Translocation of an intracellular antigen to the surface of medullary breast cancer cells early in apoptosis allows for an antigen-driven antibody response elicited by tumor-infiltrating B cells.
Tumor-infiltrating lymphoplasmacytic cells are a key feature of medullary carcinoma of the breast (MCB), a distinct subtype of human breast cancer that, despite cytologically anaplastic characteristics, has a more favorable prognosis than other types of breast cancer. Since it has been proposed that the improved clinical outcome is due at least in part to the presence of a prominent lymphoplasmacytic cell infiltrate in the tumor stroma, we recently examined the tumor-infiltrating B cell response in MCB and showed that it is oligoclonal and directed against an intracellular protein translocated to the cell surface upon MCB cell apoptosis. Human Abs cloned from MCB lymphoplasmacytic infiltrate-derived phage display libraries and reflecting the dominant part of the response were used to identify the target Ag as actin. Here, we have characterized in detail the cloned human IgG Abs and the translocation process of actin to the cell surface of apoptotic MCB cells. Our analysis shows that the cloned Abs bind specifically and with high affinity to actin, as determined by ELISA and surface plasmon resonance. Sequence analysis revealed that the Abs are highly somatically mutated, with high replacement to silent ratios, indicative of an Ag-driven, affinity-matured response. Interestingly, the tumor-infiltrating B cells in half the MCB patients mainly exhibited an IgG2 response, while IgG1 dominated in the others. To gain insight to the molecular events that may elicit such an Ab response, we examined the translocation of actin to the cell surface of apoptotic MCB cells using flow cytometry and laser scanning cytometry. Our results show that actin becomes exposed on the cell surface of a large proportion of apoptotic MCB cells as an early apoptotic event. We propose that the Ab response against actin produced by tumor-infiltrating B lymphoplasmacytic cells is Ag-driven, affinity-matured, and elicited due to the increased rate of apoptosis occurring within the MCB tumor that facilitates the translocation and proteolytic fragmentation of intracellular proteins.
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Margit H
MH
Hansen
Heidi V
HV
Nielsen
Henrik J
HJ
Ditzel
D000199:Actins / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D017209:Apoptosis / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism*; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D017104:Bacteriophage M13 / Q000235:genetics; D001943:Breast Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D018276:Carcinoma, Medullary / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010950:Plasma Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D021381:Protein Transport / Q000276:immunology; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D000199:Actins; D001324:Autoantigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library