CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Recruitment of dendritic cell progenitors to foci of influenza A virus infection sustains immunity.

Protection from infection with respiratory viruses such as influenza A virus (IAV) requires T cell­mediated immune responses initiated by conventional dendritic cells (cDCs) that reside in the respiratory tract. Here, we show that effective induction of T cell responses against IAV in mice requires reinforcement of the resident lung cDC network by cDC progenitors. We found that CCR2-binding chemokines produced during IAV infection recruit pre-cDCs from blood and direct them to foci of infection, increasing the number of progeny cDCs next to sites of viral replication. Ablation of CCR2 in the cDC lineage prevented this increase and resulted in a deficit in IAV-specific T cell responses and diminished resistance to reinfection. These data suggest that the homeostatic network of cDCs in tissues is insufficient for immunity and reveal a chemokine-driven mechanism of expansion of lung cDC numbers that amplifies T cell responses against respiratory viruses.