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1.
J Invest Dermatol ; 115(1): 48-56, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10886507

ABSTRACT

We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3'-untranslated region and is originated by alternative polyadenylation. This novel 3'-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2-8b). The protein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2-8b. Reverse transcription-polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.


Subject(s)
Intramolecular Oxidoreductases/genetics , Melanocytes/chemistry , Melanoma/genetics , RNA, Messenger/metabolism , Alternative Splicing , Base Sequence , Cell Line , Humans , Intramolecular Oxidoreductases/isolation & purification , Molecular Sequence Data , Protein Isoforms/isolation & purification , Protein Isoforms/physiology , Transcription, Genetic , Tumor Cells, Cultured
2.
J Immunol ; 163(12): 6912-23, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10586094

ABSTRACT

In this study we tested the hypothesis that loss of T cell signaling molecules in metastatic melanoma patients' T cells may affect differently T cell subsets characterized by distinct TCR variable regions. By a two-color immunofluorescence technique, expression of zeta-chain, lck, and ZAP-70 was evaluated in CD3+ T cells and in three representative T cell subsets expressing TCRAV2, TCRBV2, or TCRBV18. Partial loss of lck and ZAP-70 was found in CD3+ T cells from PBL of most melanoma patients, but not of healthy donors. The extent of zeta-chain, lck, and ZAP-70 loss depended on the TCRV region expressed by the T cells, and this association was maintained or increased during progression of disease. Coculture of patients' or donors' T cell with melanoma cells, or with their supernatants, but not with normal fibroblasts or their supernatants, down-modulated expression of zeta-chain, lck, and ZAP-70 in a TCRV region-dependent way. Immunodepletion of soluble HLA class I molecules present in tumor supernatants, but not of soluble ICAM-1, blocked the suppressive effect on T cell signaling molecule expression. T cell activation with mAbs to a single TCRV region and to CD28 led to significant and TCRV region-specific re-induction of zeta-chain expression. These findings indicate that extent of TCR signaling molecules loss in T lymphocytes from metastatic melanoma patients depends on the TCRV region and suggest that tumor-derived HLA class I molecules may contribute to induce such alterations.


Subject(s)
Melanoma/immunology , Melanoma/secondary , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Signal Transduction/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal/pharmacology , CD28 Antigens/immunology , CD3 Complex/biosynthesis , Cell-Free System/immunology , Disease Progression , Down-Regulation/immunology , Female , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Immunophenotyping , Lymphocyte Activation/immunology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/biosynthesis , Male , Melanoma/metabolism , Melanoma/pathology , Membrane Proteins/biosynthesis , Middle Aged , Protein-Tyrosine Kinases/biosynthesis , Receptors, Antigen, T-Cell/biosynthesis , Solubility , Suppressor Factors, Immunologic/metabolism , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase
3.
Cancer Immunol Immunother ; 48(1): 39-46, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10235487

ABSTRACT

It was previously found that primary melenomas from HLA-A2-matched patients display an increased expression of a few T cell receptor (TCR) variable-region beta-chain transcripts (BV) compared with autologous peripheral blood lymphocytes (PBL) and uninvolved skin. In order to see whether expansions of clonal/oligoclonal subsets of T cells occurred, complementarity-determining region 3 (CDR3) of BV transcripts overexpressed in the neoplastic infiltrate were cloned and sequenced. Dominant rearrangements were found for BV14, which were commonly increased in the neoplastic lesions of all analysed HLA-A2 melanoma patients, as well as for other overexpressed BV gene families, but none of them could be identified among autologous PBL. No identical CDR3 sequences could be detected in the dominant BV14 rearrangements obtained from the different patients. In one patient a single clonally expanded SLSGTGVNEQF CDR3 clonotype accounted for the entire BV14 relative frequency of expression (24%) in tumor-infiltrating lymphocytes (TIL). Two independent mixed lymphocyte/tumor cultures (MLTC) could be successfully established from TIL of the patient and were found to exert HLA-class-I-restricted cytotoxicity for the autologous melanoma line. BV14 T cells that constituted from 22% to 32% of all T cells present in both MLTC lines, as assessed by flow cytometry, all displayed the same CDR3 clonotype found in vivo and could be shown, by TCR down-modulation experiments, to be involved in autologous tumor recognition. These results support the hypothesis of a tumor-antigen-driven origin of clonally amplified T cells present at high frequency in the in situ neoplastic infiltrate.


