Anti-Tyrosinase-Related Protein-2 Immune Response in Vitiligo Patients and Melanoma Patients Receiving Active-Specific Immunotherapy
1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.1998.00411.x
ISSN1523-1747
AutoresTetsuro Okamoto, Shigeyuki Fujii, Sharon K.S. Huang, Dave S.�B. Hoon, Reiko F. Irie, A Nizze, Donald L. Morton,
Tópico(s)T-cell and B-cell Immunology
ResumoSeveral melanosome glycoproteins have been shown to be antigenic in humans. Correlation of antigen-specific immune responses in patients with the autoimmune disease vitiligo, therapy-induced hypopigmentation, and cutaneous melanoma has not been well studied. We examined antibody responses to a melanocyte autoantigen, tyrosinase-related protein-2 (TRP-2), as it is highly expressed in cutaneous melanoma and melanocytes. TRP-2 recombinant protein was synthesized for western blot and affinity anti-TRP-2 enzyme-linked immunosorbent assay. We demonstrated that patients with malignant melanoma, vitiligo, and active-specific immunotherapy-induced depigmentation had significant anti-TRP-2 IgG titers. The highest level of anti-TRP-2 IgG response was found in vitiligo patients. Induction and enhancement of anti-TRP-2 IgG responses were observed in melanoma patients treated with a polyvalent melanoma cell vaccine containing TRP-2. Active-specific immunotherapy could induce and/or augment the TRP-2 IgG antibody titers. Melanoma patients who developed hypopigmentation and had improved survival after polyvalent melanoma cell vaccine had significantly augmented anti-TRP-2 antibody responses compared with patients with poor prognosis. This study demonstrates that TRP-2 autoantigen is immunogenic in humans. TRP-2 antibody responses provide a linkage between autoimmune responses by vitiligo patients and melanoma patients responding to immunotherapy who have induced hypopigmentation. Several melanosome glycoproteins have been shown to be antigenic in humans. Correlation of antigen-specific immune responses in patients with the autoimmune disease vitiligo, therapy-induced hypopigmentation, and cutaneous melanoma has not been well studied. We examined antibody responses to a melanocyte autoantigen, tyrosinase-related protein-2 (TRP-2), as it is highly expressed in cutaneous melanoma and melanocytes. TRP-2 recombinant protein was synthesized for western blot and affinity anti-TRP-2 enzyme-linked immunosorbent assay. We demonstrated that patients with malignant melanoma, vitiligo, and active-specific immunotherapy-induced depigmentation had significant anti-TRP-2 IgG titers. The highest level of anti-TRP-2 IgG response was found in vitiligo patients. Induction and enhancement of anti-TRP-2 IgG responses were observed in melanoma patients treated with a polyvalent melanoma cell vaccine containing TRP-2. Active-specific immunotherapy could induce and/or augment the TRP-2 IgG antibody titers. Melanoma patients who developed hypopigmentation and had improved survival after polyvalent melanoma cell vaccine had significantly augmented anti-TRP-2 antibody responses compared with patients with poor prognosis. This study demonstrates that TRP-2 autoantigen is immunogenic in humans. TRP-2 antibody responses provide a linkage between autoimmune responses by vitiligo patients and melanoma patients responding to immunotherapy who have induced hypopigmentation. melanoma-associated antigen melanoma-associated hypopigmentation polyvalent melanoma cell vaccine tyrosinase-related protein 1 and 2 The melanogenesis biochemical pathway in melanocytes consists of a number of enzymes and substrates necessary to synthesize eumelanin. The major known enzymes in melanogenesis are tyrosinase, TRP-1 (5,6-dihidroxyindole-5-carboxylic acid oxidase), and TRP-2 (DOPAchrome tautomerase) (del Marmol and Beermann, 1996del Marmol V. Beermann F. Tyrosinase related proteins in mammalian pigmentation.FEBS Lett. 1996; 381: 165-168Abstract Full Text PDF PubMed Scopus (322) Google Scholar). These proteins are also classified as melanocyte-specific differentiation antigens due to their primary restriction to melanocytes (Naftzger et al., 1996Naftzger C. Takechi Y. Kohda H. Hara I. Vijayasaradhi S. Houghton A.N. Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity.Proc Natl Acad Sci USA. 1996; 93: 14809-14814Crossref PubMed Scopus (139) Google Scholar). There are other melanogenesis differentiation antigens such as gp100 and MART-1; however, their role in the melanogenesis pathway is unknown (Adema et al., 1994Adema G.J. de Boer A.J. Vogel A.M. Leonen Wam Figdor C.G. Molecular characterization of the melanocyte lineage-specific antigen gp100.J Biol Chem. 1994; 269: 20126-20133Abstract Full Text PDF PubMed Google Scholar;Chen et al., 1996Chen Y.T. Stockert E. Jungbluth A. Tsang S. Coplan K.A. Scanlan M.J. Old L.J. Serological analysis of Melan-A (MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.Proc Natl Acad Sci USA. 1996; 93: 5915-5919Crossref PubMed Scopus (173) Google Scholar). Because melanogenesis proteins are consistently expressed in normal melanocytes and are highly expressed in melanoma, they have recently received increased attention as detection markers and potential targets of active-specific immunotherapy (Spagnoli et al., 1995Spagnoli G.C. Schaefer C. Willimann T.E. et al.Peptide-specific CTL in tumor-infiltrating lymphocytes from metastatic melanomas expressing MART-1/Melan-A, gp100 and Tyrosinase genes: a study in an unselected group of HLA-A2.1-positive patients.Int J Cancer. 1995; 64: 309-315Crossref PubMed Scopus (55) Google Scholar;Zhai et al., 1996Zhai Y. Yang J.C. Kawakami Y. et al.Antigen-specific tumor vaccines. Development and characterization of recombinant adenovirus encoding MART-1 or gp100 for gene therapy.J Immunol. 1996; 156: 700-710PubMed Google Scholar;Sarantou et al., 1997Sarantou T. Chi Ddj Garrison D.A. Conrad A.J. Schmid P. Morton D.L. Hoon DsB Melanoma-associated antigens as messenger RNA detection markers for melanoma.Cancer Res. 1997; 57: 1371-1376PubMed Google Scholar). There are two major diseases, vitiligo and cutaneous melanoma, in which the disease etiology evolves around melanocytes. Cutaneous melanoma is a malignant tumor derived from transformed melanocytes. Despite improvements in diagnosis and treatment for melanoma, patients who have distant metastasis have poor prognosis (Barth and Morton, 1996Barth A. Morton D.L. The role of adjuvant therapy in melanoma management.Cancer. 1996; 75: 726-734Crossref Scopus (94) Google Scholar). Vitiligo is a skin disorder of depigmented patches resulting from loss of melanocytes in the lesion and is thought to be due to autoimmunity against melanocytes (Naughton et al., 1983Naughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vitiligo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (191) Google Scholar;Norris et al., 1988Norris D.A. Kissinger R.M. Naughton G.M. Bystryn J.C. Evidence for Immunologic mechanisms in human vitiligo: patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity.J Invest Dermatol. 1988; 90: 783-789Abstract Full Text PDF PubMed Google Scholar;Song et al., 1994Song Y.H. Connor E. Li Y. Zorovich B. Balducci P. Maclaren N. The role of tyrosinase in autoimmune vitiligo.Lancet. 1994; 344: 1049-1052Abstract PubMed Scopus (161) Google Scholar). Autoimmunity is not clearly implicated in vitiligo; however, there is evidence implicating that this response is associated with known autoimmune diseases. Vitiligo can be psychologically debilitating to patients as a consequence of its cosmetic effect. Activation and suppression of melanogenesis antigen-specific immune responses in vitiligo and melanoma patients, respectively, have been strongly suggested to be related to their disease etiologies (Berd et al., 1996Berd D. Mastrangelo M.J. Lattime E. Sato T. Maguire H.C. Melanoma and vitiligo: immunology's Grecian urn.Cancer Immunol Immunother. 