Recipients of SA-FasL–engineered splenocytes have reduced levels of T effectors and increased T-regulatory cells. (A) Absolute number of CD4⁺ Teff (CD4⁺FoxP3^-CD44⁺CD62L^-PD1⁺), CD8⁺ Teff (CD8⁺CD44⁺CD62L^-PD1⁺), and Treg (CD4⁺CD25⁺FoxP3⁺) cells in mesenteric lymph nodes (mLN) and the liver (cells per gram). Intrahepatic immune cells and mesenteric lymph nodes were harvested 7 and 21 days after transplantation from F1 recipients of C57BL/6 bone marrow cells (BM) and BM cells cotransplanted with SA-FasL–engineered (BM + SA-FasL-spleen) or nonengineered splenocytes (BM + spleen). Cells were analyzed for activated CD4⁺ and CD8⁺ Teff and CD4⁺ Treg cells using flow cytometry. (B) Ratios of CD4⁺ Treg cells to CD4⁺ and CD8⁺ Teff cells. (C) Representative images of H&E staining of large intestine from F1 recipients (n = 3) at day 21 after transplantation showing cellular infiltration (yellow arrowheads), epidermal cell vacuolar degeneration (red arrowheads), mucosal epithelia degeneration (blue arrowhead), and disruption of mucosal-submucosal junction (red arrow) in nonengineered splenocytes recipients (BM + spleen) as compared with that of SA-FasL-engineered splenocytes (BM + SA-FasL-spleen) recipients (n = 3). (D) Representative images of 2-color immunofluorescence staining for CD4 (red) and nucleus (blue) with infiltrated CD4⁺ T-cell counts per section per animal. Recipients of nonengineered splenocytes have significantly higher frequencies of CD4⁺ T cells within crypts and lamina propria of large intestine than the recipients of SA-FasL–engineered splenocytes. Tissues from recipients of BM cells without donor splenocytes served as control (n = 2). Data point represent averaged cell numbers from 5 random fields per section per animal. Scale bar: 50 μm (left); 25 μm (right). Data are shown as mean ± SEM. For comparisons, 1-way ANOVA with Tukey post hoc test was used in panels A,D and Mann Whitney test in panel B. ANOVA, analysis of variance; SEM, standard error mean. ∗P < .05∗∗; P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.