Notch Signaling Promotes the Differentiation of Human CLEC9A+ Dendritic Cells from Hematopoietic Stem and Progenitor Cells

Human CLEC9A+ dendritic cells (also known as CD141+ DCs, XCR1+ DCs, and cDC1) are an endogenous DC subset with constitutive ability to traffic to secondary lymphoid tissues, efficiently cross-present cellular antigens, and prime CD4+ and CD8+ antiviral and antitumor T cell responses. As such, CLEC9A+ DCs have been proposed as ideal candidates for adoptive cell immunotherapy, however their rarity in vivo and a lack of knowledge regarding their development have thus far precluded their therapeutic use. We used in vitro differentiation approaches to identify novel microenvironmental cues that regulate the development of CLEC9A+ DCs from human CD34+ hematopoietic stem and progenitor cells (HSPCs). Our findings reveal a previously unknown role for Notch signaling as a key regulatory pathway in CLEC9A+ DC differentiation from HSPCs. Exposure of HSPCs to the Notch ligand DLL1 expressed by MS5 stromal cells, together with the DC-promoting cytokines FLT3L and GM-CSF, markedly enhanced the generation of CLEC9A+ DCs relative to control cultures lacking DLL1 expression. Resulting CLEC9A+ DC frequency was on average 66% of total cells in DLL1 cultures, compared with 6% in control cultures at 20 days (p The effect of Notch signaling on CLEC9A+ DC development was conserved between HSPCs isolated from human cord blood (CB), bone marrow (BM), or G-CSF mobilized peripheral blood (MPB). Furthermore, highly purified BM monocyte/dendritic cell progenitors (MDP) or common dendritic cell progenitors (CDP) cultured in the presence of DLL1 preferentially generated CLEC9A+ DCs with loss of alternative lineage outputs, supporting a mechanism by which Notch signaling skews lineage commitment to the CLEC9A+ DC lineage at the level of multipotent DC progenitors. Notch-induced CLEC9A+ DCs derived from HSPCs were phenotypically and functionally similar to primary CLEC9A+ DCs isolated from the blood. Whole transcriptome profiling by RNAseq of Notch-induced CLEC9A+ DCs confirmed lineage-specific upregulation of key CLEC9A+ DC genes including XCR1, CADM1, BATF3, and IRF8 . Furthermore, global gene expression profiles were similar between Notch-induced CLEC9A+ DCs generated from HSPCs isolated from CB or MPB. Notch-induced CLEC9A+ DCs expressed high levels of CD62L and CCR7, and underwent efficient chemotaxis in response to CCL21 in transwell migration assays, suggesting lymph node-homing capacity. Compared to primary blood CLEC9A+ DCs, Notch-induced CLEC9A+ DCs exhibited higher basal surface expression of T cell costimulatory molecules including CD80, CD83, and CD86, reminiscent of dermal CLEC9A+ DCs, and these were further upregulated in response to the TLR agonists poly(I:C) or R848. Notch-induced CLEC9A+ DCs exhibited potent CD4+ and CD8+ immunostimulatory activity in mixed lymphocyte reactions, and induced antigen-specific CD8+ T cell responses to an HLA-A*0201-restricted NY-ESO-1 epitope through either cross-presentation of cellular antigen acquired from necrotic tumor cells, or endogenous presentation of antigen transduced at the CD34+ HSPC stage. In conclusion, we have identified a novel role for Notch signaling in the differentiation of CLEC9A+ DCs from HSPCs. We propose Notch signaling enforces CLEC9A+ DC lineage commitment in multipotent DC progenitors, and RNAseq studies addressing the molecular basis of Notch-induced CLEC9A+ DC commitment are in progress. Importantly, the identification of Notch as a critical regulatory pathway in human CLEC9A+ DC development allows robust in vitro differentiation of large numbers of highly functional CLEC9A+ DCs. Taken together our findings provide insight into the development of this important DC lineage and permit the preclinical development of next generation cellular vaccine strategies using in vitro derived CLEC9A+ DCs. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Kohn: Kite Pharma: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Biogen IDEC: Research Funding; BioMarin Pharmaceutical: Research Funding; Orchard Therapeutics Ltd.: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Crooks: Kite Pharma: Research Funding.