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 < 0.0001). Promotion of CLEC9A+ DC development was accompanied by statistically significant decreases in monocyte, CD1c+ DC, and plasmacytoid DC frequencies and cell numbers, with granulocytic differentiation relatively unaffected. Similar changes were seen when DLL1 was substituted for the Notch ligands DLL4 or JAG1, and abrogated by gamma secretase inhibition. Furthermore, promotion of CLEC9A+ DC differentiation was mimicked by transduction of HSPCs with intracellular Notch1, consistent with a cell-intrinsic role for Notch in CLEC9A+ DC development.

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.