Humoral immune responses

Project leaders: Gestur Vidarsson PhD and Prof Ellen van der Schoot MD PhD

In most immune mediated blood cell diseases and in all fetal/neonatal alloimmune cytopenias the destruction of blood cells is mediated by antibodies. We are therefore investigating 1) the B cells producing these antibodies, 2) the characteristics of these antibodies and 3) the interaction of antibodies with the FcRn, the receptor responsible for placental transport.

B cells

We have previously developed a culture method in which B cells can be cultured and stimulated to Ig production at the single cell level. Using this method we found that in hyperimmune anti-D donors the majority of antigen-specific memory cells resides in the IgM-positive B cells. Upon antigen challenge the number of IgG-positive cells increases, whereas the IgM positive cells remains stable. We found that the BCL6 mutational status of the IgM memory cells was lower than of their CD27+IgG+ counterparts. In analogy with recent studies in mice, we postulate that the IgM memory cells will not class switch in the presence of IgG in the serum, but can replenish the memory pool once the titer has dropped and in case of infectious agents the antigenic make-up of the pathogen might have changed.

Antibodies

By analyzing the Fc-glycosylation of the pathogenic, affinitypurified IgG1 alloantibodies formed during pregnancy against antigens of the fetus (HPA-1 or RhD) at the glycopeptide level using mass spectrometry, we found markedly decreased levels of core-fucosylation as well as increased levels of galactosylation and sialylation as compared to glycosylation patterns of total serum IgG1 of the same patients. Because IgG1 Fc-core-fucosylation is known to influence ADCC activity, modulation of core-fucosylation may have a profound effect on disease severity and prognosis. To correlate Fc-glycosylation with biological activity we have developed an assay to determine the induction of FcRIII mediated respiratory burst by anti-HPA opsonized platelets. Remarkably, this assay was found to be dependent on the presence of C-reactive protein. This serum protein was found to be increased in cord blood samples of FNAIT (Fetal Neonatal Alloimmune Thrombocytopenia) affected neonates.

FcRn

Human IgG3 displays the strongest effector functions of all human IgG subclasses but has a short half-life, suggesting FcRn-mediated IgG salvage to be defective for IgG3. We have previously observed that human IgG1 inhibited FcRn-mediated transport of IgG3 at the level of receptor binding. The observed inhibition was due to a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other known IgG species, as H435-engrafted IgG3 inhibited FcRn-mediated transport and rescue of R435-IgG1. An arginine in position 435 in IgG may theoretically affect (see figure) binding to FcRn in two ways – either through less sensitivity to deprotonation and enhanced binding at pH 7.4 and/or through steric hindrance because of the longer sidechain. We found indeed that both factors may play a role. Both IgG1 and IgG3 bound FcRn in a pH dependent manner, with similar high affinity at pH 6.0 but residual binding at pH 7.4. However, R435-containing IgG bound better to FcRn at pH 7.4 than H435-containing IgG1, an advantage lost at pH 6.0 as seen by ELISA. Using an FcRn-coated biosensor we observed H435-containing IgG to bind FcRn slightly better at pH6.0, confirming previous findings showing inferior binding of IgG3 to FcRn and hinting at a possible allotypic variation affecting the binding affinity for IgG3. Our observation of the increased binding of IgG3 at pH 7.4 but decreased binding at pH 6.0 compared to IgG1, suggests a decreased capacity of IgG3 to release FcRn at neutral pH and decreased competitiveness for FcRn binding at pH 6.0. This is in line with the functional evidence presented here on the decreased recycling rate, and short biological half life of IgG3. Importantly we showed that the half lives of H435-containing IgG3 allotypes in humans are comparable to IgG1. This H435-IgG3 also proved better suited for protection against pneumococcal challenge in mice, demonstrating that H435-IgG3 is a formidable candidate for monoclonal antibody therapies in patients.

Inhibition of IgG3 transport by IgG1 is due to R435 in IgG3

Inhibition of IgG3 transport by IgG1 is due to R435 in IgG3
A) Mutating the amino acid at position 435 in IgG1 (H435) and in IgG3 (R435) to an alanine reduces transport, while exchanging the histidine native to IgG1 and the arginine native to IgG3 on each others backbone had no effect on their transport rate when offered separately to FcRntransfected A375 cells.

B) While transport of IgG3-WT was inhibited in the presence of IgG1-WT, IgG1 bearing an alanine or an arginine at position 435 had no effect on IgG3 transport. C) Transport of IgG3 with a histidine at position 435 was not inhibited by WT IgG1. When the amino acids found at position 435 in IgG1 and IgG3 were swapped, IgG1-H435R transport was inhibited by IgG3-R435H. A-

C) The +/- indicate the presence or absence of IgG (10 μg/ml/subclass), IgG1 is represented by open bars, IgG3 by hatched bars. The presence of mutated variants (435H, 435A, 435R) is indicated by the corresponding letter. The data represent mean and standard deviation from 3 independent experiments. Transport of WT IgG was compared to transport of mutant IgG by one-way ANOVA with Dunnett’s multiple comparison test and significance is indicated as described in the Materials and Methods section.

 

Key publications