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BACKGROUND: Antibodies produced against the D antigen make use of IGHV genes restricted to the IGHV3 superfamily. These findings are based on the IGHV gene analysis in anti-D–producing B cells from hyperimmunized donors, however, and therefore the restriction might be due to the hyperimmunization. In this study the IGHV gene usage of anti-Rh–producing B cells in a woman who was immunized in the last trimester of her pregnancy was analyzed. STUDY DESIGN AND METHODS: Serologic analysis was performed by absorption and elution. Antibody-dependent cellular cytotoxicity (ADCC) of the different anti-Rh was determined. A phage display library was constructed from 2.2 × 106 isolated B cells and pannings were performed with red cells of the r′r, R1R1, and R2R2 phenotype. RESULTS: A plasma sample of the immunized person showed high levels of both anti-D and anti-G and low levels of anti-C. Anti-D and anti-G contributed equally strong to the ADCC whereas anti-C did not. Eighteen anti-D–, 5 anti-G–, and 1 anti-C–specific phage clones were found, of which 16, 2, and 1 used the IGHV3s genes, respectively. CONCLUSION: For the first time a restriction to the IGHV3s genes in anti-D in a naturally immunized pregnant woman is shown. Moreover, the use of IGHV3s genes appears to be present in anti-C and anti-G as well. Therefore, it is concluded that restricted IGHV3s gene usage in anti-D is not due to hyperimmunization but due to characteristics of the Rh antigens and the intrinsic binding capacities of IGHV3s genes, supporting the common Rh footprint hypothesis.

Summary 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.

The Ig-genes of the heavy chains in anti-D-specific hybridomas and Fab/scFv-fragments selected from phage-display libraries are restricted to a group of closely related genes (IGHV3s genes). We analyzed the Ig-gene repertoire in anti-D-specific B cells of two hyperimmunized donors using a completely different method. Single B cells were cultured for 10 days in an EL4.B5 culture system. mRNA from anti-D-producing B cells was reverse transcribed into cDNA. Heavy- and light-chain gene rearrangements were amplified by PCR reactions, sequenced and cloned into a pNUT-vector system, thereby allowing the production of complete IgG and IgM. Eleven anti-D-specific B-cell clones were isolated and analyzed. Eight of these clones (including IgM-producing clones) had IGHV3s genes. We demonstrated that functional anti-D-specific IgM (4 clones) and IgG (2 clones) was produced. Using a new method, we analyzed the IGHV gene repertoire of anti-D-specific B cells of hyperimmunized donors and showed that it is indeed restricted. Moreover, we found a high frequency (1:100 and 1:500) of anti-D-specific B cells in the peripheral B cells of hyperimmunized donors. We suggest that this approach could be applied for the selection of human mAbs from immunized donors and for the analysis of Ig-gene repertoires at the single-B cell level. © 2005 Elsevier B.V. All rights reserved

B cell memory to T cell–dependent (TD) Ags are considered to largely reside in class-switched CD27+ cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D+ erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid–specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27− B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27+IgG+ B cells was observed. Next, B cells were enriched with D+ erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27−IgM+ and CD27+IgM+ B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27−IgM+, CD27+IgM+, and CD27+IgG+ B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM+ B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27+ B cells from Ag-experienced precursors.

ObjectivesThe mechanism of action of treatment with tumour necrosis factor (TNF) blockers in rheumatoid arthritis (RA) is still not completely understood. The aim of this study was to test if adalimumab treatment could affect the influx of monocytes into the synovium.MethodsA novel technique was used to analyse the migration of labelled autologous monocytes before and 14 days after initiation of adalimumab treatment using scintigraphy. CD14 monocytes were isolated from patients with RA, using a positive selection procedure with magnetic-activated cell sorting, and labelled with technetium-99m-hexamethylpropylene-amino-oxime. Scintigraphic scans were made 1, 2 and 3 h after re-infusion.ResultsAs early as 14 days after the start of treatment with adalimumab a significant decrease in disease activity score evaluated in 28 joints was shown. There was no significant decrease in the influx of monocytes into the joint at this time.ConclusionsThis study indicates that adalimumab treatment does not reduce the influx of monocytes into the synovium early after initiation of treatment. As previous studies showed a rapid decrease in macrophage infiltration after TNF-antibody therapy, which could not be explained by increased cell death, this points to an important role for enhanced efflux of inflammatory cells from the synovium.