Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding

A Robin Temming,Arthur E.H Bentlage,Steven W De Taeye,Gerlof P Bosman,Suzanne N Lissenberg-Thunnissen,Ninotska I.L Derksen,Giso Brasser,Juk Yee Mok,Wim J.E Van Esch,Heather L Howie,James C Zimring,Gestur Vidarsson

doi:10.1016/j.molimm.2020.08.015

A Robin Temming, Arthur E.H Bentlage + Show 10 more

Open Access

https://doi.org/10.1016/j.molimm.2020.08.015

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Abstract

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs.

Highlights

Immunoglobulins (Ig) play a pivotal role in adaptive immune re sponses and are produced and secreted by plasma B cells
Amino acid sequences were derived from UniProt database with entries: P01857, P01859, P01860, P01861, P01868, P01863, P01867, A0A0A6YY53, P03987 for Immunoglobulin G (IgG) subclasses and P12314, P12318, P31994, P31995, P08637, O75015, P26151, P08101, P08508, A0A0B4J1G0 for FcγRs
Full profiles of binding reactivities of human IgG (hIgG) with mouse and macaque FcγR were published (Chan et al, 2016; Dekkers et al, 2017a; Derebe et al, 2018). We extended this knowledge by providing for the first time detailed quantifiable information on cross-reactivity between the com plete repertoire of mouse IgG (mIgG) and all human FcγR (hFcγR) classes

Summary

Introduction

Immunoglobulins (Ig) play a pivotal role in adaptive immune re sponses and are produced and secreted by plasma B cells. Each Ig molecule has a dimeric structure with two antigen-binding fragment (Fab) domains symmetrically linked via the hinge region to one crys tallizable fragment (Fc) domain This Fc portion enables antibodies to exert effector functions like activation of the complement system through interaction with C1q, or activation of myeloid and natural killer (NK) cell-mediated effector functions through IgG-Fc receptors (FcγRs). Immunoglobulins comprise several classes (IgM, IgD, IgA, IgE, IgG) of which IgG make up the main fraction in serum and can, in humans, be further divided into IgG1, IgG2, IgG3 and IgG4 These IgG subclasses show highly conserved amino acid sequences (more than 90% homol ogy) (Vidarsson et al, 2014) but each having its unique functional characteristics, e.g. acting through different FcγRs (de Taeye et al., 2019; Vidarsson et al, 2014). Some strains of mice express IgG2c instead of IgG2a (e.g. in C57BL/6 mice), likely as an allotypic variation (Martin et al, 1998)

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