Kidney Week

Abstract: PO1832

Single-Cell Transcriptomic Profiling Reveals Aberrant Signaling Responses to Tfh Cytokines in IgA₁-Secreting Cells from IgA Nephropathy Patients

Session Information

• Glomerular Diseases: IgA, C3G, and FSGS
  October 22, 2020 | Location: On-Demand
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Reily, Colin, University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

Colin Reily,

• Nakazawa, Shigeaki, Osaka University Graduate School of Medicine, Department of Urology, Osaka, Japan

Shigeaki Nakazawa,

• Kamata, Masakazu, University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

Masakazu Kamata,

• Crossman, David K., University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

David K. Crossman,

• Rizk, Dana, University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

Dana Rizk,

• Julian, Bruce A., University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

Bruce A. Julian,

• Novak, Jan, University of Alabama at Birmingham, Departments of Medicine, Microbiology and Genetics, Birmingham, Alabama, United States

Jan Novak,

Background

IgA nephropathy (IgAN), a common primary glomerulonephritis, is characterized by glomerular IgA1 immunodeposits enriched for galactose-deficient IgA1 (Gd-IgA1). These immunodeposits are likely derived from Gd-IgA1-containing immune complexes that are elevated in the circulation of IgAN patients. Gd-IgA1 is produced by IgA1-secreting cells due to abnormal expression of several glycosyltransferases in IgAN. Furthermore,some cytokines can enhance production of Gd-IgA1 by IgA1-secreting cells from IgAN patients but not those from healthy controls. We hypothesize that pro-inflammatory factors, such as those identified by GWAS or produced during episodes of synpharyngitic hematuria in IgAN patients, may further dysregulate IgA1-secreting cells and lead to enhanced Gd-IgA1 production.

Methods

As an experimental model, we used T-follicular helper (Tfh) cell-derived cytokines (IL-4, IL-6, IL-21, CD40L) to stimulate for 30 min immortalized Ig-producing cells derived from peripheral blood of IgAN patients and healthy controls. Then, single-cell transcriptomic analysis was performed. IgA1 splice variants were integrated into the hg38 reference genome to identify IgA1-secreting cells and the data analyzed using Seurat and Alteryx workflow. siRNA knock-down (k/d) was used to confirm involvement of candidate regulatory elements in cytokine-mediated overproduction of Gd-IgA1. Production of Gd-IgA1 was assessed after 72 h.

Results

Single-cell transcriptomics identified discrete populations of IgA1-secreting cells with differentialC1GALT1expression after Tfh-cytokine stimulation. Furthermore, these subpopulations exhibited reduced expression of cytokine-signaling regulatory elements, SOCS3,SOCS1, PTPN2, and PTPN11(p = 0.07, 0.04, 0.02, 0.07), indicating dysregulation of cytokine-signaling JAK/STAT pathways in IgAN-derived cells. Involvement of SOCS3 in Gd-IgA1 production was further supported by the observation that SOCS3siRNA k/d increased Gd-IgA1 production in healthy-control cells (p = 0.01).

Conclusion

Single-cell transcriptomics with stratification of IgA1-secreting cells based on C1GALT1expression and siRNA k/d experiments revealed that aberrant regulation of Tfh cytokine signaling was associated with enhanced Gd-IgA1 production in IgAN.

Funding

• NIDDK Support