Delta-Like Notch Ligands Expressed By Host Non-Hematopoietic Radioresistant Cells Regulate Graft-Versus-Host Disease and Extrathymic T Cell Development After Bone Marrow Transplantation

Allogeneic bone marrow transplantation (allo-BMT) is limited by graft-versus-host disease (GVHD). We previously reported an essential role for Notch signaling in alloreactive T cells mediating GVHD after allo-BMT, demonstrating a profound decrease in GVHD incidence and severity with genetic Notch inhibition in donor T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands (Zhang et al., Blood 117(1), 2011; Sandy et al., J Immunol 190(11), 2013; Tran et al., JCI123(4), 2013). However, the cellular source of these critical Notch ligands remains unknown. While host hematopoietic antigen-presenting cells (APCs) seem a likely source, recent evidence indicates that these cells are not solely responsible for donor T cell activation in GVHD. We considered three alternative sources of Notch ligands: donor-derived hematopoietic cells, host hematopoietic APCs surviving lethal irradiation, and host non-hematopoietic radioresistant cells.

To test these possibilities, we used complementary genetic and biochemical approaches to inactivate Dll1/Dll4 in specific compartments, or to provide ubiquitous systemic blockade of these ligands. Bone marrow (BM) chimeras were created by transplanting BM from poly(I:C)-induced Mx-Cre⁺ x Dll1^f/fDll4^f/f B6-CD45.2 mice into lethally irradiated B6-CD45.1 mice, generating chimeric mice without Dll1 and Dll4 only in the host hematopoietic compartment. After 14 weeks of reconstitution, CD45.2 → CD45.1 turnover was near complete in hematopoietic progenitors and professional APCs. Dll1/Dll4 excision was >99% by qPCR. WT (wild-type) BM chimeras subjected to MHC-mismatched allo-BMT (BALB/c → [Mx-Cre^- x Dll1^f/fDll4^f/f (B6-CD45.2) → B6-SJL CD45.1]) had evidence of severe aGVHD and poor survival, as expected. Interestingly, BM chimera mice lacking Dll1 and Dll4 expression in the host hematopoietic compartment had equally severe GVHD and impaired survival following allo-BMT (BALB/c → [Mx-Cre⁺ x Dll1^f/fDll4^f/f (B6-CD45.2) → B6-SJL CD45.1]). In contrast, both WT and Dll1/Dll4-deficient BM chimeras treated with anti-Dll1/Dll4 antibodies (i.p. x4 over days 0-10 of transplant) had markedly decreased GVHD and improved survival (log rank Χ²=30.6, p<0.0001). Anti-Dll1/Dll4 antibodies blocked cytokine production by alloreactive T cells even after transplantation of purified T cells into irradiated recipients, ruling out dominant presentation of Notch ligands by donor-derived professional APCs. We are now backcrossing our Mx-Cre⁺ x Dll1^f/fDll4^f/f mice onto a BALB/c background to allow for simultaneous elimination of both donor and host hematopoietic Dll1 and Dll4 expression during allo-BMT. In addition to Notch-dependent regulation of GVHD, we studied immature pre-T cells that arise at extrathymic sites after BMT. We and others have observed Notch-dependent T cell development in lymphoid organs during early T cell reconstitution after transplantation (Lancrin et al., J Exp Med 195(7), 2002; Maillard et al., Blood 107(9), 2006; Holland et al., JCI122(12), 2013). Thus, this phenomenon is an alternative readout for exposure to Notch ligands in the post-BMT environment. Systemic blockade of Dll1 but not Dll4 with neutralizing antibodies completely blocked the development of these cells, indicating strict Dll1-dependence. In contrast, Dll1 elimination in host, donor or both host/donor hematopoietic compartments did not abrogate extrathymic pre-T cell development, consistent with a source of Notch ligands in host radioresistant cells.

Altogether, these findings suggest that Notch ligands expressed by radio-resistant non-hematopoietic host tissues have important immunobiological functions during GVHD and T cell reconstitution. Identifying the cellular source of Delta-like ligands is critical to understand the effects of Notch signaling after bone marrow transplantation.