Sietse Q. Nagelkerke

Immunomodulation by IVIg and the Role of Fc-Gamma Receptors: Classic Mechanisms of Action after all?

Intravenous IgG (IVIg) contains polyclonal immunoglobulin G (IgG) from thousands of donors. It is administered at a low dose at regular intervals as antibody replacement therapy and at a higher dose as immunomodulatory treatment in various auto-immune or auto-inflammatory diseases. The working mechanism of immunomodulation is not well understood. Many different explanations have been given. During the last decade, we have focused on classical antibody binding via the Fc-domain of the IgG molecules to the common IgG receptors, i.e. the Fcγ receptors (FcγRs). Variation in the genes encoding human FcγRs determines function as well as expression among immune cells. As described here, NK cells and myeloid cells, including macrophages, can express different FcγR variants, depending on the individual’s genotype, copy number variation (CNV), and promoter polymorphisms. B-cells seem to only express the single inhibitory receptor. Although these inhibitory FcγRIIb receptors are also expressed by monocytes, macrophages, and only rarely by NK cells or neutrophils, their presence is unlikely to explain the immunomodulatory capacity of IVIg, nor does the sialylation of IgG. Direct IVIg effects at the level of the activating FcγRs, including the more recently described FcγRIIc, deserve renewed attention to describe IVIg-related immunomodulation.

Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene–matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1–4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.

Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis

OBJECTIVE To determine relevant Fc-gamma receptor (FcγR) polymorphisms in relation to susceptibility to SLE and LN, and to determine the functional consequences of genetic associations found. METHODS Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and relevant known functional single nucleotide polymorphisms of FcγRII and FcγRIII were determined in a LN-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow cytometry. RESULTS In multivariable analysis, low copy number of CNR1 (including FCGR3B; odds ratio (OR) 2.04; 95% CI: 1.29, 3.23), FCGR2A-131RR (OR 2.00; 95% CI: 1.33, 2.99), and the 2B.4 haplotype of FCGR2B (OR 1.59; 95% CI: 1.13, 2.24), but not FCGR2C open reading frame, were significantly (all P < 0.01) and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with LN and led to surface expression of FcγRIIb on neutrophils and monocytes. CONCLUSION This study is the first to investigate the most relevant and functional single nucleotide polymorphisms and copy number variations of FcγRII and FcγRIII polymorphisms in one study population, enabling the determination of the individual contribution of each polymorphism in multivariable analysis. Three polymorphisms were shown to be independently associated with susceptibility to SLE. The novel findings of a negative association of the 2B.4 haplotype with LN, and increased expression of FcγRIIb on neutrophils and monocytes as a result of this 2B.4 haplotype warrant future research in the role of these cells and FcγRs in the pathogenesis of SLE and LN.

FcγRIIa cross-talk with TLRs, IL-1R, and IFNγR selectively modulates cytokine production in human myeloid cells

Myeloid antigen-presenting cells (APCs) tailor immune responses to the pathogen involved through the production of specific pro- and anti-inflammatory cytokines. It is becoming increasingly clear that the ultimate cytokine profile produced by myeloid APCs crucially depends on interaction between multiple pathogen recognizing receptors. In this respect, we recently identified an important role for cross-talk between Fc gamma receptor IIa (FcγRIIa) and Toll-like receptors (TLRs) in human dendritic cells (DCs), which induces anti-bacterial immunity through the selective induction of TNFα and Th17-promoting cytokines. Here, we show that FcγRIIa-TLR cross-talk is not restricted to DCs, but is a common feature of various human myeloid APC subsets including monocytes and macrophages. Interestingly, FcγRIIa-TLR cross-talk in monocytes resulted in the induction of a cytokine profile distinct from that in DCs and macrophages, indicating that FcγRIIa stimulation induces cell-type and tissue specific responses. Surprisingly, we show that the FCGR2A H131R single nucleotide polymorphism (SNP), which is known to greatly affect FcγRIIa-mediated uptake of IgG2-opsonized bacteria, did not affect FcγRIIa-dependent cytokine production, indicating that these processes are differently regulated. In addition, we demonstrate that FcγRIIa selectively synergized with TLRs, IL-1R, and IFNγR, but did not affect cytokine production induced by other receptors such as C-type lectin receptor Dectin-1. Taken together, these data demonstrate that FcγRIIa-dependent modulation of cytokine production is more widespread than previously considered, and indicate that cross-talk of FcγRIIa with various receptors and in multiple cell types contributes to the induction of pathogen and tissue-specific immunity.

Failure to detect functional neutrophil B helper cells in the human spleen.

