The Notch Ligand DLL4 Derived from Human Dendritic Cells Is Critical for Promoting T Helper (Th)1 and Th17 Cell Differentiation

Dendritic cells (DCs) are important for primary T cell responses, and cytokines produced by DCs are thought to be essential for promoting T helper (Th)1  and Th17 differentiation. However, DCs can drive effector differentiation independent of cytokines. In mouse models of graft-versus-host-disease (GVHD), which is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT), we found that DC-derived Notch ligand Dll4 was important for CD4⁺ Th1 and Th17 cell differentiation. Blocking Dll4 led to decreased production of IFN-g and IL-17 in mice receiving allo-HSCT and inhibition of GVHD. However, the human counterparts of murine DLL4⁺ DCs and their function in alloreactive T cells have never been investigated.

      We report here the identification of human DLL4⁺ DCs and the critical role of DLL4 in DC-regulation of Th1 and Th17 cell differentiation. Flow cytometric analysis revealed that CD1c⁺ DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors (n=18) did not express DLL4. However, 24 hours after stimulation with Toll-like receptor (TLR) agonists, PB DCs from healthy donors produced high levels of DLL4 on their surface. Pam3 (TLR1/2 stimulus), Poly I:C (TLR3 stimulus), LPS (TLR4 stimulus) and R848 (TLR7/8 stimulus) induced high levels of DLL4 expression on the surface of 50% to 80% of CD1c⁺ DCs. CpG oligodeoxynucleotides (TLR9 agonists) did not increase DLL4 in CD1c⁺ DCs, likely due to their lacking of TLR9. pDCs increased DLL4 expression when activated by R848 (16.0% ± 2.7%) and to a less extent by CpG oligodeoxynucleotides  (8.6% ± 0.8%). Thus, activation of TLR signaling induces high levels of DLL4 in CD1c⁺ DCs and pDCs, with R848 being the most potent stimulus. Functional analysis using mixed lymphocyte reaction revealed that R848-activated CD1c⁺ DCs and pDCs induced greater proliferation of allogeneic CD4⁺ T cells and production of more IFN-g- and IL-17-producing effector cells compared to unstimulated CD1c⁺ DCs and pDCs. Blocking DLL4 using a neutralizing antibody decreased Notch signaling in T cells stimulated with activated DCs and led to production of 2- and 3-fold less Th1 cells and Th17 cells compared to IgG control, suggesting the importance of DLL4 in DC-regulation of effector differentiation. Molecular mechanism investigation revealed that SATAT3 and NFkB were crucial for inducing DLL4 in human DCs. Inhibiting STAT3 alone using its specific inhibitor S31-201 dramatically decreased DLL4 expression in activated PB DCs. Promoter reporter assays showed that STAT3 was required for activating DLL4 transcription. Inhibiting NFkB using its inhibitor PDTC also decreased the expression of DLL4 on the surface of R848-stimulated PB DCs. However, DCs derived from monocytes induced by GM-CSF and IL-4, which had activation of NFkB but did not express active STAT3 following stimulation by R848 + LPS, were DLL4 negative despite their upregulation of costimulatory molecules (e.g., CD40, CD80, CD83, and CD86). Thus, activation of STAT3 is critical for inducing DLL4 in human DCs, whereas active NFkB is important but not sufficient for inducing DLL4 in PB DCs. Finally, given the importance of alloreactive Th1 and Th17 cells in mediating GVHD in human allogeneic HSCT recipients, we further obtained PB from patients (n=7) undergoing allo-HSCT between 21 and 39 days after transplantation when these patients were fully engrafted and no longer pancytopenic. As compared to healthy donors, HSCT recipients had an averaged 12-fold higher frequency of DLL4⁺CD1c⁺ DCs. These results indicate that upregulation of DLL4 on the surface of DCs is associated with alloreactive inflammatory conditions in HSCT patients.

       In summary, our findings show that DLL4 surface expression on human DCs is critical for the priming of human Th1 and Th17 responses and may have significant implication in better understanding of T cell-mediated inflammatory conditions such as chronic infection, autoimmune diseases, tumor rejection and GVHD after allo-HSCT.