Cross-Species Analysis of FcγRIIa/b (CD32a/b) Polymorphisms at Position 131: Structural and Functional Insights into the Mechanism of IgG-Mediated Phagocytosis in Human and Macaque

William Tolbert¹Paula B. Nhan¹Haleigh E Conley²XIAOXUAN GE³Monika Chandravanshi¹Madeleine Lee¹Julianna Veilleux¹Marek Korzeniowski¹Suneetha Gottumukkala¹Margaret E Ackerman³Justin Pollara²Marzena E Pazgier^1,4*

• ¹Uniformed Services University of the Health Sciences, Bethesda, United States
• ²Duke University School of Medicine, Durham, United States
• ³Dartmouth College Thayer School of Engineering, Hanover, United States
• ⁴Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, United States

Antibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques (Macaca mulatta), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2. We determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species and linked these structures to affinity and activity. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc. Through analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species-and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P131 variant showing uniformly reduced IgG affinity. These insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.