Introduction: The Fas pathway is critical for immune homeostasis and tolerance to self-antigens, as a deficiency in this pathway is associated with excessive autoimmunity in humans. Allogeneic Teff cells upregulate Fas following activation and undergo FasL-mediated apoptosis. We previously reported the generation of a novel form of recombinant FasL, SA-FasL, that has robust apoptotic activity on cells expressing Fas receptor. We herein tested systemic treatment with a short course of SA-FasL protein for tolerance induction to cardiac allografts in mice.
Method:  C57BL/6 mice were transplanted with allogeneic BALB/c heart grafts. Four groups of transplant recipients were used for this study. The first group (n=9) was treated starting on the day of transplantation with SA-FasL protein (1 µg) and rapamycin (0.2 mg/kg) daily for 15 days.  The second group (n=6) was treated with SA-FasL alone, the third group (n=6) with rapamycin alone, and the fourth group (n=6) was left untreated. Recipients were monitored for graft survival for 100 days.  At the experimental end-point, a cohort was transplanted with donor and third-party skin grafts, while another was euthanized to collect the graft and lymphoid tissues for immune monitoring. The percentages and absolute numbers of CD4+FoxP3+ Treg cells were assessed using flow cytometry.
Results: Treatment with SA-FasL and rapamycin resulted in the survival of all heart allografts over the 100-day observation period.  In marked contrast, the median survival time for the control group was 8 days, SA-FasL only group 28.3 days, and rapamycin only group 19.2 days. Over 70% of long-term cardiac graft recipients accepted donor skin grafts while rejecting third-party skin grafts within 10-12 days. There was a significant increase in the percentages and absolute numbers of CD4+FoxP3+ Treg cells in the spleen, draining lymph node, and cardiac graft in the SA-FasL plus rapamycin treatment group as compared with the other groups. Depletion of CD4+FoxP3+ Treg cells at the induction (day 15) or maintenance (day 60) phase of tolerance resulted in prompt heart graft rejection, demonstrating the critical role these cells play in the observed tolerance.
Conclusion: Treatment with SA-FasL protein is an effective strategy for modulating alloreactive responses for tolerance induction to solid organs with significant translational potential.