OA26 Paired bulk and single cell analysis of transitional B cells in systemic sclerosis highlights defective peripheral tolerance

Abstract Background/Aims Transitional B cells comprise a distinct population of B cells that have recently migrated to the periphery from the bone marrow. In systemic sclerosis (SSc), there are differences in the number and function of transitional B cells compared with healthy controls (HCs). In addition, we have previously shown that transitional B cells evade peripheral tolerance from SSc patients who are seropositive for anti-topoisomerase I autoantibodies (ATA). To investigate mechanisms that underpin this defect, we performed a paired bulk and single-cell RNA sequencing study of transitional B cell subsets in treatment-naïve ATA-positive patients with diffuse cutaneous SSc (dcSSc) and HCs. Methods CD19+CD24hiCD38hi transitional B cells (5000) were sorted from four HCs and four treatment-naïve ATA-positive dcSSc patients. Single-cell RNA-sequencing was performed on the sorted B cells and the data was analysed using Seurat in RStudio. Paired bulk RNA-sequencing of the sorted transitional T1 (CD19+CD24hiCD38hi CD27-IgMhiIgDmed) and T2 (CD19+CD24hiCD38hiCD27-IgMhiIgDhi) subsets was undertaken in a subset of this cohort. Results From the sorted CD19+CD24hiCD38hi lymphocytes single cell transcriptomics identified six B cell clusters in SSc patients and HCs with differential expression of key genes such as CD19 and TCL1A. This included a cluster of marginal zone precursor cells expressing PLD4 and MZB1 which is expanded in SSc. We also identified an interesting memory-like B cell cluster which was diminished in the SSc group and expresses genes such as TNFRSF13B (BAFF), AICDA (AID) and S100A10. In the HCs, this cluster also expresses genes such as CD27 and IGHA1 which are absent in SSc indicating that B cell development is impaired in the disease. Subsequent pathway analyses provided evidence for the molecular bases of defective tolerance in SSc patients. This appears to be related to dysregulated NF-κB signalling and aberrant extrafollicular B cell development as evidenced through pseudotime trajectory analysis. Differential gene expression analyses using bulk RNA-sequencing of sorted T1 and T2 transitional B cells further identified five candidate gene signatures such as MYC-targets and DNA repair which are significantly enriched in the T2 subset. This latter finding is in congruence with the single cell data and provides further evidence to support the existence of a peripheral tolerance/selection checkpoint at the transitional B cell stage. Conclusion We have identified four transitional B cell subsets and have characterised their transcriptomic profile in SSc. Additionally, we provide new evidence for a potential breach in peripheral tolerance of SSc patients. Further studies are underway to identify key pathways that underpin B cell dysregulation in SSc and explore the relevance of such findings for better targeted therapies. Disclosure C. Beesley: Grants/research support; C.B. is supported through Versus Arthritis (grant number 558800). N. Goldman: None. D. Abraham: None. C. Denton: None. R. Mageed: None. V.H. Ong: None.