Highlights • FcγR polymorphisms affect binding to IgG and thereby strength of immune reactions. • Likewise, FcγR-IgG binding is affected by which IgG subclasses are involved. • Biological differences ...between IgG allotypes also exist, particularly for IgG3. • IgG effector functions are also strongly affected by IgG-Fc glycosylation patterns. • IgG-Fc glycan type is antigen dependent, ultimately governed on the B-cell level.
A central dogma in immunology is that an antibody's in vivo functionality is mediated by 2 independent events: antigen binding by the variable (V) region, followed by effector activation by the ...constant (C) region. However, this view has recently been challenged by reports suggesting allostery exists between the 2 regions, triggered by conformational changes or configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. Here we review the types of cooperativity in IgG molecules by examining evidence for and against allosteric cooperativity in both Fab and Fc domains and the characteristics of associative cooperativity in effector system activation. We investigate the origin and the mechanism of allostery with an emphasis on the C-region-mediated effects on both V and C region interactions, and discuss its implications in biological functions. While available research does not support the existence of antigen-induced conformational allosteric cooperativity in IgGs, there is substantial evidence for configurational allostery due to glycosylation and sequence variations.
Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by ...IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.
Antibodies are essential mediators of immunological defense mechanisms, are clinically used as therapeutic agents, but are also functionally involved in various immune-mediated disorders. Whereas IgG ...antibodies accomplish some of their biological tasks autonomously, many functions depend on their binding to activating and inhibitory Fcγ receptors (FcγR). From a qualitative point of view expression patterns of FcγR on immunologically relevant cell types are well-characterized both for mice and humans. Surprisingly, however, there is only quite limited information available on actual quantities of FcγR expressed by the different leukocyte populations. In this study we provide a comprehensive data set assessing quantitatively how many individual human and mouse FcγRs are expressed on B cells, NK cells, eosinophils, neutrophils, basophils and both classical, and non-classical monocytes under steady state conditions. Moreover, among human donors we found two groups with different expression levels of the inhibitory FcγRIIb on monocytes which appears to correlate with haplotypes of the activating FcγRIIIa.
Summary
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.
Long-lived “memory-like” NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have ...shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.
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•NK cells with multiple protein deficiencies are present in HCMV-infected individuals•SYK deficiency is associated with hyper-methylation of the gene promoter•Memory-like NK cells have protein deficiencies in combination with FcRγ deficiency•FcRγ-deficient NK cells expand preferentially in an antibody-dependent manner
Long-lived “memory-like” NK cells have been identified in HCMV-infected individuals at variable frequencies, but little is known about how this NK cell pool is formed. Kim and colleagues show data that support epigenetic modifications and antibody-dependent expansion as mechanisms underlying the formation of this memory-like NK cell pool.
The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a ...glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')
blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of
causing different levels of surface FcγRIIIb expression. Individuals with one copy of
showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG
heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.
The placental transfer of maternal IgG is critical for infant protection against infectious pathogens. However, factors that modulate the placental transfer of IgG remain largely undefined. ...HIV-infected women have impaired placental IgG transfer, presenting a unique “disruption model” to define factors that modulate placental IgG transfer. We measured the placental transfer efficiency of maternal HIV and pathogen-specific IgG in US and Malawian HIV-infected mothers and their HIV-exposed uninfected and infected infants. We examined the role of maternal HIV disease progression, infant factors, placental Fc receptor expression, IgG subclass, and glycan signatures and their association with placental IgG transfer efficiency. Maternal IgG characteristics, such as binding to placentally expressed Fc receptors FcγRIIa and FcγRIIIa, and Fc region glycan profiles were associated with placental IgG transfer efficiency. Our findings suggest that Fc region characteristics modulate the selective placental transfer of IgG, with implications for maternal vaccine design and infant health.
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•Maternal HIV progression factors are associated with poor placental IgG transfer•Binding to placental Fcγ receptors can predict placental IgG transfer efficiency•Fc region glycan profiles can predict the efficiency of placental transfer of IgG
The impaired transfer of maternal IgG from HIV-infected mothers to their infants is associated with altered binding to Fc receptors FcγRIIa and FcγRIIIa as well as glycan modifications in the Fc region.
House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific ...immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles.
Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success.
Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score.
Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities.
Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.
CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody ...clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody–CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ε receptor γ–chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell–derived CD16a but yielded N-glycoforms different from NK cell–derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein’s structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.
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