Single cell rna sequencing of circulating leukocytes fromiga nephropathy patients identifies an important role for innate immune cells

Abstract Background and Aims IgA-nephropathy (IgAN) is a slowly progressive disease characterized by mesangial deposition of IgA1 that causes kidney injury and can eventually lead to end stage renal disease. A key factor underlying the development of IgAN is the presence of galactose-deficient IgA1 (Gd-IgA1) in the circulation. The formation of IgA and IgG antibodies against Gd-IgA1 results in the formation of immune complexes which deposit in the kidney mesangium. Production of Gd-IgA1 hints to defects in B cell regulation. However, the exact immunological dysregulation leading to the formation of Gd-IgA1, and the subsequent antibody formation against it, remains elusive. Here we use single cell RNA sequencing (scRNA-seq) of circulating leukocytes for in depth analysis of immune cell populations in patients with active IgAN to gain a better understanding of the pathogenesis of this disease. Method Peripheral blood mononuclear cells were isolated for scRNA-seq from the blood of four patients with active IgAN, and four controls, matched for age and estimated glomerular filtration rate (eGFR). Cell hashing (Biolegend) of individual samples was performed before pooling samples for scRNA-seq. scRNA expression libraries were performed using the chromium X machine (10xgenomics) according to 10XGenomics single cell 5’ V2 user guide. Libraries were sequenced on an Illumina Nextseq500 and reads were mapped to GrCh38 using CellRanger. Differential gene expression analysis was performed for each immune cell type (FDR < 0.05). Reactome pathway enrichment analysis was performed on the differentially expressed genes. Results Identification of immune cell subtypes in IgAN patients and matched controls did not show a significant difference in cell numbers or ratios. B cells showed only minor changes in gene expression between IgAN patients and controls, and T cell populations showed changes in a limited set of genes, whereas interestingly, most differentially expressed genes were found in monocytes and NK cells (Figure 1A). Analysis of the differentially expressed genes showed enrichment for genes in interferon signaling pathways, mostly in monocytes and NK cells (Figure 1B). Gene enrichment analysis showed differential expression of type I IFN genes and antigen presentation genes, also mainly in monocytes and NK cells. Conclusion Our data shows the specific endotype of circulating immune cells from patients with active IgAN. While gene expression changes in T and B cells are discrete, changes in monocytes and NK cells appear especially prominent. In these cell types, we observed an important role for interferon signaling. Further analysis of scRNA-seq data in a larger patient cohort will follow, as well as analysis of T and B cell receptor clones in IgAN patients, with which we aim to gain new insights into immune dysregulation in IgAN.