1996; 42: 263-267Crossref PubMed Scopus (25) Google Scholar). Melanoma patients receiving active-specific immunotherapy such as vaccines have been shown to have elevated antigen-specific immune responses that associate with more favorable prognosis (Jones et al., 1981Jones P.C. Sze L.L. Liu P.Y. Morton D.L. Irie R.F. Prolonged survival for melanoma patients with elevated IgM antibody to oncofetal antigen.J Natl Cancer Inst. 1981; 66: 249-254PubMed Google Scholar;Livingston et al., 1994Livingston P.O. Wong Gyc Adluri S. et al.Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside.J Clin Oncol. 1994; 12: 1036-1044Crossref PubMed Scopus (486) Google Scholar). Enhancement and induction of both antigen-specific antibody and cell-mediated immunity to melanoma-associated antigen (MAA) have been demonstrated in vaccinated patients (Morton et al., 1992Morton D.L. Foshag L.J. Hoon Dsb et al.Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine.Ann Surg. 1992; 216: 463-482Crossref PubMed Scopus (369) Google Scholar;Hayashi et al., 1993Hayashi Y. Hoon D.S.B. Foshag L.J. Park M.S. Terasaki P.I. Morton D.L. A preclinical model to assess the antigenicity of an HLA-A2 melanoma cell vaccine.Cancer. 1993; 72: 750-759Crossref PubMed Scopus (12) Google Scholar;Hoon et al., 1995aHoon Dsb Yuzuki D. Hayashida M. Morton D.L. Melanoma patients immunized with melanoma cell vaccine induce antibody response to recombinant MAGE-1 antigen.J Immunol. 1995 a; 154: 730-737PubMed Google Scholar). Vitiligo-like depigmentation called melanoma-associated hypopigmentation (MAH) has been reported in melanoma patients responding to immunotherapy (Mattes et al., 1983Mattes M.J. Thomson T.M. Old L.J. Lloyd K.O. A pigmentation-associated, differentiation antigen of human melanoma defined by a precipitation antibody in human serum.Int J Cancer. 1983; 32: 717-721Crossref PubMed Scopus (71) Google Scholar;Richards et al., 1992Richards J.M. Mehta N. Ramming K. Skosey P. Sequential chemoimmunotherapy in the treatment of metastatic melanoma.J Clin Oncol. 1992; 10: 1338-1343Crossref PubMed Scopus (243) Google Scholar;Rosenberg, 1997Rosenberg S.A. Cancer vaccines based on the identification of genes encoding cancer regression antigens.Immunology Today. 1997; 18: 175-182Abstract Full Text PDF PubMed Scopus (374) Google Scholar). The onset of MAH can also occur in patients having received therapy other than immunotherapy or in the absence of therapy. The mechanism of concomitant depigmentation is thought to involve immune responses against autoantigens found in melanomas and melanocytes. Both vitiligo and melanoma patients have been found to have antibodies to melanoma cells in their sera (Naughton et al., 1983Naughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vitiligo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (191) Google Scholar). Appearance of MAH in melanoma patients who respond to treatment has been linked to a better prognosis (Mattes et al., 1983Mattes M.J. Thomson T.M. Old L.J. Lloyd K.O. A pigmentation-associated, differentiation antigen of human melanoma defined by a precipitation antibody in human serum.Int J Cancer. 1983; 32: 717-721Crossref PubMed Scopus (71) Google Scholar;Richards et al., 1992Richards J.M. Mehta N. Ramming K. Skosey P. Sequential chemoimmunotherapy in the treatment of metastatic melanoma.J Clin Oncol. 1992; 10: 1338-1343Crossref PubMed Scopus (243) Google Scholar); however, linkage between immune responses to autoantigen in both diseases has not been well characterized. Tyrosinase is one of the melanoma autoantigens and the presence of anti-tyrosinase antibody in sera of both melanoma and vitiligo patients has been demonstrated (Merimsky et al., 1996Merimsky O. Baharav E. Shoenfeld Y. Chaitchik S. Tsigelman R. Cohen-Aloro D. Fishman P. Anti-tyrosinase antibodies in malignant melanoma.Cancer Immunol Immunother. 1996; 42: 297-302Crossref PubMed Scopus (29) Google Scholar). Another well-characterized melanoma autoantigen is TRP-1 (Naftzger et al., 1996Naftzger C. Takechi Y. Kohda H. Hara I. Vijayasaradhi S. Houghton A.N. Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity.Proc Natl Acad Sci USA. 1996; 93: 14809-14814Crossref PubMed Scopus (139) Google Scholar). Immune responses to TRP-1 have been demonstrated in melanoma patients (Thomson et al., 1985Thomson T.M. Mattes M.J. Roux L. Old L.J. Lloyd K.O. Pigmentation-associated glycoprotein of human melanomas and melanocytes: a definition with a monoclonal antibody.J Invest Dermatol. 1985; 85: 169-174Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Anti-TRP-1 responses in a murine melanoma model system have been linked to melanoma regression and autoimmunity (depigmentation) (Takechi et al., 1996Takechi Y. Hara I. Naftzger C. Xu Y. Houghton A.N. A melanosomal membrane protein is a cell surface target for melanoma therapy.Clin Cancer Res. 1996; 2: 1837-1842PubMed Google Scholar). TRP-2 is a relatively newly discovered melanogenesis synthesis related enzyme and has recently been characterized and cloned (Bouchard et al., 1994Bouchard B. del Marmol B. Jackson I.J. Cherif D. Dubertret L. Molecular characterization of a human tyrosinase-related protein-2 cDNA patterns of expression in melanocytic cells.Eur J Biochem. 1994; 219: 127-134Crossref PubMed Scopus (65) Google Scholar). TRP-2 is DOPAchrome tautomerase involved in the conversion of DOPAchrome to 5,6-dihydroxyindole-5-carboxylic acid (Bouchard et al., 1994Bouchard B. del Marmol B. Jackson I.J. Cherif D. Dubertret L. Molecular characterization of a human tyrosinase-related protein-2 cDNA patterns of expression in melanocytic cells.Eur J Biochem. 1994; 219: 127-134Crossref PubMed Scopus (65) Google Scholar). TRP-2 (peptide) was recently shown to be recognized by CTL from melanoma patientsin vitro (Wang et al., 1996Wang R.F. Appella E. Kawakami Y. Kang X. Rosenberg S.A. Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.J Exp Med. 1996; 184: 2207-2216Crossref PubMed Scopus (271) Google Scholar); however, no studies to our knowledge have assessed TRP-2 immunogenicityin vivo nor immune responses in melanoma and vitiligo patients. In this study, TRP-2 recombinant protein was synthesized for assessment of anti-TRP-2 antibody responses in melanoma and vitiligo patients' sera. Recombinant TRP-2 protein allows detection of multiple antibody responses to multiple antigenic epitopes that are not defined. Melanoma patients receiving therapy with polyvalent melanoma cell vaccine (PMCV) expressing TRP-2 were evaluated for anti-TRP-2 antibody responses. The PMCV is a whole cell vaccine that has been previously characterized to induce significant antibody and T cell responses to multiple MAA, and has been shown to improve melanoma patient prognosis in a phase II clinical trial (Morton et al., 1992Morton D.L. Foshag L.J. Hoon Dsb et al.Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine.Ann Surg. 1992; 216: 463-482Crossref PubMed Scopus (369) Google Scholar). In this study, we demonstrated anti-TRP-2 antibody (IgG) responses in melanoma and vitiligo patients. Melanoma patients who developed MAH after PMCV treatment had significantly higher anti-TRP-2 antibody responses. The study demonstrates the significance of anti-TRP-2 immune responses in melanoma patients with possible linkage to autoimmunity in vitiligo patients. Most importantly the study demonstrates that the autoantigen TRP-2 is immunogenic in humans and related to improved prognosis. All melanoma patients (American Joint Committee on Cancer stage III) were clinically and pathologically diagnosed. After the surgical removal of tumor(s), melanoma patients were started with PMCV as previously described (Morton et al., 1992Morton D.L. Foshag L.J. Hoon Dsb et al.Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine.Ann Surg. 1992; 216: 463-482Crossref PubMed Scopus (369) Google Scholar). Briefly, patients received PMCV consisting of three well-defined irradiated allogeneic melanoma cell lines (8 × 106 cells per injection) three times biweekly and then monthly for a year, followed by every 3–6 mo thereafter. PMCV was given intradermally at multiple sites in the axillary and inguinal region. BCG (Tice strain, 3 × 106 CFU) was given with PMCV in the first two treatments. Follow-up clinical and laboratory evaluations were repeated monthly, which included a chest X-ray every 2–3 mo and if necessary MRI/PET and/or CT scans every 6–12 mo. Melanoma patients who developed hypopigmentation (MAH) during PMCV treatment were diagnosed at the John Wayne Cancer Clinic. Blood was collected from melanoma, MAH, and vitiligo patients. Normal blood was collected from volunteers who were age- and sex-matched with melanoma and vitiligo patients. Sera were processed, aliquoted, and cryopreserved at –80°C until used as previously described (Hoon et al., 1995aHoon Dsb Yuzuki D. Hayashida M. Morton D.L. Melanoma patients immunized with melanoma cell vaccine induce antibody response to recombinant MAGE-1 antigen.J Immunol. 1995 a; 154: 730-737PubMed Google Scholar). All sera were coded and tested in a blind fashion by the individuals performing the affinity anti-TRP-2 enzyme-linked immunosorbent assay (ELISA) analysis. Melanoma cell line, M12, and PMCV lines 1, 2, and 3 were separately grown in RPMI 1640 with 10% fetal calf serum (heat inactivated) plus antibiotics (Hoon et al., 1993Hoon Dsb Wang Y. Sze L. et al.Molecular cloning of a human monoclonal antibody reactive to ganglioside GM3 antigen on human cancers.Cancer Res. 1993; 53: 5244-5250PubMed Google Scholar). A primary normal melanocyte line was obtained from Clonetics (San Diego, CA) and cultured in melanocyte growth medium according to the manufacturer's instruction. The breast cancer cell line, BT-20 (ATCC HTB19) was obtained form ATCC (Rockvillle, MD) and grown in Eagle's modified essential medium with 10% fetal calf serum (heat inactivated). When the cell lines attained 75% confluency, total RNA was isolated, purified, and prepared for reverse transcriptase polymerase chain reaction (RT-PCR) analysis as previously described (Doi et al., 1996Doi F. Chi Ddj Charuworn B.B. Conrad A.J. Russell J. Morton D.L. Hoon D.S.B. Detection of β;-human chorionic gonadotropin mRNA as a marker for cutaneous malignant melanoma.Int J Cancer. 1996; 65: 454-459Crossref PubMed Scopus (31) Google Scholar). Recombinant human TRP-2 protein was genetically engineered into a plasmid vector and expressed inE. coli. Briefly, total RNA was isolated from the melanoma cell line M12, and cDNA was made by reverse transcription as previously described (Hoon et al., 1995bHoon Dsb Wang Y. Dale P.S. et al.Detection of occult melanoma cells in blood with a multiple-marker polymerase chain assay.J Clin Oncol. 1995 b; 13: 2109-2116PubMed Google Scholar). Then the cDNA of TRP-2 (1356 bp,Bouchard et al., 1994Bouchard B. del Marmol B. Jackson I.J. Cherif D. Dubertret L. Molecular characterization of a human tyrosinase-related protein-2 cDNA patterns of expression in melanocytic cells.Eur J Biochem. 1994; 219: 127-134Crossref PubMed Scopus (65) Google Scholar) was amplified by PCR, and restriction enzyme sites were incorporated for cloning. The PCR product obtained was cloned into the expression vector pGEX/HIS for expression of glutathione S-transferase (GST) fusion protein and a 6x-HIS affinity tag. The cloned cDNA sequence of TRP-2 was verified by nucleic acid sequencing. The pGEX/HIS vector was reengineered from the pGEX-2T vector (Pharmacia, Piscataway, NJ) by inserting into theSma-I site a DNA fragment for expression of a 6x-HIS affinity tag. The vector construct was transformed intoE. coli BL21 strain and selected on ampicillin-containing plates. One clone, confirmed by DNA sequencing, was induced by isopropyl β-D-thiogalactoside (IPTG) and purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B (Pharmacia, Piscataway, NJ). The GST fusion recombinant TRP-2 protein was bound to the affinity column, cleaved in the column by thrombin digestion, and then eluted. Protein was further purified by gel electrophoresis and Ni2+-NTA affinity chromatography. This affinity purification was repeated twice. The yield of affinity-purified recombinant TRP-2 protein ranged from 4 to 5 mg per liter of bacterial