Transcriptional programs underlying human monocyte differentiation and diversity.

Classical monocytes (CD14hiCD16-) differentiate into intermediate monocytes (CD14+CD16+), which in turn yield nonclassical monocytes (CD14-CD16hi). To investigate the transcriptional regulation underlying this differentiation or conversion, we analyzed 3 single-cell RNA-sequencing datasets of peripheral mononuclear blood cells from healthy individuals using the single-cell regulatory network inference and clustering package. Cells were re-annotated into classical monocytes, intermediate monocytes, nonclassical monocytes, classical dendritic cells (cDCs), and plasmacytoid dendritic cells (pDCs) based on gene signatures. Regulon activity was analyzed, revealing 220 shared regulons across datasets. Distinct regulons characterized most myeloid subsets except intermediate monocytes, which appeared as a transitional state, sharing regulons with both classical and nonclassical monocytes. Regulons such as HMGB2, CREB5, and FOSB were enriched in classical monocytes, while TCF7L2 and POU2F2 were specific to nonclassical monocytes. Plasmacytoid DCs showed the greatest divergence, possessing many unique regulons, including AR and RUNX2, whereas cDCs shared more regulons with monocytes than pDCs, with RUNX1 specific to cDCs. All mononuclear phagocytes shared a common core of active regulons, including RELB, ID1, CLOCK, BACH1, and FLI1. Notably, FLI1 was expressed across all myeloid subclasses but emerged as a key regulator influencing monocyte gene regulatory networks. Pseudotime modeling using regulon activity demonstrated that monocyte conversion is a continuous process. Differential regulon analysis identified distinct biological processes that were enriched in monocyte subsets, highlighting that regulon activity analysis provides novel insights into myeloid cell biology. Our findings underscore the key role of transcriptional regulatory programs in defining mononuclear phagocyte identity and reveal novel signatures associated with monocyte diversity and differentiation.