Abstract: FR-PO819
Cell-Surface Glycosylation Identifies Subpopulations of B Cells in IgA Nephropathy with Distinct Signaling Corresponding to the Production of the Main Autoantigen, Galactose-Deficient IgA1
Session Information
- Glomerular Diseases: Inflammation and Immunology
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Person, Taylor, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Rizk, Dana V., 2Department of Medicine, Nephrology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Novak, Jan, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Reily, Colin, 2Department of Medicine, Nephrology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States
Background
Patients with IgA nephropathy (IgAN) have elevated levels of circulating autoantigen, galactose-deficient IgA1 (Gd-IgA1), that is recognized by IgG autoantibodies. Some of the resultant immune complexes deposit in the glomeruli and induce kidney injury. Based on the polymeric form of Gd-IgA1 in the circulating immune complexes and synpharyngitic hematuria at disease onset/activity, we hypothesize that only a subpopulation(s) of IgA1-secreting cells is responsible for Gd-IgA1 production, and that abnormal cellular responses further enhance Gd-IgA1 levels in circulation. However, as the autoantigen is released from B cells, identification of those subpopulations is difficult. Here we report that distinct cell-surface glycosylation and cellular responses to CpG-ODN (oligodeoxynucleotides) stimulation, are associated with Gd-IgA1 production and distinct intracellular signaling.
Methods
Immortalized IgA1-producing cells derived from peripheral blood of IgAN patients and healthy controls were stimulated with CpG-ODN (2 ug/mL) for 30 min and then stained with HPA and PNA to detect cell-surface GalNAc and GalNAc-Gal glycoconjugates and anti-IgA antibody to identify IgA-positive cells. Intracellular signaling was assessed using fluorochrome-conjugated antibodies specific for pSTAT1, pSTAT3, pSTAT5, pSTAT6, p38-MAPK, pERK1/2, p65-NF-kB, with flow cytometry readout using a BD FACSymphony with spectral compensation. Cells were incubated for 48 h for analyses of IgA and Gd-IgA1 in cell-culture supernatants by ELISA.
Results
CpG stimulation decreased the number of HPA+/PNA+ cells (p<0.05) as well as the amount (detected as median fluorescence index; MFI) (p<0.05). Cells with high HPA vs. low HPA differed in baseline and CpG-induced pSTAT3 (p<0.02), pSTAT5 (p<0.01), and pMEK1/2 (p<0.01). Cells with high HPA+/PNA+ showed significant correlation between Gd-IgA1 production and p38-MAPK (p<0.01), pSTAT3 (p<0.01), and p65 NF-kB (p=0.05) after CpG stimulation.
Conclusion
Differential cell-surface glycosylation identified differential cellular signaling, both baseline and CpG-induced, that correlated with Gd-IgA1 production. Further characterization of these cell subpopulations will provide insight into mechanisms involved in IgAN pathobiology.
Funding
- NIDDK Support