Integrated  single cell multiomic profiling and functional validation reveal distinct cellular routes to human plasma cell differentiation.
Upon activation, B cells undergo profound transcriptional, epigenetic, and metabolic reprogramming to form antibody secreting plasma cells bearing little resemblance to their progenitors. Here, we used single cell RNA and ATAC sequencing of primary and in vitro differentiating human B cells to identify multiple distinct plasma cell subsets and differentiation routes depending on the starting cell type. In primary tonsils, we observed two distinct plasma cell subsets distinguished by expression of CD44 variant 9 (CD44v9), CD38, CD31, and CD10. A transient and rare CD30+ intermediate was identified both in primary tonsils and in vitro. CD40, TLR9, and cytokine stimulation of naïve and memory B cells formed CD30+ cells, which then formed plasma cells that were exclusively CD44v9+. CD30+ intermediates were not generated from primary germinal center B cells as they differentiated into plasma cells, which mostly lacked expression of CD44v9. Analysis of single cell multiomic data and pharmacological inhibition experiments demonstrated that the CD30+ intermediate was promoted by the transcription factor MEF2C. BAFF and APRIL promoted further maturation of these cells to CD44v9+ plasma cells. These data suggest that human germinal center-independent and -dependent ontogenies are biased towards distinct differentiation routes and terminal plasma cells. Overall design: Multiome-seq: Single nuclei were isolated from tonsil, bone marrow, and in vitro differentiating samples. Joint scRNA- and scATAC libraries were prepared using the 10x Genomics platform.