VCOM Research Day Program Book 2023

Graduate Student Research Biomedical

01 Single-Cell RNA Sequencing Reveals Disease Stage-Dependent Transcriptomic Profile of Regulatory B Cells in Systemic Lupus Erythematous

Razan Alajoleen 1 ; Prakash R. Timilesena 2 ; Song Li 2 ; Xin Luo 1 Corresponding author: arazan@vt.edu

1 Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg 2 School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg

Systemic lupus erythematosus, known as SLE, is an autoimmune disease that is associated with an abnormal activation of immune cells. Regulatory B (Breg) cells are a specific B cell subset that negatively regulate immune responses via secretion of immunoregulatory cytokines interleukin (IL)-10, IL-35, and transforming growth factor (TGF)- ß . We had previously shown that pre-disease Breg cells were more potent in suppressing autoimmunity than active-disease Breg cells. In this study, using single cell RNA (scRNA) sequencing, we profiled ~10,000 Breg cells from female MRL/ lpr mice at the pre disease (6-8 weeks of age) vs. active-disease (10-12 weeks of age) stages. We identified 18 gene clusters with the cell number in each cluster varied from 15 cells to 4855 cells. Based on known markers of Breg

subsets, scRNA analysis identified clusters C5, C6, C7, C10, C13 and C16 as transtional-2 marginal zone precursor B cells (T2-MZP), C14 as marginal zone B cells (MZB), C0 and C1 as transitional 1 B cells (T1), C17 as germinal center B cells (GC), and C18 as B1 B cells. Our data showed that the pre-disease Breg cells were predominantly T2-MZP and MZB cells. Follicular B cells outside the marginal zone, on the other hand, are significantly increased in the active-disease stage. Our analysis also identified long noncoding RNAs (lncRNAs) that can regulate gene expression and play a significant role in autoimmunity and inflammation. Two lncRNAs, Malat1 and Xist , were significantly increased in active-disease Breg cells, where the expression of Lglas3, Slpi , and Spp1

were also increased. Collectively, our findings suggest that the SLE disease stage-dependent functions of Breg cells are regulated at the transcriptional level. In future experiments, we will determine whether the immunosuppressive functions of active-disease Breg cells can be restored by knocking down the differentially overexpressed genes in these cells.

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