culture. Protein concentration was measured using a BCA Protein Assay Kit based on the bicinchoninic acid procedure (Pierce, Rockford, IL). Purified recombinant TRP-2 was analyzed by western blotting using Ni2+-NTA conjugate (Qiagen, Chatsworth, CA) to verify the presence of 6x-HIS tag, and an anti-GST antibody conjugate (Pharmacia) was used to confirm the absence of GST tag. The respective controls were used in assessment of the proteins in western blotting. Western blotting was performed as previously described (Okamoto et al., 1997Okamoto T. Kaneda Y. Yuzuki D. Huang Sks Chi Ddj Hoon DsB Induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine.Gene Ther. 1997; 4: 969-976Crossref PubMed Scopus (35) Google Scholar). Briefly, 5 μg of purified TRP-2 was run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and blotted to nitrocellulose membrane. Patients' sera were diluted to 1:50 or 1:100 and incubated for 1 h using a multiscreen apparatus (BioRad, Hercules, CA). This was followed by incubation with 1:1000 dilution of alkaline phosphatase-conjugated anti-human IgG (γ-chain specific) or anti-human IgM (μ-chain specific) (Boehringer, Indianapolis, IN), and then developed by using 5-bromo-4-chloro-3-indolyl-1-phosphate and nitro blue tetrazolium (Promega, Madison, WI) according to the manufacturer's procedure. As a control, recombinant HOJ-1 protein, a 15 kDa tumor-associated protein with a 6x-HIS tag, was expressed inE. coli, induced, isolated, and purified in a similar manner to TRP-2 (Hoonet al. to be published). No IgG or IgM antibody response was detected against HOJ-1 protein by western blotting analysis using anti-TRP-2 positive sera and at the same protein concentration of TRP-2. Anti-TRP-2 positive patients' sera were also assessed for anti-MAGE-3 antibodies using recombinant MAGE-3 protein (Okamoto et al., 1997Okamoto T. Kaneda Y. Yuzuki D. Huang Sks Chi Ddj Hoon DsB Induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine.Gene Ther. 1997; 4: 969-976Crossref PubMed Scopus (35) Google Scholar). MAGE-3 recombinant protein with a 6x-HIS tag was purified in a similar manner. Anti-MAGE-3 IgG and IgM antibodies were found infrequently and only at very low antibody dilutions (data not shown). These analyses indicated that the anti-TRP-2 responses were not directed towards the 6x-HIS tag or potential bacterial contaminating molecules. Affinity ELISA was performed with purified recombinant TRP-2 protein containing a 6x-HIS tag on the N-terminus as reported previously (Okamoto et al., 1997Okamoto T. Kaneda Y. Yuzuki D. Huang Sks Chi Ddj Hoon DsB Induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine.Gene Ther. 1997; 4: 969-976Crossref PubMed Scopus (35) Google Scholar). Recombinant protein was incubated overnight at room temperature in Ni2+ chelate-coated ELISA microwell plates (Xenopore, Hawthorne, NJ) according to the manufacturer's instructions. After blocking with specific buffer, sera were added in 2-fold dilutions in phosphate-buffered saline from 1:40 to 1:1280 and incubated for 2 h at room temperature in duplicate. Goat anti-human IgG (γ-chain specific) or IgM (μ-chain specific) horseradish peroxidase-conjugate were used to detect antibodies, respectively. ELISA was developed and read using a Molecular Device ELISA Reader and analyzed with the instruments' software (San Francisco, CA). Antibody titers were defined as the highest serum dilution yielding an absorbance reading greater than the mean plus 2 SD of healthy donor volunteer background absorbance. Respective positive controls (positive serum samples) and negative controls (no serum, antihuman IgG, or IgM conjugate alone) were carried out. Assays were performed at least twice for verification of results. The RT-PCR assay was carried out as previously described (Sarantou et al., 1997Sarantou T. Chi Ddj Garrison D.A. Conrad A.J. Schmid P. Morton D.L. Hoon DsB Melanoma-associated antigens as messenger RNA detection markers for melanoma.Cancer Res. 1997; 57: 1371-1376PubMed Google Scholar). One