A novel role for human neutrophilic granulocytes was recently described, showing that these cells, upon entering the spleen, can be reprogrammed into a distinct B cell-helper neutrophil phenotype that is capable of eliciting B cell responses such as immunoglobulin secretion, class switch recombination and somatic hypermutation. Using similar protocols, we detected a homogeneous population of CD15highCD16high neutrophils in fresh human spleen samples, which did not differ in phenotype and function from blood neutrophils. No phenotypic characteristics of costimulatory nature were detected on splenic or circulating neutrophils, nor could we reproduce the immunoglobulin production of splenic B cells in the presence of splenic neutrophils, although B cell function and neutrophil activity were normal. Independent confirmation of a role for NBH cells is required.

Haplotypes of FcγRIIa and FcγRIIIb Polymorphic Variants Influence IgG-Mediated Responses in Neutrophils

Human blood neutrophils normally express two FcγRs (FcγRIIa and FcγRIIIb) that, upon multivalent binding of IgG in immune complexes or on opsonized targets, mediate responses such as phagocytosis, Ab-dependent cellular cytotoxicity, and respiratory burst. Allelic variants have been described for both FcγRIIa (131H/R) and FcγRIIIb (NA1/NA2/SH), with different binding affinity for IgG subclasses. Because neither of these variants acts alone, we have set out to systematically analyze in a large cohort of healthy FCGR2/3-genotyped volunteers how the different haplotypes of neutrophil FcγRs functionally interact. Maximal IgG-induced H2O2 production by neutrophils from individuals with different (homozygous) haplotypes was observed in the following order: 131HH-NA2NA2 > 131RR-NA1NA1 > 131HH-NA1NA1 > 131RR-NA2NA2. Although FcγRIIa 131H is known to bind IgG1 and IgG2 more avidly, no such differences in affinity are known for FcγRIIIb variants. Nonetheless, a remarkable impact of the FcγRIIIb variants on IgG-mediated neutrophil activity was thus demonstrated, which was not explained by differences in FcγR surface expression. The FcγR expression profile was changed by overnight G-CSF/IFN-γ activation of the neutrophils and eliminated any haplotypic impact on the respiratory burst. To our knowledge, our results are the first to provide an integrated functional analysis of neutrophil FcγR haplotypes and suggest that particularly the early phase of IgG-mediated neutrophil reactivity is influenced by FCGR2/3 genotypic variation.

Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Nonclassical FCGR2C haplotype is associated with protection from red blood cell alloimmunization in sickle cell disease

Red blood cell (RBC) transfusions are of vital importance in patients with sickle cell disease (SCD). However, a major complication of transfusion therapy is alloimmunization. The low-affinity Fcγ receptors, expressed on immune cells, are important regulators of antibody responses. Genetic variation in FCGR genes has been associated with various auto- and alloimmune diseases. The aim of this study was to evaluate the association between genetic variation of FCGR and RBC alloimmunization in SCD. In this case-control study, DNA samples from 2 cohorts of transfused SCD patients were combined (France and The Netherlands). Cases had a positive history of alloimmunization, having received ≥1 RBC unit. Controls had a negative history of alloimmunization, having received ≥20 RBC units. Single nucleotide polymorphisms and copy number variation of the FCGR2/3 gene cluster were studied in a FCGR-specific multiplex ligation-dependent probe amplification assay. Frequencies were compared using logistic regression. Two hundred seventy-two patients were included (130 controls, 142 cases). The nonclassical open reading frame in the FCGR2C gene (FCGR2C.nc-ORF) was strongly associated with a decreased alloimmunization risk (odds ratio [OR] 0.26, 95% confidence [CI] 0.11-0.64). This association persisted when only including controls with exposure to ≥100 units (OR 0.30, CI 0.11-0.85) and appeared even stronger when excluding cases with Rh or K antibodies only (OR 0.19, CI 0.06-0.59). In conclusion, SCD patients with the FCGR2Cnc-ORF polymorphism have over a 3-fold lower risk for RBC alloimmunization in comparison with patients without this mutation. This protective effect was strongest for exposure to antigens other than the immunogenic Rh or K antigens.

RhIg-prophylaxis is not influenced by FCGR2/3 polymorphisms involved in red blood cell clearance

To the editor: Red blood cell (RBC) alloimmunization against the RhD antigen may occur in RhD-negative women during pregnancy or at delivery of a RhD-positive child.[1][1] The administration of Rh-immunoglobulin (Rh-Ig) prevents anti-D alloimmunization and subsequent hemolytic disease of the fetus

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing

The efficacy of anticancer monoclonal antibodies (mAbs) is limited by the exhaustion of cellular effector mechanisms. The combination of IgG and IgA to two different tumor targets leads to enhanced cytotoxicity, providing a basis for therapeutic mAb improvements. Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy. Cancer Immunol Res; 3(12); 1316–24. ©2015 AACR.