Single-cell transcriptomic analysis of PBMCs from IgAN patients

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is a significant cause of chronic kidney disease. While previous studies have highlighted the role of immune cells in IgAN development, the disease's heterogeneous clinical presentation and prognosis have hindered a comprehensive understanding of its specific pathogenesis. This study aimed to identify circulating immune cell subsets linked to the progression of IgAN. Peripheral blood samples were obtained from 9 healthy controls and 17 biopsy-proven IgAN patients, and stratified into early- and late-stage groups based on their estimated glomerular filtration rate (eGFR) at the time of blood sampling. To characterize IgAN-specific immune cell profiles and underlying regulatory mechanisms, single-cell RNA sequencing was conducted to observe distinct immune cell profiles and transcriptomic differences between the IgAN groups. Notably, CD8+ T cells, particularly IL-7Ralow effector memory, exhibited a significant correlation with the disease progression. Trajectory analysis highlighted distinct CD8+ T-cell developmental patterns, with the early-stage group showing unique enrichment of memory CD8+ T cells. Gene expression profiling of CD8+ T-cell clusters indicated differences in activation and differentiation, emphasizing the critical role of CD8+ T cells in IgAN progression. In contrast, CD4+ T cells and B cells exhibited limited correlation with disease progression. The present transcriptome profiling underscores the prognostic significance of CD8+ T-cell subpopulations in IgAN progression. The findings highlight their potential as therapeutic targets and offers novel insights into the role of circulating immune cells in the progression of IgAN. Overall design: Blood samples were collected from a cohort of 26 donors consisting of 17 IgAN patients and 9 volunteer healthy donors and peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll–Histopaque gradient (1.077 g/mL; GE Healthcare Life Sciences, Piscataway, NJ, USA). The samples were stored in liquid nitrogen until analysis, preserved in a freezing medium containing 50% fetal bovine serum. Live PBMCs were subsequently isolated using BD FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA). Among the 26 subjects, 8 scRNA-Seq libraries were prepared using Chromium Next GEM Single Cell 5' Reagent Kits v1.1 (10x Genomics, Pleasanton, CA, USA) (refer to first batch), while the remaining 18 were processed using Chromium Next GEM Single Cell 5' Reagent Kits v2.0 (10x Genomics, Pleasanton, CA, USA) (refer to second batch). Libraries were sequenced on Novaseq 6000 platform (Illumina, San Diego, CA, USA). All cell barcodes and gene expression levels were compiled in a table format using Cell Ranger (10x Genomics, Pleasanton, CA, USA).