Engineering donor graft with SA-FasL abrogates lethal aGVHD and shows efficient immune reconstitution. (A) Survival of lethally irradiated (1000 cGy) F1(C57BL/6xBALB/c) recipients grafted with a mixture of C57BL/6 allogeneic bone marrow cells (1 × 10⁷) and splenocytes (2 × 10⁷). F1 animals underwent transplantation with nonengineered C57BL/6 bone marrow cells with or without GVHD-causing spleen cells engineered with the indicated doses of SA-FasL protein (ng/10⁶ cells) or a SA dose (12.5 ng/10⁶) equimolar to the highest dose of SA-FasL. Animals were monitored for survival, clinical GVHD scores, and body weight. (B) Donor chimerism (H-2K^b+H-2K^d-) in the indicated tissues and frequency of CD4⁺ Treg (CD4⁺CD25⁺FoxP3⁺), Teff (CD4⁺CD44^hiCD62L^-), and NK (NK1.1⁺CD3^-) cells. (C) Frequency of CD4⁺ Treg (CD4⁺CD25⁺FoxP3⁺), Teff (CD4⁺CD44^hiCD62L^-), and NK (NK1.1⁺CD3^-) cells, FoxP3 MFI, and Treg/Teff ratios in the spleen of long-term (>100 days) animals compared with that of bone marrow only recipients and unmanipulated naïve F1 animals. (D) Skin graft survival. Long-term survivors (100 days after transplantation) with bone marrow cells only and bone marrow cells along with SA-FasL–engineered splenocytes were challenged simultaneously with donor-matched (BALB/c, H-2^d) and third-party (C3H, H-2^k) skin grafts. All long-term recipients accepted donor-matched skin allografts while rejecting third-party grafts in an acute fashion. For comparison of survival curves, log-rank (Mantel-Cox) test was used in panels A,D. Mann Whitney test in panel B and 1-way ANOVA with Tukey post hoc test in panel C was used for mean comparison. Data are represented as mean ± SEM. ANOVA, analysis of variance; SEM, standard error mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.