SA-FasL transiently displayed on the surface of T cells results in their elimination in response to alloantigens in vitro. (A) In vitro proliferation assay. SA-FasL– or SA-engineered 4C T cells (H-2K^b) were stimulated with irradiated BALB/c splenocytes (H-2K^d) for 48 or 72 hours. Cultures were pulsed with [³H]thymidine for the last 16 hours of incubation and harvested using a beta plate counter. DNA-incorporated radioactivity is plotted as an assessment of cell proliferation. Data were pooled from 2 independent experiments. (B) Frequencies of live total CD4⁺ and CD8⁺ T cells (top panels) and Vβ13⁺ transgenic CD4⁺ and CD8⁺ T cells (bottom panels) in mixed lymphocyte cultures. Experimental conditions are the same as in (A), except instead of pulsing with [³H]thymidine, cultures were harvested at 72 hours, stained with the Abs to indicated markers, and analyzed using flow cytometry. Data were pooled from 2 independent experiments. (C) SA-FasL induces autocrine death in alloreactive T cells. CTV-labeled nonengineered 4C cells were mixed 1:1 ratio with CFSE-labeled SA-FasL-4C cells and used as responders at the indicated ratios against a fixed number of irradiated BALB/c cells as stimulators. Cells were harvested after 72 hours of incubation and analyzed for live cells using flow cytometry (left panel). Representative flow dot plots of proliferating 4C cells (right panel). Data sets pooled from 2 independent experiments. One-way ANOVA with Tukey multiple comparison was used in panels A-B. Unpaired two-tailed t test was used in panel C. Data are shown as mean ± SEM. ANOVA, analysis of variance; SEM, standard error mean; cpm, counts per minute. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001