Notch Signaling Mediated By Dll1/4 Notch Ligands Controls the Pathogenesis of Both Multi-Organ System Non-Sclerodermatous and Sclerodermatous Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT) with limited success of current therapeutic options. We have shown that Notch inhibition in donor T cells or systemic neutralization of the Notch ligands Delta-like1 and Delta-like4 (Dll1/4) prevents acute GVHD in multiple mouse allo-HCT models (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., JCI 2013). However, the role of Notch signaling in cGVHD remains unknown. To address this question, we used genetic and antibody-mediated strategies of Notch inhibition in two established and pathophysiologically distinct mouse models of cGVHD characterized by dominance of sclerodermatous changes (Scl) or non-Scl multi-organ system disease that includes bronchiolitis obliterans (BO), respectively.

In the B10.D2→BALB/c MHC-matched, minor antigen mismatched model of Scl-cGVHD, transgenic expression of the pan-Notch inhibitor DNMAML in T cells induced high level protection from cGVHD as assessed using clinical and pathological scoring criteria. Systemic antibody-mediated blockade of Dll1/4 Notch ligands prevented cGVHD when given early after allo-HCT, with dominant effects of Dll4 inhibition alone. However, Dll4 blockade provided no therapeutic benefits if delayed by 48 hours after allo-HCT or when targeting fully established Scl-cGVHD, suggesting that Dll4-mediated Notch signals are critical in this model when delivered to T cells early after allo-HCT. To understand the impact of Notch inhibition during priming of alloantigen-specific T cells, we studied immunodominant Vβ3⁺CD4⁺ T cells that expand in response to a host superantigen encoded by Mtv6 in BALB/c recipients. Both control and Notch-inhibited Vβ3⁺CD4⁺ T cells upregulated activation markers and expanded to comprise >80% of donor T cells in lymphoid and target organs by day 6. However, Notch inhibition markedly decreased IFN-γ, TNF-α, and IL-17 production in these cells, while expanding Vβ3⁺CD4⁺FoxP3⁺ regulatory T cells. Thus, Notch inhibition preserved in vivo T cell proliferation and expansion, but tilted the balance in favor of alloantigen-specific regulatory T cells over highly inflammatory effector T cells. In the B6→B10.BR MHC-mismatched model of allo-HCT, characterized by prominent germinal center (GC) responses and BO-cGVHD, genetic pan-Notch inhibition in T cells with DNMAML blocked GC formation and provided long-lasting high level protection from BO, as assessed using pulmonary function tests to measure resistance, elastance and compliance, as well as pathological examination (day 56 after allo-HCT). Flow cytometric evaluation showed that follicular helper T cells (CD4⁺PD-1^hiCXCR5⁺) and cells with a GC B cell phenotype (CD19⁺GL7⁺CD95^hi) were markedly decreased in numbers. Genetic deletion of Notch1 or Notch2 in donor T cells also prevented the immunological manifestations of cGVHD. Antibody-mediated blockade of the Notch ligands Dll1, Dll4, or both in the peri-transplant period protected recipients from BO-cGVHD. In contrast to our observations in Scl-cGVHD, delayed Dll1/4 blockade starting at day 28 after allo-HCT preserved pulmonary function, decreased GC formation and alloantibody deposition in target tissues, and ameliorated BO-cGVHD. Thus, Dll1/4 inhibition could provide therapeutic benefits even in established BO-cGVHD.

Altogether, we identified a key role for early Notch signals in alloantigen-specific T cells that define subsequent cGVHD pathogenesis. Interference with Notch signaling early after allo-HCT provided long-lasting protection from cGVHD. In addition, our observations in the multi-organ system BO-cGVHD model suggest a therapeutic potential for delayed inhibition of Dll1 or Dll4 Notch ligands in an active disease setting driven by alloantibody formation. Our preclinical data suggest that Notch signaling should be explored as a novel druggable target to prevent or treat different forms of cGVHD.