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Trained immunity, or innate immune memory, has been attributed to the long-term retention of stimulus-induced histone post-translational modifications (PTMs) following clearance of the initial stimulus. Yet, it remains unknown how this epigenetic memory can persist for months in dividing cells given the lack of any known mechanism for stimulus-induced histone PTMs to be directly copied from parent to daughter strand during DNA replication. Here, using time course RNA-seq, ChIP-seq, and infection assays, we find that trained macrophages are transcriptionally, epigenetically, and functionally re-programmed for at least 14 cell divisions after stimulus washout. However, the epigenetic changes observed after multiple rounds of cell division do not result from the self-sustained propagation of stimulus-induced epigenetic changes through cell division. Instead, long-lasting epigenetic differences between trained and non-trained cells are always coupled with changes in transcription factor (TF) activity, emphasizing the central role played by TFs, and gene expression changes more broadly, in driving the transmission of stimulus-induced epigenetic changes across cell divisions.

The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.