Background: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small-vessel autoimmune vasculitis which can result in life-threatening kidney failure or pulmonary haemorrhage. The most prevalent phenotypes are granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), mainly associated with anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO), respectively. Besides the presence of ANCA, increased BAFF levels and alterations in the peripheral B cell compartment, the beneficial clinical effects of peripheral B cell depletion using rituximab (anti-CD20) therapy ultimately confirmed the importance of B cells in AAV. However, this treatment results in impaired humoral immune responses and permits the survival of (autoreactive) long-lived plasma cells. Hitherto, characterization of the B lineage in AAV relies mainly on the peripheral blood, but it is imperative to also study the difficult-to-access plasma cell niches in other compartments of the immune system, such as lymph nodes and bone marrow, as well as in the target tissues (i.e. lungs/kidneys), to fully determine the role of B lineage cells in the pathogenesis of AAV.

Objectives: To profile immune cells in kidney biopsies from AAV patients with active disease and a nephrectomy control using a single-cell RNA sequencing (scRNAseq) approach, and characterize the different clusters of B lineage cells in more detail.

Methods: Needle-core kidney biopsies were obtained from 4 patients with AAV glomerulonephritis and one nephrectomy control. Live CD45 ⁺ kidney immune cells were sorted from the kidney single-cell suspensions and processed for single-cell data generation and analysis, performing the adequate filtering and pre-processing steps. Mapping the dataset to the mature immune kidney cell atlas was used for annotation.

Results: We identified 24 clusters based on the reference at resolution 1, from which 2 B cell enriched clusters (based on CD19/CD20 expression; 5.07% of total) and one plasmablast/plasma cell cluster (based on CD38/CD138 expression; 0.45% of total) were selected for further analysis. These 3 clusters were re-clustered at low and high resolutions, rendering 4 to 9 different subpopulations. The 4 clusters obtained at low resolution suggest the presence of memory B cells ( MS4A1, CD19, CD27, PAX5 ), naïve B cells ( MS4A1, CD19, IGHD, PAX5 ), a plasmablast-like population ( MS4A1, CD19, CD27, TNFRSF13C, XBP1 ) and plasma cells ( CD38, IGHG1, SDC1, IRF4, XBP1, PRDM1 ). Generally, the relative mean frequencies of the B lineage populations were 48.4% memory B cells, 41.7% naïve B cells, 4.1% plasmablasts, and 5.8% plasma cells. With higher resolution analysis, 4 different subtypes of memory B cells were observed, in addition to 3 naïve/germinal center-like B cells, and the 2 previously well-defined plasmablast-like and plasma cell clusters, which need to be characterized in more detail.

Conclusion: These preliminary data characterise for the first time cells of the B and plasma cell lineage in kidney biopsies from AAV patients with glomerulonephritis using scRNAseq. Subsequent in-depth study of the resulting clusters may allow identification of subsets potentially specific for AAV (or even ANCA subtype) that could be actively involved in the immune response in the affected kidneys. Finally, gene set enrichment analysis of the data may reveal differential gene expression patterns and intracellular signalling pathways involved, which may provide novel targets for more directed therapies.

REFERENCES NIL. Acknowledgements: NIL.

Disclosure of Interests: None declared.