Cooperative Elicitation of Complement-Dependent Cytotoxicity by Anti-PLA2R Autoantibodies in Primary Membranous Nephropathy
- Glomerular Diseases: Podocyte Biology - II
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1403 Podocyte Biology
- Wu, Tsai-yi, Chang Gung University, Taoyuan, Taoyuan, Taiwan
- Tu, Kun-Hua, Chang Gung University, Taoyuan, Taiwan
- Ku, Cheng-Lung, Chang Gung University, Taoyuan, Taoyuan, Taiwan
Group or Team Name
- Laboratory of Human Immunology and Infectious Diseases.
Primary membranous nephropathy (pMN) is an autoimmune disease causing abnormalities in the glomerular basement membrane and podocytes, leading to proteinuria. These changes relate to autoantibodies and accumulated activated complement proteins on the glomerulus. Autoantibodies, primarily against phospholipase A2 receptor 1 (PLA2R) found in 80% of pMN patients, are thought to activate the complement system and contribute to pMN. Surprisingly, the predominant PLA2R autoantibodies in pMN patients are IgG4, which doesn't activate the complement, while the complement-activating IgG1 and IgG3 are less common, questioning the role of PLA2R autoantibodies in pMN pathogenesis.
We isolated monoclonal anti-PLA2R antibodies (mAbs) from pMN patients' PBMC using single B cell sorting, then amplified the IgG gene for cloning into F293 cell vectors. We confirmed the mAbs' characteristics via Western blot, ELISA, and a BLI system. These mAbs were found binding to the membrane-bound PLA2R on human podocyte cells through immunocytochemistry. By using a Lentivirus system, we created a PLA2R-overexpressing podocyte line for a disease model. We assessed the mAbs' ability to trigger complement-dependent cytotoxicity (CDC) using a CellTiter-Glo assay to measure cell viability and complement-induced lysis.
We studied PLA2R-reactive IgGs' role in complement activation in pMN, using a single-cell method to generate 16 anti-PLA2R mAbs from five patients. Our analysis revealed variable binding affinity across mAb groups, independent of binding sites. Using a CDC assay on PLA2R-overexpressing podocytes, we discovered that the combination of CTLD1 with another mAb significantly boosted CDC activation. After modifying the mAbs to IgG4, we found that a single IgG1 mAb was critical for CDC activation, but IgG4 could enhance it. Intriguingly, polyclonal IgGs from pMN patients, not from healthy volunteers, induced CDC. Furthermore, some patients with only CysR-IgG3 showed increased CDC activation with added CTLD1-IgG1 mAb.
These results identify an essential role of complement-reactive IgGs, presumably IgG1 and IgG3 synergies between single-domain epitopes CysR and CTLD1, which underlies pMN.
- Government Support – Non-U.S.