Introduction: Alloreactive donor T effector (Teff) cells play a significant role in causing acute graft-versus-host disease (aGVHD) in the context of hematopoietic stem cell transplantation. Non-specific depletion of T cells from the graft hampers stem cell engraftment and post-transplant immune reconstitution.  Activated Teff cells exhibit an increase in Fas receptor expression and undergo restimulation-induced cell death (RICD) mediated by Fas ligand (FasL). In this study, we leveraged RICD as a method to specifically eliminate alloreactive Teff cells in vivo, proposing it as a prophylactic approach to prevent aGVHD.
Methods: We have previously reported a novel construct of FasL fused with streptavidin, known as SA-FasL. In this study, donor human peripheral blood mononuclear cells (PBMCs) were successfully modified to transiently display this unique form of FasL protein (SA-FasL) on their surface, with no adverse effects on cell viability or engraftment capability. For the invivo experiment, female NSG mice (8-10 weeks old) underwent 200 cGy total body irradiation and, four hours later, were intravenously injected with 10x106 PBMCs that were either engineered with SA-FasL, or SA-protein (as a control for FasL), or non-engineered. Throughout the study, animals were regularly assessed for changes in body weight, and was monitored for the development of xenogeneic acute GVHD.
Results: Upon examination five days post-infusion, recipients of SA-engineered human PBMCs exhibited significantly higher numbers of human total CD45+ cells, as well as CD3+, CD4+ and CD8+ T cells in the spleen compared to those who received SA-FasL-engineered human PBMCs. A similar pattern was also evident in the liver, a primary target for xenogeneic acute GVHD mediated by human T cells. The infusion of 10x106 SA-engineered human PBMCs into sublethally irradiated NSG mice resulted in severe acute GVHD, with all animals expiring within 25 days, displaying significantly higher clinical scores and a median survival time of 15 days. In marked contrast, recipients of SA-FasL-engineered PBMCs demonstrated significantly enhanced survival rates, accompanied by a significant reduction in clinical GVHD scores and the maintenance of body weight.
Conclusion: Modifying donor cells to transiently display SA-FasL on their surface offers a practical and efficient strategy to prevent acute graft-versus-host disease, holding considerable translational promise.