in the rat. Br J Haematol 1999;105:478–485.
Kappers-Klunne MC, de Haan M, Struijk PC et al. Serum thrombopoietin levels in relation to disease status in patients with immune thrombocytopenic purpura. Br J Haematol 2001;115:1004–1006.
Kuter DJ. Future directions with platelet growth factors. Semin Hematol 2000;37:41–49.
Vadhan-Raj S, Verschraegen CF, Bueso-Ramos C et al. Recombinant human thrombopoietin attenuates carboplatin-induced severe thrombocytopenia and the need for platelet transfusions in patients with gynecologic cancer. Ann Intern Med 2000;132:364–368.
Vadhan-Raj S, Patel S, Bueso-Ramos C et al. Importance of predosing of recombinant human thrombopoietin to reduce chemotherapy-induced early thrombocytopenia. J Clin Oncol 2003;21:3158–3167.
Ohkouchi S, Yamada A, Imai N et al. Non-mutated tumor-rejection antigen peptides elicit type-I allergy in the majority of healthy individuals. Tissue Antigens 2002;59:259–272.
Kawamoto N, Yamada A, Ohkouchi S et al. IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients. Tissue Antigens 2003;61:352–361.
Mine T, Sato Y, Noguchi M et al. Humoral responses to peptides correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses. Clin Cancer Res 2004;10:929–937.
Matsueda S, Kobayashi K, Nonaka Y et al. Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2+ patients with hormone-refractory prostate cancer. Cancer Immunol Immunother 2004;53:479–489.
Emmons RV, Reid DM, Cohen RL et al. Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. Blood 1996;87:4068–4071.
Sasaki Y, Takahashi T, Miyazaki H et al. Production of thrombopoietin by human carcinomas and its novel isoforms. Blood 1999;94:1952–1960.
Ishihara Y, Harada M, Azuma K et al. HER2/neu-derived peptides recognized by both cellular and humoral immune systems in HLA-A2+ cancer patients. Int J Oncol 2004;24:967–975.
Feese MD, Tamada T, Kato Y et al. Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proc Natl Acad Sci U S A 2004;101:1816–1821.
Kuroki R, Hirose M, Kato Y et al. Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody. Acta Crystallogr D Biol Crystallogr 2002;58:856–858.
Gurunath R, Beena TK, Adiga PR et al. Enhancing peptide antigenicity by helix stabilization. FEBS Lett 1995;361:176–178.
Jager D, Taverna C, Zippelius A et al. Identification of tumor antigens as potential target antigens for immunotherapy by serological expression cloning. Cancer Immunol Immunother 2004;53:144–147.
Muro Y. Autoantibodies in atopic dermatitis. J Dermatol Sci 2001;25:171–178.
Craft J, Fatenejad S. Self antigens and epitope spreading in systemic autoimmunity. Arthritis Rheum 1997;40:1374–1382.
James JA, Gross T, Scofield RH et al. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B3-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity. J Exp Med 1995;181:453–461.
Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 1998;393:474–478.
Schoenberger SP, Toes RE, van der Voort EI et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 1998;393:480–483.
Bristol JA, Orsini C, Lindinger P et al. Identification of a ras oncogene peptide that contains both CD4+ and CD8+ T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization. Cell Immunol 2000;205:73–83.
Harada M, Gohara R, Matsueda S et al. In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide. J Immunol 2004;172:2659–2667.
El Andaloussi AE, Duchez P, Rieffers J et al. Expression of thrombopoietin by umbilical vein endothelial cells. Eur Cytokine Netw 2001;12:268–273.
Dormady SP, Bashayan O, Dougherty R etal. Immortalized multipotential mesenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res 2001;10:125–140.(Hiroko Takedatsua, Kohji )
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@med.kurume-u.ac.jp
ABSTRACT
Thrombopoietin (TPO), the c-Mpl ligand, is a key regulator of platelet production . 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 . The production of TPO is elevated in patients with several thrombocytopenic disorders but not in those with immune thrombocytopenic purpura (ITP) . 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 . 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 . 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 . 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 . 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 . Immunoglobulin G (IgG) reactive to certain CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients . 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 . 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.
MATERIALS AND METHODS
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 . 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.
Table 3. Induction of TPO peptide–reactive PBMCs in thrombocytopenic patients
Table 4. Induction of TPO peptide–reactive PBMCs in healthy donors
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.
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.
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.
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.
DISCUSSION
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.).
REFERENCES
Kaushansky K. Thrombopoietin. N Engl J Med 1998;339:746–754.
Kuter DJ, Begley CG. Recombinant human thrombopoietin: basic biology and evaluation of clinical studies. Blood 2002;100:3457–3469.
Yang C, Li YC, Kuter DJ. The physiological response of thrombopoietin (c-Mpl ligand) to thrombocytopenia in the rat. Br J Haematol 1999;105:478–485.
Kappers-Klunne MC, de Haan M, Struijk PC et al. Serum thrombopoietin levels in relation to disease status in patients with immune thrombocytopenic purpura. Br J Haematol 2001;115:1004–1006.
Kuter DJ. Future directions with platelet growth factors. Semin Hematol 2000;37:41–49.
Vadhan-Raj S, Verschraegen CF, Bueso-Ramos C et al. Recombinant human thrombopoietin attenuates carboplatin-induced severe thrombocytopenia and the need for platelet transfusions in patients with gynecologic cancer. Ann Intern Med 2000;132:364–368.
Vadhan-Raj S, Patel S, Bueso-Ramos C et al. Importance of predosing of recombinant human thrombopoietin to reduce chemotherapy-induced early thrombocytopenia. J Clin Oncol 2003;21:3158–3167.
Ohkouchi S, Yamada A, Imai N et al. Non-mutated tumor-rejection antigen peptides elicit type-I allergy in the majority of healthy individuals. Tissue Antigens 2002;59:259–272.
Kawamoto N, Yamada A, Ohkouchi S et al. IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients. Tissue Antigens 2003;61:352–361.
Mine T, Sato Y, Noguchi M et al. Humoral responses to peptides correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses. Clin Cancer Res 2004;10:929–937.
Matsueda S, Kobayashi K, Nonaka Y et al. Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2+ patients with hormone-refractory prostate cancer. Cancer Immunol Immunother 2004;53:479–489.
Emmons RV, Reid DM, Cohen RL et al. Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. Blood 1996;87:4068–4071.
Sasaki Y, Takahashi T, Miyazaki H et al. Production of thrombopoietin by human carcinomas and its novel isoforms. Blood 1999;94:1952–1960.
Ishihara Y, Harada M, Azuma K et al. HER2/neu-derived peptides recognized by both cellular and humoral immune systems in HLA-A2+ cancer patients. Int J Oncol 2004;24:967–975.
Feese MD, Tamada T, Kato Y et al. Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proc Natl Acad Sci U S A 2004;101:1816–1821.
Kuroki R, Hirose M, Kato Y et al. Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody. Acta Crystallogr D Biol Crystallogr 2002;58:856–858.
Gurunath R, Beena TK, Adiga PR et al. Enhancing peptide antigenicity by helix stabilization. FEBS Lett 1995;361:176–178.
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