Functional consequences of allotypic polymorphisms in human immunoglobulin G subclasses

Abstract

Heritable polymorphisms within the human IgG locus, collectively termed allotypes, have often been linked by statistical associations, but rarely mechanistically, to a wide range of disease states. One potential explanation for these associations is that IgG allotype alters host cell receptors' affinity for IgG, dampening or enhancing an immune response depending on the nature of the change and the receptors. In this work, a panel of allotypic antibody variants were evaluated using multiplexed, label-free biophysical methods and cell-based functional assays to determine what effect, if any, human IgG polymorphisms have on antibody function. While we observed several differences in FcγR affinity among allotypes, there was little evidence of dramatically altered FcγR-based effector function or antigen recognition activity associated with this aspect of genetic variability.

Keywords: Allotype; Effector function; Fc receptor; IgG; Immunuglobulin; Polymorphism.

Figure 1:. Summary of IgG subclass and allotypic diversity

A. In this schematic representation of the human IgG heavy chain polymorphisms, the position of each amino acid identity relative to the residue number of each wheel corresponds to the allotype indicated at the same point on the wheel in the key at the top left of each subclass. The color of each wedge reflects the side chain chemistry, outlined at the top right of part B. The locations indicated by the pointers represent the best approximation of a residue’s location in the three-dimensional structure (PDB: 1HZH). B. Adapted from de Taeye et al, Frontiers in Immunology, 2020 (de Taeye et al., 2020). The names of each allotype are given using (from left to right) the numerical designation of the antigenic marker (or the simplified designation in the case of IgG3, where an asterisk replaces all subsequent components of the numerical designations), the allele name, and the older alphabetical designation. Color is used to denote side chain chemistry, and shading of a cell is used to call attention to amino acid identities that are not in the majority for that position in that subclass. ^aThese alleles represent the closest, but not exact, matches to the versions of the antibodies cloned and expressed for this work. The nature of the discrepancy is noted within each row. ^bThe produced versions do not include an isoallotypic substitution of Lys392. ^cThe native version of IGHG*03 contains only two core hinge units, not the three used in this work to maintain consistency with other IgG3s. ^dThe version of IGHG3*17 produced does not include the isoallotypic substitutions of Asn192 and Phe193

Figure 2:. Reactivity to common secondary reagents among Human IgG allotypes.

A-C. Specificity of isotype and subclass-specific antibody detection reagents. Fluorescently coded microspheres with a polyclonal anti-human IgG (A), monoclonal anti-human IgG subclass (B), or anti-human IgA and IgM (C) capture antibodies were incubated in allotype-switched mAbs across a titration range, and binding detected with a polyclonal anti-human IgG conjugate. Capture reagents are indicated at the top of each graph, and mAbs subclass and allotype are indicated with shape and color, respectively.

Figure 3:. Antigen recognition and virus neutralization properties across allotypes

A. Allotypic variants were tested over a range of concentrations to ensure that they retained binding to trimeric HIV-1 envelope protein (JRFL SOSIP) presented on beads. Median Fluorescent Intensity (MFI) is reported in Arbitrary Units (AU). B. Affinities of each allotypic variant to a monomeric gp120 envelope protein from the BaL strain of HIV-1 as defined by SPR. C. Neutralization potency across diverse HIV-1 strains was measured as the reduction in relative light units (RLU) from a reporter virus – target cell culture in the presence of differing VRC01 dilutions. Reported neutralization titers reflect the IgG concentration that reduced infectivity by 50% (IC₅₀). Statistical significance was defined using a one-way ANOVA with Sidek’s test for multiple comparisons, *p<0.05, **p<0.005.

Figure 4:. FcγR binding sensorgrams for IgG1 allotypes

Low affinity FcγR (rows) were evaluated over a range of concentrations for binding to IgG1 allotypic variants (columns). Typical rapid on and rapid off kinetics were observed. Association data was collected for 300s and dissociation for 350s.

Figure 5:. FcγR binding sensorgrams for IgG2 and IgG4 allotypes

Low affinity FcγR (rows) were evaluated over a range of concentrations for binding to IgG2 (red) and IgG4 (purple) allotypic variants (columns). Typical rapid on and rapid off kinetics were observed. Association data was collected for 300s and dissociation for 350s.

Figure 6:. FcγR binding sensorgrams for IgG3 allotypes

Low affinity FcγR (rows) were evaluated over a range of concentrations for binding to IgG3 allotypic variants (columns). Typical rapid on and rapid off kinetics were observed. Association data was collected for 300s and dissociation for 350s.

Figure 7:. Relative binding affinities for FcγR for allotypes by subclass

The affinity of each allotype replicate relative to the subclass mean for each receptor is plotted for each allotypic variant. Equilibrium binding affinities were calculated using the average response at equilibrium and a maximum response projected for each combination of spots and receptor series. The dotted line indicates the average affinity for each subclass and receptor pair. The significance of the affinity differences was tested using a one-way ANOVA Kruskal-Wallis test with Dunn’s test for multiple comparisons.

Figure 8:. In vitro effector functions of IgG allotypes

A-E. Allotypic variants were evaluated for their ability to promote effector functions in vitro. Those functions were antibodydependent cellular phagocytosis (ADCP) by THP-1 monocytes (A) and primary neutrophils (B); deposition of complement component C3 on IgG capture beads (C); and NK-92 degranulation (D) and target cell killing (E) as measures of antibodydependent cellular cytotoxicity (ADCC). Dotted vertical line in A indicates the midpoint of the x-axis scale. Dotted horizonal line in E indicates the level of killing observed in a technical control.