Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti-RhD (anti-D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG-Fc-receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically-applied monocyte-based antibody-dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N-linked glycan at position 297 in the IgG-Fc-region, consisting of several different glycoforms. We therefore systematically analysed IgG-derived glycopeptides by mass spectrometry from 70 anti-D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG-fucosylation to be an important pathological feature in HDFN with diagnostic potential.

RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered.

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.