Single-cell sequencing-based analysis of CD4 + T-cell and B-cell heterogeneity in patients with lupus nephritis.

objectiveSystemic lupus erythematosus (SLE) is a prevalent chronic autoimmune disorder that can affect various organs and tissues throughout the body, with the kidneys being the most commonly involved, often leading to lupus nephritis (LN). Immune cells, notably CD4 + T cells and B cells, are crucial in the development of SLE. Investigating the heterogeneity of CD4 + T cells and B cells in patients with LN using single-cell technology is of significant importance.

methodsWith informed consent, this study explored the profiles of LN CD4 + T and B cells through single-cell transcriptome sequencing of peripheral blood mononuclear cell (PBMC) samples from three LN patients and three Normal Control(NC). Initially, the cells were segregated into immune cell clusters using cellular lineage-based clustering. Subsequently, CD4 + T and B cells were further categorized, and marker genes influencing LN CD4 + T and B cells were identified using differential analysis and publicly available data. The differentially expressed genes in LN patients were then functionally analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analyses. Additionally, cell communication analysis was conducted to examine the primary secretory pathways of immune cells, along with the profiles of numerous specific cell subtypes and ligand-receptor pairs.

resultsIn this study, we successfully mapped PBMC cells through single-cell sequencing of LN, and 69,069 single cells were clustered into 8 immune cell subpopulations using dimensionality reduction. These subpopulations included Neutrophils, NK cells, CD4 + T cells, CD8 + T cells, B cells, Macrophages, and Dendritic cells. Further subdivision of CD4 + T cells and B cells into subpopulations revealed that CD4 + T cells were divided into seven subpopulations, namely Tn, Th2, Th1, Tem, Treg, Tm, and Th17 subpopulations. The marker genes of each subpopulation mainly included CCR7, SELL, LEF1, TCF7, MALAT1, GZMA, IL7R, TGFB1, CCL5, KLRB1, CD4, and STAT3, among others. B cells were further divided into six subpopulations, namely Fo-B, AIM2 + Bcell, TCF4 + Bcell, Unknown (to be defined), B1-ATMBCs, and Trans-Bcell, with marker genes including TCL1A, IGHD, FCER2, AIM2, IGHA1, and JCHAIN.The GO enrichment analysis of each cell subpopulation focused on the regulation of lymphocyte activation, cellular response to type I interferon, and other processes. The KEGG analysis concentrated on the chemokine signaling pathway and cytokine-cytokine receptor interactions. Cell communication analysis revealed that the ligand-receptor pairs of the eight immune cell subpopulations were distributed across 11 signaling pathways, including ANNEXIN, BAFF, BAG, CCL, CXCL, FLT3, GALECTIN, GRN, IL16, MIF, and RESISTIN pathways. Three of these important ligand-receptor pairs were CD74 + CXCR4, CD74 + CD44, and IL16-CD4.Communication analysis of T and B cells revealed two-by-two interactions between CD4 + T, CD8 + T, and B cells, with the strongest interaction strengths observed between CD4 + T and CD8 + T, and between CD8 + T and B cells. CellChat identified 22 ligand-receptor pairs in 3 cell subsets, distributed across 12 signaling pathways, including ADGRE5, CD22, CD45, CD99, CLEC, ICAM, ITGB2, LCK, MHC-I, MIF, SELPG, and SEMA4 pathways. The most prominent signaling pathways were MHC-I, MIF, and CLEC, with major ligand-receptor pairs being HLA-B - CD8B, MIF - (CD74 + CXCR4), and CLEC2C - KLRB1.

conclusionIn summary, we clarified the immune cell profile of PBMCs from LN patients, with a focus on characterizing the heterogeneity of CD4 + T cells and B cells. We identified several specific cellular subtypes and ligand-receptor pairs, which suggest potential therapeutic targets for lupus erythematosus.