Subject(s)
Lymphocyte Activation/immunology , Melanoma/immunology , T-Lymphocytes/immunology , Clone Cells , DNA, Complementary/genetics , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , HLA-A2 Antigen/immunology , Humans , Lymphocyte Culture Test, Mixed , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/cytology , T-Lymphocytes, Cytotoxic/immunology
4.
J Exp Med ; 188(6): 1005-16, 1998 Sep 21.
Article in English | MEDLINE | ID: mdl-9743519

ABSTRACT

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.


Subject(s)
Antigens, Neoplasm/biosynthesis , Intramolecular Oxidoreductases/genetics , Introns , Melanocytes/immunology , Melanoma/immunology , Protein Biosynthesis , RNA, Messenger/genetics , T-Lymphocytes, Cytotoxic/immunology , Alleles , Animals , Antigen Presentation/genetics , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Base Sequence , COS Cells , Cloning, Molecular , DNA, Complementary/isolation & purification , Epitopes/biosynthesis , Epitopes/immunology , Gene Expression Regulation, Neoplastic/immunology , HLA-A3 Antigen/genetics , Histocompatibility Testing , Humans , Melanocytes/metabolism , Melanoma/genetics , Molecular Sequence Data , Peptides/genetics , Peptides/immunology , Peptides/metabolism , Polymerase Chain Reaction , Tumor Cells, Cultured
5.
Lab Invest ; 78(5): 619-27, 1998 May.
Article in English | MEDLINE | ID: mdl-9605186

ABSTRACT

The T-cell receptor beta-chain variable (TCRBV) region repertoire expressed by tumor-infiltrating lymphocytes was characterized by immunohistochemical analysis using a panel of 18 monoclonal antibodies on cryosectioned specimens of 14 primary vertical growth phase (VGP) melanomas with a T-cell infiltrate histopathologically defined as brisk or nonbrisk. T lymphocytes present in the VGP of all patients displayed a restricted T-cell receptor usage, with a pattern of reactivity similar in brisk versus nonbrisk infiltrates. No evidence of restriction was found in the extra-VGP lymphocytic infiltrates, when available, within the same specimen. Furthermore, the repertoire of TCRBV expressed in nodal metastases was similar to that of the corresponding primary melanomas in the two cases tested. The results obtained by this in situ analysis indicate that the TCRBV repertoire in VGP is determined by a preferential migration of T lymphocytes, possibly indicative of an immune response to melanoma-associated antigens.


Subject(s)
Melanoma/metabolism , Melanoma/pathology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal , Cell Movement/physiology , Female , Humans , Immunohistochemistry/methods , Lymphocytes, Tumor-Infiltrating/metabolism , Male , Middle Aged , T-Lymphocytes/physiology
6.
Mol Microbiol ; 23(5): 997-1007, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9076736

ABSTRACT

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1-8 of mature glucoamylase greatly enhance excretion of both glucoamylase and beta-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro. Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro.


Subject(s)
Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Peptides/genetics , Peptides/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , Biological Transport/genetics , Blotting, Western , Cell Extracts/analysis , Cloning, Molecular , Culture Media/analysis , Culture Media/metabolism , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Glucan 1,4-alpha-Glucosidase/immunology , Mutagenesis, Site-Directed , Plasmids , Sequence Deletion , Signal Transduction/genetics , beta-Galactosidase/immunology
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