Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers.
Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.
Mulder
Arend
A
Department of Immunohaematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands. a.mulder@lumc.nl
Eijsink
Chantal
C
Kardol
Marrie J
MJ
Franke-van Dijk
Marry E I
ME
van der Burg
Sjoerd H
SH
Kester
Michel
M
Doxiadis
Ilias I N
II
Claas
Frans H J
FH
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000097:blood; D016175:B-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D018985:Epitopes, B-Lymphocyte / Q000032:analysis / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000032:analysis / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000097:blood; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000097:blood; D008297:Male; D009842:Oligopeptides / Q000276:immunology*; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D009842:Oligopeptides