The pathogenic anti-PLA2R autoantibodies cooperatively elicit the activation of complement in primary membranous nephropathy

Abstract Primary membranous nephropathy (pMN) is an autoimmune disease associated with autoantibody-mediated immune complexes and the deposition of activated complement proteins on the glomerulus. The phospholipase A2 receptor 1 (PLA2R) has been identified as a major antigen for autoantibodies in 80% of pMN patients. Antibodies of IgG1 and IgG3, but not IgG4, can activate complement. Unexpectedly, in pMN patients, the PLA2R autoantibodies are IgG4 predominance, and those of IgG1 and IgG3 are less abundant in the affected tissues, arguing the role of PLA2R autoantibodies in the pathologies of pMN. To study the molecular basis of individual PLA2R-reactive IgGs mediating complement activation in pMN, we employed a single-cell capture method and generated 16 anti-PLA2R monoclonal antibodies (mAbs). We characterize mAbs properties including epitope, affinity and gene feature. Then we adopted the CDC assay with PLA2R-podocytes to investigate the complement activation of the mAbs. Our data show that the combination of CTLD1 and any other epitope showed a synergistic effect that enhanced CDC activation, which single epitope anti-PLA2R mAb cannot achieve. Surprisingly, IgG1 mAb can still promote the CDC when pair with IgG4 mAb in synergistic effects. Moreover, polyclonal anti-PLA2R IgGs from pMN patients but not healthy volunteers can induce CDC. Next, we found that a group of patients only have CysR-IgG3, neither CTLD1-IgG1 nor IgG3; when we add CTLD1-IgG1 mAb can enhance CDC activation. Our data identify an essential role of complement-reactive IgGs, presumably IgG1 and IgG3 synergies between epitopes synergistic effects, and highlight the importance of immune complexes in complement activation by PLA2R autoantibodies, which underlies pMN.