Engineering donor lymphocytes with Fas ligand protein effectively prevents acute graft-versus-host disease.

Alloreactive T effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Ex vivo non-specific depletion of T cells from the donor graft impedes stem cell engraftment and post-transplant immune reconstitution. Teffs upregulate Fas following activation and undergo FasL-mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs in vivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes taking the advantage of high affinity interaction between biotin and streptavidin. SA-FasL-engineered mouse and human T cells underwent apoptosis following activation in response to alloantigens in vitro and in vivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients following cotransplantation with bone marrow cells, whereas all controls transplanted with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL-engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represent a practical approach with significant translation potential for the prevention of aGVHD.