Single cell RNA sequencing-based comparison of mouse splenic pDC, pDC-like cells, tDC, cDC1 and cDC2 at steady state and during a viral infection

Plasmacytoid dendritic cells (pDC) are the main source of type I interferon (IFN) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DC (tDC), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. To overcome this bottleneck, we designed, generated and validated a pDC-Tomato reporter mouse. We bred pDC-Tomato with Zbtb46 GFP mice to yield the he ZeST mouse strain that enabled transcriptomic profiling of all splenic DC types, by single cell RNA sequencing, both at steady state and during the course of the infection with mouse cytomegalovirus (MCMV). Analyses of the transcriptomic dataset unraveled diverging activation of pDC-like cells vs tDC during the infection. This dataset and the associated specific gene modules will be useful to delineate the physiological functions of pDC versus other DC types. pDC, pDC-like cells, tDC, cDC1 and cDC2 were sorted from ZeST reporter mice at 0, 36 or 48 hours after MCMV infection. Single-cells were FACS-sorted into fourteen 96-well plates (4 plates for 0 hours, 5 plates for each of the two other time points). All plate maps were identical, encompassing at the time of sorting: 14 individual cells for each of the following populations, pDC expressing Tomato, pDC negative for Tomato, pDC-like cells, tDC, and cells positive for both Ly6D and Zbtb46; 12 cDC1, 12 cDC2 and 2 empty control wells. Gene expression was profiled by 5'-end single-cell RNAseq following the FB5P-seq protocol.