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Notch signaling promotes T-cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like ligand DLL4. To assess if Notch’s effects are evolutionarily conserved and identify key mechanisms, we studied antibody-mediated DLL4 blockade in a non-human primate model similar to human allo-HCT. Short-term DLL4 blockade improved post-transplant survival with striking, durable protection from gastrointestinal GVHD, out of proportion to other disease sites. Unlike prior immunosuppressive strategies, anti-DLL4 interfered with a T-cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin a4b7 in conventional T-cells via b1 competition for a4 binding, while preserving a4b7 in regulatory T-cells. Thereby, DLL4/Notch blockade decreased effector T-cell infiltration into the gut, with increased regulatory to conventional T-cell ratios early after allo-HCT. Our results identify a conserved, biologically unique and targetable role of DLL4/Notch signaling in GVHD.One Sentence SummaryNotch signaling promotes pathogenic effector T cell infiltration of the intestine during acute graft-versus-host disease.

A TPP1 mutation known to cause telomere shortening and bone marrow failure in humans recapitulates telomere loss but results in severe germline defects in mice without impacting murine hematopoiesis., Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Notch signaling is an evolutionarily conserved cell-to-cell signaling pathway that regulates cellular differentiation and function across multiple tissue types and developmental stages. In this review, we discuss our current understanding of Notch signaling in mammalian innate and adaptive immunity. The importance of Notch signaling is pervasive throughout the immune system, as it elicits lineage and context-dependent effects in a wide repertoire of cells. Although regulation of binary cell fate decisions encompasses many of the functions first ascribed to Notch in the immune system, recent advances in the field have refined and expanded our view of the Notch pathway beyond this initial concept. From establishing T cell identity in the thymus to regulating mature T cell function in the periphery, the Notch pathway is an essential, recurring signal for the T cell lineage. Among B cells, Notch signaling is required for the development and maintenance of marginal zone B cells in the spleen. Emerging roles for Notch signaling in innate and innate-like lineages such as classical dendritic cells and innate lymphoid cells are likewise coming into view. Lastly, we speculate on the molecular underpinnings that shape the activity and versatility of the Notch pathway.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.

Studies of hematopoietic stem cells (HSCs) have advanced our understanding of the intracellular signals and microenvironmental interactions that influence HSC fate. Our lab is interested in the role of orphan nuclear receptor NR4A1, an immediate response gene sensitive to external stimuli, in HSC development. NR4A1 regulates the development of specific, mature hematopoietic cell lineages from both the innate and adaptive immune system including patrolling monocytes, a mature myeloid cell subset. More recently, we have shown that NR4A1 expression also identifies a subpopulation of myeloid-biased long-term HSCs in the bone marrow. Given that NR4A1 directs both immature and differentiated cell types, we investigated its role in myeloid cell maturation. Within the bone marrow and spleen progenitor cell compartments, we find that NR4A1 is exclusively expressed by myeloid progenitors and not by more restricted megakaryocyte, erythroid, or common monocyte progenitors. Nr4a1−/−mice exhibit skewed myeloid progenitor cell populations, exhibiting a 2-fold increase in CD105+CD150− erythroid progenitors and cKit+Ter119+CD71+ pro-erythroblasts in the spleen but not in the bone marrow. In vitro cultures of Nr4a1−/−splenic myeloid progenitors also show that NR4A1 restricts the frequency of erythroid cells, yet bone marrow transplant studies show similar erythroid progenitor reconstitution from both WT and Nr4a1−/− donors. Taken together, our data reveal a new role for NR4A1 as both a direct and indirect modulator of erythropoiesis.