Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.
The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.
Kotlan
Beatrix
B
National Medical Center/Institute of Haematology and Immunology, Budapest, Hungary.
Simsa
Peter
P
Teillaud
Jean-Luc
JL
Fridman
Wolf Herman
WH
Toth
Jozsef
J
McKnight
Michael
M
Glassy
Mark C
MC
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000473:pathology; D001666:Binding Sites, Antibody / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry / Q000276:immunology* / Q000473:pathology; D019556:COS Cells; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry / Q000276:immunology / Q000473:pathology; D018276:Carcinoma, Medullary / Q000737:chemistry / Q000276:immunology* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002999:Clone Cells; D004252:DNA Mutational Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005732:Gangliosides / Q000737:chemistry* / Q000276:immunology*; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007133:Immunoglobulin Joining Region / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification; D007146:Immunoglobulin lambda-Chains / Q000235:genetics / Q000302:isolation & purification; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000473:pathology; D009361:Neoplasm Invasiveness; D019151:Peptide Library; D017422:Sequence Analysis, DNA
D000951:Antigens, Neoplasm; D005732:Gangliosides; D007140:Immunoglobulin Fab Fragments; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains; D019151:Peptide Library; C026226:ganglioside, GD3