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1.
J Immunol Methods ; 365(1-2): 87-94, 2011 Feb 28.
Article in English | MEDLINE | ID: mdl-21194532

ABSTRACT

Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary (CHO) ldlD cell line stably transfected with MUC1. This cell line has a glycosylation defect, which can be reversed by addition of different monosaccharides to the cell culture and enables the production of cells expressing the MUC1-Tn glycoforms. After validation with glycospecific antibodies, the CHO-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique to detect antibodies binding to the underglycosylated MUC1 protein. This cellular system is complementary to the previously published methods to detect MUC1 serum antibodies, since the antibodies to the native protein are evaluated and therefore it can be effectively used for MUC1 antibody monitoring in vaccination studies as well as for functional assays.


Subject(s)
Antibodies, Neoplasm/blood , Antigens, Neoplasm/chemistry , Flow Cytometry/methods , Mucin-1/chemistry , Mucin-1/immunology , Amino Acid Sequence , Animals , Antigens, Neoplasm/genetics , Breast Neoplasms/immunology , Breast Neoplasms/therapy , CHO Cells , Cancer Vaccines/administration & dosage , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Case-Control Studies , Cricetinae , Cricetulus , Female , Glycosylation , Humans , Molecular Sequence Data , Mucin-1/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Transfection , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology
2.
J Immunol ; 174(12): 7764-72, 2005 Jun 15.
Article in English | MEDLINE | ID: mdl-15944279

ABSTRACT

Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which leads to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors. We have investigated whether a mucin released by tumor cells could be involved in causing these immunomodulating effects on DCs. We used a recombinant purified form of the MUC1 glycoprotein, an epithelial associated mucin that is overexpressed, aberrantly glycosylated, and shed during cancer transformation. The O-glycosylation profile of the recombinant MUC1 glycoprotein (ST-MUC1) resembled that expressed by epithelial tumors in vivo, consisting of large numbers of sialylated core 1 (sialyl-T, ST) oligosaccharides. When cultured in the presence of ST-MUC1, human monocyte-derived DCs displayed a modified phenotype with decreased expression of costimulatory molecules (CD86, CD40), Ag-presenting molecules (DR and CD1d), and differentiation markers (CD83). In contrast, markers associated with an immature phenotype, CD1a and CD206 (mannose receptor), were increased. This effect was already evident at day 4 of DC culture and was dose dependent. The modified phenotype of DCs corresponded to an altered balance in IL-12/IL-10 cytokine production, with DC expressing an IL-10(high)IL-12(low) phenotype after exposure to ST-MUC1. These DCs were defective in their ability to induce immune responses in both allogeneic and autologous settings, as detected in proliferation and ELISPOT assays. The altered DC differentiation and Ag presentation function induced by the soluble sialylated tumor-associated mucin may represent a mechanism by which epithelial tumors can escape immunosurveillance.


Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/pathology , Immunosuppressive Agents/pharmacology , Mucin-1/physiology , Peptide Fragments/physiology , Recombinant Fusion Proteins/physiology , Amino Acid Sequence , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , CHO Cells , Cell Differentiation/genetics , Cells, Cultured , Cricetinae , Dendritic Cells/metabolism , Down-Regulation/genetics , Down-Regulation/immunology , Glycosylation , Humans , Immunophenotyping , Immunosuppressive Agents/metabolism , Molecular Sequence Data , Monocytes/cytology , Monocytes/immunology , Monocytes/metabolism , Mucin-1/genetics , Mucin-1/metabolism , N-Acetylneuraminic Acid/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
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