Systemic Immunomodulation with SA-FasL Protein-Engineered Donor Splenocytes Under Transient Cover of Rapamycin Induces Robust Tolerance to Cardiac Allografts

Abstract

Introduction: We previously demonstrated that donor splenocytes engineered to display on their surface a novel form of FasL molecule chimeric with streptavidin (SA-FasL) induced tolerance in ˜ 60% of ACI rats transplanted with allogeneic WF heart grafts. The main objective of this study is to further improve the efficacy of this tolerance protocol by using different routes of injections, a short course of rapamycin as immunosuppressant, and bone marrow cells engineered with SA-FasL as a clinically practical approach. Methods: A heterotopic cardiac allograft model using BALB/c as donors and C57BL/6 mice as recipients was used. Donor splenocytes (Spl) or bone marrow cells (BMCs) were biotinylated under physiological conditions followed by incubation with SA-FasL protein (70 ng/106 cells). After extensive washing to remove the unbound protein, the level of SA-FasL on the cell surface was assessed by flow cytometry. Five million of SA-FasL-engineered cells were injected intraperitoneally or intravenously into C57BL/6 mice on day 1, 3, 5, post cardiac transplantation performed on day 0. Graft recipients were also treated with a short course of rapamycin (0.2 mg/kg) administered daily from day 0 to 14 post-transplantation. Ventricular contractions were assessed by daily abdominal palpation and rejection was defined as cessation of heartbeat verified by autopsy and pathology. Results: Intraperitoneal treatment with donor SA-FasL-engineered splenocytes induced tolerance in all cardiac graft recipients (n=15; >100 days), as compared to SA-FasL-engineered BMCs treated group [n=3, mean survival time (MST) = 22.7±3.5 days], or streptavidin-engineered control group (n=7; MST = 20.9±2.2 days). Intravenous treatment exhibited the same efficacy in inducing tolerance with SA-FasL-engineered splenocytes (n=5; >100 days). Interestingly, only live allogeneic splenocytes, but not syngeneic or irradiated allogeneic splenocytes, induced tolerance, as the grafts from the latter two groups were rejected at a similar tempo to SA control group (n=4, MST = 28±0.6 days and n=5, MST = 26 days). Multilineage phenotypic analysis of splenocytes and BMCs showed high percentages of lymphocytes (58% T cells and 26% B cells) in splenocytes, while BMCs primarily included granulocytes (74%), suggesting that either B or T cells or the combination of both of these cell types are responsible for the tolerogenic effect of splenocytes. Conclusions: SA-FasL-engineered live allogeneic splenocytes, but not BMCs, induce robust tolerance to allogeneic cardiac grafts. Ongoing studies are aimed at identifying the cell type in the spleen that is responsible for tolerance to expedite the application of this approach to the clinic. Supported in parts by AHA 09GRNT2380136, KDR-PP09-23,and NIH T32 AI055456.