Single Cell Rnaseq Identifies Chemokine Responses to BK Polyomavirus Infection and Novel Contribution of MAIT Cells

Introduction BK polyomavirus (BKPyV) is associated with cystitis, nephropathy and transplant-associated thrombotic microangiopathy (TA-TMA) in hematopoietic stem cell transplant (HSCT) recipients. Massive BKPyV propagation alone is insufficient for disease and many patients are asymptomatic despite high viral loads. We previously identified intracellular BKPyV in circulating endothelial cells from HSCT recipients with BKPyV viremia, confirming BKPyV infects the endothelium. In this study, we hypothesized that differences in endothelial cell and host immune responses to BKPyV viremia influence BKPyV disease. Objectives We sought to identify biological pathways that influence BKPyV disease in HSCT recipients. Methods We performed single cell RNA-seq (scRNAseq) in two settings. First, we used an in vitro model to study BKPyV infection in primary kidney endothelial cells. Cells were cultured with BKPyV and harvested at 1 and 5 days post-infection (DPI). We then studied the transcriptomes of peripheral blood mononuclear cells (PBMCs) from HSCT recipients with high BKPyV viremia (>10,000 copies/mL, n=3) and without BKPyV viremia or viruria (n=3) at day 60 after HSCT. Results Cellular BKPyV infection was measured with microscopy (Fig. 1A) and scRNAseq. At 1 DPI endothelial cells were 37% infected and 99% at 5 DPI. We compared the transcriptomes from the top quartile of infected endothelial cells to the bottom quartile of infected cells (including uninfected cells, Fig. 1B) at 5 DPI. Pathway analysis of differentially expressed genes (DEGs) found hypoxia, TNFα signaling, PI3 kinase signaling and complement pathways were significantly different (Fig. 1C). Two cell clusters were identified (Fig. 1B) and we compared “high” infected cells in cluster 1 to “high” infected cells in cluster 2 (Fig. 1D). Pathway analysis showed PI3K signaling was significantly different. Both cell clusters in this comparison had high levels of BKPyV transcript which means PI3K signaling was not related to BKPyV infection alone.Single cell RNAseq of PBMCs from patients with and without BKPyV viremia identified DEGs across all cell types (Fig.2A). NK cells and mucosal-associated invariant T-cells (MAIT cells) had notable differences on UMAP (Fig. 2B) and cell composition analysis (Fig. 2C/E). Pathway analysis of NK and MAIT cells showed similar pathway changes observed in our model, including TNFα signaling, hypoxia, complement and PI3K signaling (Fig. 2D/F). These pathways and cell types are likely involved in the in vivo response to BKPyV viremia and may contribute to symptomatic disease. Conclusion This study identified novel yet plausible innate and cellular immunity pathways that are likely involved in the host response to BKPyV. Furthermore, the role of MAIT cells in BKPyV has not been described. Complement and PI3K activation may be integral to BKPyV-related diseases such as cystitis and TA-TMA.