SINGLE-CELL MENDELIAN RANDOMIZATION REVEAL THE REGULATORY MECHANISMS OF CD4+ T CELL ACTIVATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, and the kidney is a commonly affected organ in SLE. CD4+ T cells play a central role in the pathogenesis of SLE. Their abnormal activation not only promotes the production of autoantibodies by B cells but also directly infiltrates tissues such as the kidneys, contributing to damage. To gain deeper insights into the molecular mechanisms underlying CD4+ T cell activation in SLE, this study integrated single-cell expression quantitative trait locus (eQTL) data, Mendelian randomization (MR), and colocalization analysis. Based on single-cell transcriptomic data from 10 lupus nephritis patients, we systematically investigated the regulatory mechanisms of CD4+ T cell activation in SLE at the single-cell level.

Using single-cell eQTL data as exposure variables and combining summary data from SLE genome-wide association studies (GWAS), we performed two-sample MR analyses to evaluate the causal relationship between eQTLs and SLE risk. Methods such as LD check and Coloc were used for colocalization validation. Significantly associated genes were subjected to functional enrichment analysis, protein-protein interaction network construction, tissue-specific expression analysis, and mouse phenotype association analysis. Additionally, we conducted MR analyses using dynamic single-cell eQTL data from the CD4+ T cell activation process and collected single-cell transcriptomic data from peripheral blood mononuclear cells (PBMCs) of 10 lupus nephritis patients to validate expression differences of candidate genes. The regulatory mechanism of RNF5 in mediating abnormal CD4+ T cell activation in SLE was further elucidated at the molecular and protein levels.

MR and colocalization analyses based on three SLE GWAS cohorts identified 269 genes significantly associated with SLE risk across 14 immune cell types. Among these, CD4+ T cells contained the highest number of associated genes, with nearly half exhibiting cell-type-specific regulatory patterns. Functional enrichment analysis revealed that these genes were significantly enriched in SLE-related pathways, immune response activation, and B cell receptor signaling pathways. Single-cell transcriptomic validation analysis showed differential expression of candidate genes in CD4+ T cells of SLE patients. Further MR analysis of CD4+ T cell activation data identified key genes regulating CD4+ T cell activation, among which RNF5 showed a significant and consistent association with SLE risk. Finally, both single-cell transcriptomic and qPCR data from 10 lupus nephritis patients indicated downregulated expression of RNF5 in CD4+ T cells. Western Blot results further suggested that RNF5 downregulation may promote STING pathway activation, influencing interferon production and mediating abnormal activation in CD4+ T cells of SLE patients.

By integrating single-cell eQTL data, multi-cohort Mendelian randomization, and colocalization analysis, this study revealed the genetic regulatory mechanisms of systemic lupus erythematosus and highlighted the critical role of specific immune cell subsets, such as CD4+ T cells, in disease pathogenesis. A total of 269 genes significantly associated with SLE risk were identified. More importantly, through dynamic eQTL analysis and single-cell transcriptomic validation, this study uncovered the regulatory role of RNF5 in abnormal CD4+ T cell activation. RNF5 may influence interferon responses via the STING pathway, providing a new molecular perspective for understanding the functional role of CD4+ T cells in SLE.