Assigning Immunoglobulin Class from Single-Cell Transcriptomes in IgA1-Secreting Versus Membrane Subpopulations

Abstract

IgA nephropathy (IgAN) is an autoimmune disease characterized by renal glomerular immunodeposits enriched for galactose-deficient IgA1 (Gd-IgA1; autoantigen) with the corresponding IgG autoantibodies. Despite the known contribution of Gd-IgA1 to IgAN, little is known concerning IgA1-secreting subpopulations responsible for autoantigen production. The goal of this study is to identify IgA1-secreting and membrane subpopulations from single-cell transcriptomic analysis. We developed a novel single-cell analytics workflow to discern cells expressing IgA1 secreted isoform or membrane-bound isoform. Multiple approaches were compared to assess immunoglobulin-isotype identity in single cells, and multiple immunoglobulin heavy-chain genes expressed in the same cells were found. To better identify specific immunoglobulin heavy-chain transcripts, we merged a software platform called Alteryx with the existing single-cell R toolkit program Seurat. This process allowed for improved calls on IgA1-secreting subpopulations based on secreting versus membrane splice-variant expression levels.

Phosphatase control of cytokine-mediated overproduction of galactose-deficient IgA1, the main autoantigen in IgA nephropathy

“…Further processing used an open-source R software tool from Dr. Satija’s lab, Seurat V4.01 ( https://satijalab.org/seurat/ ), and a software package from Alteryx for the flexible design of large-scale database analysis ( www.alteryx.com ). In addition, scaffold gene data (non-contiguous gene sequences not in hg38) from published sources on splice variants of IGHA1 (immunoglobulin heavy chain A1) that determine secreted ( IGHA1s ) vs. membrane-bound ( IGHA1m ) IgA1 isoforms were inserted into the hg38 gene reference database [ 25 ]. Based on this approach, we identified 1164 and 1400 IgA1-secreting cells ( IGHA1s) from healthy-control- and IgAN-derived cells, respectively.…”