Identification of Previously Uncharacterized Delta 4-Positive Inflammatory Plasmacytoid Dendritic Cells That Play Important Roles in Eliciting Graft-Versus-Host Disease in Mice

Abstract 337

Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Host antigen-presenting cells (APCs) are known to be essential for presenting alloantigens to activate donor T cells to become effector cells mediating GVHD after allo-HSCT. However, APCs are heterogeneous populations. The identity of APC subset(s) that directs effector differentiation of alloantigen-activated T cells and by which mechanism this effect may be achieved remain largely unknown. The Notch signaling pathway controls cell proliferation, differentiation and survival. Upon interaction with Notch ligands of the δ-like family (Dll1, Dll3 and Dll4) and Jagged family (J1, J2), Notch receptors (Notch 1, 2, 3, and 4) are cleaved by γ-secretase and translocate into the nucleus to modify gene transcription. We have recently demonstrated that activation of Notch receptors in donor T cells is critical to the production of alloreactive effector T cells producing multiple inflammatory cytokines (e.g., IFN-γ, TNF-α and IL-17) during GVH reaction (Blood 2011). Building on these findings, we hypothesized that: 1) Notch ligand(s) derived from APCs may be important for directing effector differentiation of alloantigen-activated T cells, and 2) the expression of Notch ligand(s) may differentiate the capability of APCs to prime GVH responses. Using mouse models of GVHD, here we report the identification of previously uncharacterized Dll4-positive (Dll4⁺) inflammatory plasmacytoid dendritic cells (i-pDCs) and their roles in eliciting allogeneic T-cell responses. Host-derived Dll4⁺ i-pDCs occurred in the spleen of allo-HSCT recipients one day after transplantation, peaked by three days and declined by seven days. In contrast, host-derived inflammatory conventional DCs (i-cDCs) were Dll4-negative (Dll4⁻) and rapidly diminished by three days after transplantation. Notably, donor-derived DCs which occurred seven days after HSCT did not express Dll4. In vitro mixed lymphocyte-reaction (MLR) assay showed that these host-derived Dll4⁺ i-pDCs induced approximately 2.5-fold and 7-fold more IFN-γ- and IL-17-producing effector T cells than Dll4⁻ i-cDCs, respectively. Addition of neutralizing antibody specific to Dll4 to the MLR cultures markedly reduced the production of IFN-γ and IL-17 in donor T cells stimulated by host Dll4⁺ i-pDCs, but had minimal impact on donor T cells cultured in the presence of Dll4⁻ i-cDCs. These results suggest that Dll4⁺ i-pDCs may play important roles in directing effector differentiation of alloantigen-activated T cells. Further characterization of biological properties of Dll4⁺ i-pDCs revealed that as compared to unstimulated host pDCs at steady state conditions, Dll4⁺ i-pDCs expressed higher levels of antigen-presenting and costimulatory molecules, upregulated other Notch ligands (e.g.,J1 and J2) on their surface and produced more Ifnb and Il23. Notably, Dll4⁺ i-pDCs were mainly located in the spleen and intestine of mice receiving allogeneic HSCT. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cell producing IFN-γ, IL-17 and TNF-α in GVHD target organs (in particular of the intestine), leading to reduction of GVHD and significantly improved survival of mice after allogeneic HSCT. Furthermore, adoptive transfer of in vitro generated Dll4⁺ i-pDCs caused severe GVHD in MHC-II-deficient mice (in which host DCs are incapable to elicit GVHD). Our findings identify that Dll4⁺ i-pDCs may represent a previously uncharacterized inflammatory APC population developed during GVH reaction. These Dll4⁺ i-pDCs and their-derived Dll4 are critical for directing differentiation of alloreactive effector T cells and may be beneficial therapeutic targets for modulating GVHD.