NOTCH Inhibition Separates GVL from GVHD Via Modulating Host Dendritic Cell and Donor T-Cell Activation

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eradicate chemorefractory leukemia and reduce the relapse rate of high risk cases through the graft versus-leukemia (GVL) activity. However, graft-versus host disease (GVHD) limited the overall clinical benefit of allo-HSCT. Efforts are made to separate GVL from GVHD but few approaches are confirmed effective. Notch is a highly conserved signaling system important for lymphocyte effector and memory differentiation. Previous studies have shown that inhibition of Notch signaling could reduce aGVHD ex vivo and in vivo by downregulating both activated antigen presenting cells (APCs) and CD8⁺ T cells. It is less established whether Notch inhibition could also maintain GVL activity while reducing GVHD. The aim of the current work was to further study whether GVL activity could be maintained or enhanced by Notch inhibition.

Methods: Murine models of GVHD and GVL were established (C57BL/6→BALB/c). γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) was used to block Notch1 signaling. The recipients were monitored daily for survival, clinical signs of GVHD and tumor size. GVHD histopathology were systematically examined and evaluated based on the scoring system. In order to study the mechanism of Notch inhibition on both GVHD and GVL, phenotypes of APCs and T cells in recipients were analyzed by flow cytometry. Expression of FasL by PCR, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by ELISA and cytotoxicity of T cells were compared in recipients with or without Notch inhibition.

Results: Reduced GVHD was observed in Notch inhibited recipients, which is similar to the results of previous study. However, in GVL model, both the median time when tumor volume is over 0.5cm³ and the change of tumor volume had no difference with or without Notch inhibition. Suppressed differentiation of CD8⁺ cytotoxic T cells (CTL) and decreased levels of TNF-α did not result in attenuated GVL activity. Further mechanism studies showed that Notch inhibition up-regulates CD11c⁺CD80⁺ DCs and central memory T cells (CD4⁺CD62L⁺ and CD8⁺CD62L⁺ T cells) while suppressing CD8+ CTL differentiation. We then compared levels of TNF-α and IFN-γ which are important in mediating aGVHD. Levels of TNF-α were decreased but IFN-γ were increased rather than decreased after Notch inhibition. We hypothesized that the contradictory results may ascribe to the difference between TNF-α and IFN-γ in inducing GVHD and GVL. Reduced TNF-α and increased IFN-γ levels could be the causes for down-regulated donor CTL activity. Moreover, FasR-FasL interaction plays a critical role in GVL instead of aGVHD. FasL expression were similar in in DAPT groups and the controls, suggesting similar GVL activity in recipients with or without Notch inhibition.

Conclusions: Notch inhibition reduces GVHD and does not impair GVL effect. Increased number of central memory T cells and higher levels of interferon-γ (IFN-γ) could be the causes for enhanced GVL.