title: The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor.
abstract: Healthy volunteers are hyperimmunized with RhD-positive red cells in order to obtain plasma containing high titres of anti-D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti-D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti-D titres despite re-immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig-gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD-binding phages from both libraries, except one, had heavy chains with IGH-VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3-positive B cells. We show the presence of clonally related RhD-specific B cells in a hyperimmunized anti-D donor who had declining anti-D titres and who was unresponsive to re-immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti-D donors.
authors.affiliation: Sanquin Research, Amsterdam and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 10066 CX Amsterdam, the Netherlands.
authors.forename: S E
authors.initials: SE
authors.lastname: Dohmen
authors.forename: O J H M
authors.initials: OJ
authors.lastname: Verhagen
authors.forename: S M
authors.initials: SM
authors.lastname: de Groot
authors.forename: L M
authors.initials: LM
authors.lastname: Stott
authors.forename: R C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: S J
authors.initials: SJ
authors.lastname: Urbaniak
authors.forename: C E
authors.initials: CE
authors.lastname: van der Schoot
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D002999:Clone Cells / Q000276:immunology; D004247:DNA / Q000235:genetics; D016172:DNA Fingerprinting; D005260:Female; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics / Q000276:immunology; D050438:Genes, Immunoglobulin Heavy Chain / Q000235:genetics / Q000276:immunology; D050439:Genes, Immunoglobulin Light Chain / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D007155:Immunologic Factors / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics; D018029:Rho(D) Immune Globulin / Q000235:genetics / Q000276:immunology*
chemical_list: D007155:Immunologic Factors; D012333:RNA, Messenger; D018029:Rho(D) Immune Globulin; D004247:DNA