Delta-like Ligands Expressed By Stromal Cells in Secondary Lymphoid Organs Deliver an Early Pulse of Notch Signaling and Drive T Cell Pathogenicity in Acute Graft-Versus-Host Disease

Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands results in near-complete protection from acute GVHD in mouse models of allogeneic bone marrow transplantation. Notch-deprived alloreactive T cells proliferate and accumulate in vivo, but produce dramatically reduced levels of the proinflammatory cytokines IFNγ, TNFα and interleukin-2 (IL-2) (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., J Clin Invest 2013). In this study, we sought to: 1) determine the kinetic requirements for Notch signaling in the pathogenesis of acute GVHD; 2) identify the essential cellular compartment that delivers Dll1 and/or Dll4 ligands to incoming alloreactive T cells. In the B6 anti-BALB/c major histocompatibility complex-mismatched model, a single dose of Dll1 and Dll4 blocking antibodies at the time of transplantation abolished alloreactive T cell production of IFNγ, TNFα, and IL-2, increased regulatory T cell numbers (as assessed at day 10), and conferred long-term protection from GVHD. Conversely, delaying antibody administration by only two days after transplantation resulted in persistent T cell cytokine production, no changes in regulatory T cell numbers, and loss of long-term protection from GVHD. These findings identify a critical early window of Notch activity that promotes the pathogenesis of acute GVHD. To identify the dominant cellular source of Dll1 and Dll4, we assessed the impact of Cre-mediated Dll1 and Dll4 inactivation within host hematopoietic, donor hematopoietic, or host non-hematopoietic tissues. Bone marrow chimeras that lacked Dll1 and Dll4 solely within the host hematopoietic system were generated from poly(I:C)-induced Mx1-Cre;Dll1^fl/fl;Dll4^fl/fl donor mice. Both donor chimerism and Cre-mediated excision efficiency were >97%. Unlike systemic Dll1/4 blockade, Dll1 and Dll4 inactivation within the host hematopoietic system failed to decrease GVHD mortality or severity. Likewise, Mx1-Cre-mediated deletion of Dll1 and Dll4 within the donor hematopoietic system had minimal effects on T cell proinflammatory cytokines. In contrast, Ccl19-Cre-mediated Dll1 and Dll4 inactivation within host stromal cells profoundly impaired donor T cell production of IFNγ, TNFα, and IL-2, and resulted in long-term protection from GVHD. Lineage tracing in Ccl19-Cre x ROSA26-YFP mice revealed Cre activity within a small subset of CD45-negative lymph node and spleen stromal cells, but not in professional hematopoietic antigen-presenting cells. These data suggest that a specialized subset of non-hematopoietic stromal cells delivers an early pulse of Notch signaling to alloreactive T cells during acute GVHD. To our knowledge, these results provide the first in vivo evidence for non-motile secondary lymphoid-resident stromal cells as critical drivers of T cell-mediated immune pathology, with a central role for Notch signaling in this process. Transient interference with Notch ligand function or with their expression by the stromal cell niche in the peri-transplant period could serve as a novel therapeutic strategy for GVHD.