Single-cell multiomics reveals activation of the STAT3-PIM1 axis in T-cell subsets in proteinase 3-ANCA-positive granulomatosis with polyangiitis.

Abstract

Granulomatosis with polyangiitis (GPA) is an autoimmune disease marked by relapses and treatment-related morbidity. CD4⁺ T cells play a central role in GPA pathogenesis, but their molecular profile remains incompletely defined. This study aimed to characterise CD4⁺ T cells in patients with proteinase 3-antineutrophil cytoplasmic antibody (ANCA)-positive GPA (PR3-GPA) using single-cell multiomics to identify molecular signatures associated with disease activity. Peripheral blood mononuclear cells were obtained from patients with PR3-GPA during active disease, remission, and healthy controls. Single-cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) were used to define transcriptional and protein expression profiles of CD4⁺ T cell subsets. Findings were validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry-based assays, including phospho-flow cytometry. Serum cytokines were measured by a Luminex assay, and CD4⁺ T-cell responses to Pim kinase inhibition were assessed. Spatial transcriptomics data from kidney biopsies from patients with PR3-ANCA-positive-associated crescentic glomerulonephritis were analysed. scRNA-seq and CITE-seq revealed upregulated PIM1 expression in CD4⁺ T-cell subsets in active GPA compared with controls. RT-qPCR and flow cytometry-based analyses confirmed elevated expression of PIM1, SOCS3, and STAT3 in CD4⁺ T cells from patients with active disease. STAT3 phosphorylation and serum interleukin-6 (IL-6) were increased in active GPA. Pim kinase inhibition reduced CD4⁺ T-cell activation, proliferation, and cytokine production. Spatial transcriptomics showed upregulated STAT3 and PIM kinase expression in inflamed regions of PR3-ANCA-associated glomerulonephritis biopsies. These findings indicate that circulating CD4⁺ T cells in active PR3-GPA are characterised by activation of the STAT3-PIM1 axis, potentially driven by upstream IL-6 signalling. This supports further investigation of Pim kinase inhibition and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway modulation as potential therapeutic strategies in PR3-GPA.