microgram of RNA was reverse transcribed using oligo (dT)15 primers. Specific pairs of TRP-2 primers (sense, 5′-GAGGTGCGAGCCGACACAAG-3′; anti-sense, 3′-CGGTGCCAGGTAACAAATGC-5′) were used in the PCR reaction at 95°C for 5 min for 1 cycle; 95°C for 1 min; 60°C for 1 min; 72°C for 1 min for 35 cycles; and 72°C for 10 min. Specific PCR cDNA product was assessed by using 2% agarose gel electrophoresis and ethidium bromide. The predicted size of the amplified gene for TRP-2 was 476 bp. Positive and negative controls were incorporated for the RT-PCR assays. β-actin RT-PCR was carried out on all samples, as a control for the presence of intact mRNA. Affinity ELISA data were analyzed by the Wilcoxon rank sum test to compare the difference between diseases and by a generalized McNemar test to compare before and after PMCV treatment data (Fleiss, 1981Fleiss J. The analysis of data from matched samples.Statistical Methods for Rates and Proportions. John Wiley, New York1981: 112-132Google Scholar). p values < 0.05 (two-sided) were considered significant. The purified recombinant TRP-2 protein had an estimatedMr of 55–57 kDa as analyzed by gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This is similar to the predicted molecular mass of the protein:translation product (estimated at 52 kDa) of the native protein with 452 amino acids. Analysis of protein size by gel electrophoresis is usually slightly larger compared with the predicted size due to protein charge, amino acid composition, and migration ability in the gel. Purified recombinant TRP-2 protein allowed us to evaluate anti-TRP-2 antibody responses of patients' sera using western blot and affinity ELISA. A representative western blot of anti-TRP-2 antibody response is shown inFigure 1. Anti-TRP-2 IgG antibodies were not detected in sera from 21 healthy donors at 1:50 dilution; however, seven of 15 melanoma, seven of 12 MAH, and 10 of 15 vitiligo patients were shown to have anti-TRP-2 IgG in their sera. Similar analyses were performed for detection of anti-TRP-2 IgM. No anti-TRP-2 IgM antibody was detected in 15 melanoma and 15 vitiligo patients by western blot analysis at 1:50 dilution. Therefore, in the study we focused on analysis of anti-TRP-2 IgG responses. Specificity of the anti-TRP-2 response against recombinant TRP-2 protein was verified using control recombinant proteins with 6x-HIS tag (HOJ-1 and MAGE-3) that were prepared in an identical manner. Anti-TRP-2 positive sera did not have any significant reactivity to the control recombinant proteins, therefore indicating that the potential reactivity to 6x-HIS or bacterial contaminants was not present. To determine whether the anti-TRP-2 antibody response could recognize melanoma native TRP-2 and not an artifact response to recombinant TRP-2 conformation, we performed several specific adsorption studies. High titer anti-TRP-2 patient sera was adsorbed with melanoma tissue containing abundant TRP-2 protein and compared with adsorption with a TRP-2 negative tissue colon carcinoma tumor biopsy. The latter does not express TRP-2 as assessed by mRNA expression using RT-PCR. Adsorption with a highly melanotic melanoma tumor specimen significantly reduced (>70%) anti-TRP-2 antibody reactivity (anti-TRP-2 affinity ELISA), whereas absorption with control colon tissue did not significantly affect anti-TRP-2 antibody reactivity. This suggested that the anti-TRP-2 IgG antibodies in patients' sera can recognize and be adsorbed out with native TRP-2. To assess anti-TRP-2 IgG response in a large number of patients, we developed an affinity anti-TRP-2 ELISA. As shown inTable 1, 98% (34 of 35) of healthy donors were negative at 1:40 serum dilution. Anti-TRP-2 IgG responses in 40% of melanoma, 55% of MAH, and 67% of vitiligo patients' sera were positive, and significantly higher than in normal sera (p = 0.0001, <0.0001, and 80% of melanomas and in 50% of hypopigmented melanomas (Sarantou et al., 1997Sarantou T. Chi Ddj Garri
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