Dll1 and Dll4 Notch Ligands Prime T Cell Alloimmunity and Are Expressed in Non-Overlapping Populations of Fibroblastic Stromal Cells in Spleen and Lymph Nodes at the Onset of Gvhd
Authors: Brandstadter Joshua D , Perkey Eric , Gaudette Brian T. , Carrington Léolène , Gomez-Atria Daniela , Blazar Bruce R. , Ludewig Burkhard , Tikhonova Anastasia , Aifantis Iannis , Allman David , Maillard Ivan 
Published: February 2020

Notch signaling drives graft-versus-host-disease (GVHD) pathogenesis in preclinical models of allogeneic hematopoietic cell transplantation (allo-HCT). Short-term systemic blockade of the Notch ligands Delta-like1 (Dll1) and Delta-like4 (Dll4) within two days of allo-HCT conferred long-term protection from acute GVHD lethality in mice, with a dominant impact of Dll4. Surprisingly, critical Notch ligands in GVHD were provided by radioresistant non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene in secondary lymphoid organs (SLO) (Chung, JCI 2017). Our discovery revises prevailing GVHD models and identifies a pathogenic role for a stromal cell niche expressing Notch ligands in SLOs. However, little is known about the distribution and regulation of Dll1/Dll4-expressing cells among SLO stromal cells. To address this question, we deployed genetic and biochemical tools to map expression of Notch ligands in SLO stromal cells, characterize the transcriptional landscape of these cells, and identify putative regulators of Notch ligand expression.
To map Dll1 and Dll4 expression, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele and Dll1-mCherry or Dll4-mCherry BAC reporters. Using this model, we fluorescently traced all cells derived from Ccl19-Cre+ cells which contain the essential Dll1/Dll4 source in GVHD, while detecting Dll1 vs. Dll4 expression with high sensitivity. Within the lymph node (LN) Ccl19-Cre+ROSA26-YFP+ compartment, Dll4-mCherry was present predominantly among CD157hi CD45-Ter119-CD31-gp38+ fibroblastic stromal cells, including fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs) and follicular dendritic cells (FDCs). In the spleen, Dll4-mCherry was detected among both CD157+ cells (predominant in the T-zone) and CD157- cells. In contrast, Dll1-mCherry was abundant among spleen CD157+ and CD157- cells, but not detected in the corresponding LN compartments. Thus, Dll1 and Dll4 are expressed with a non-overlapping distribution pattern in different SLOs and stromal cell subsets. Next, we studied expression of the dominant Dll4 ligand after MHC-mismatched allo-HCT (BALB/c into C57BL/6). Although Dll4-mCherry fluorescence did not increase, we detected a rise in surface Dll4 protein, peaking 12 hrs after allo-HCT in LN Ccl19-Cre+ CD157hi FRCs, MRCs and FDCs. Increased Dll4 abundance coincided with the critical timing of pathogenic Notch signals within days after allo-HCT and required irradiation conditioning as well as infusion of allogeneic T cells, suggesting a crosstalk between alloreactive T cells and Dll4+ stromal cells. The mechanisms underlying increased surface Dll4 abundance are under investigation, but could involve post-transcriptional effects as the Dll4-mCherry transcriptional reporter did not change.
We next characterized transcriptomic features of Dll4+ fibroblastic stromal cells. We performed RNA-Seq on rare SLO stromal cells sort-purified from LNs at baseline and 12 hrs after allo-HCT. We compared CD157hi FRCs that do or do not express Dll4-mCherry, while including CD157lo FRCs that do not express Dll4-mCherry and lymphatic endothelial cells that express Dll4 at high levels as controls. These populations were sort-purified for RNA-Seq analysis, using the data to produce a comprehensive picture of gene expression and regulatory machinery in the SLO niche in alloimmunity, as well as identify new putative regulators of Notch ligand expression. Interestingly, we found only 13 differentially expressed genes between Ccl19-Cre-expressing CD157hi fibroblastic stromal cells that do and do not express the Dll4-mCherry reporter 12 hrs after transplant (log-fold change ≥ 1, corrected p ≤ 0.01). The list of up-regulated transcripts included Cxcl13, encoding a chemokine produced by FDCs, and Rankl, encoding a TNF-family member important in LN development and homeostasis.
In summary, our data uncover key new features of the specialized fibroblastic stromal cells expressing Delta-like Notch ligands in SLOs. Ongoing work is testing how qualitative and quantitative differences in Dll1 and Dll4's effects during GVHD may relate to their distribution, regulated expression, or biochemical properties as they interact with Notch receptors in T cells. Our ultimate goal is to design strategies that target Notch ligand expression in SLOs as new prophylactic approaches in GVHD.

Disclosures
Blazar: Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Maillard:Genentech: Consultancy; Regeneron: Consultancy.