Revised porcine IgG subclass classification based on innate immune cell activation

The current classification of porcine immunoglobulin G (IgG) subclasses relies on phylogenetic analysis of gene sequences, resulting in a nomenclature that is inapt to predict biological function. Here, we propose a revised, functionality-based nomenclature derived from a comprehensive re-analysis of Fc domain interaction motifs and in vitro effector function assays. We produced twelve recombinant porcine IgG subclasses and evaluated their ability to induce reactive oxygen species (ROS) production in porcine monocytes and neutrophils. Our results reveal a striking functional dichotomy: only the subclasses re-classified here as the pIgG4 family (IgG4a, IgG4b, IgG4c and IgG4d) induced a significant respiratory burst. In silico analysis suggests this unique ability is linked to a conserved Glycine-Proline (“GP”) motif in the lower hinge, which is absent in other subclasses. Furthermore, we predicted the structural determinants of complement activation, identifying a putative role for a “PAP” motif and Lysine-320 in the predicted C1q binding site. Subclasses with a K320E mutation (IgG4d, IgG5 family) or sequence deviations in the “PAP” motif displayed reduced (IgG5 family) or abolished (pIgG3) complement activity. Additionally, the ancestral pIgG3 Fc domain was confirmed to be functionally inert, a phenotype most-likely driven by a P329L mutation that disrupts the hydrophobic Fc domain/Fcγ receptor interface. These findings challenge the previous assumption that pIgG1 is a potent activator of all porcine myeloid cells and highlight that discrete amino acid motifs play a role in IgG effector functions. The proposed functional nomenclature provides a predictive framework for porcine immunology that enables a better understanding of IgG subclass diversification in health and disease.