activated syngeneic Fas CD8 T cells mediated by B6/gld DNTC was significantly reduced compared with that of B6/lpr DNTC. This result indicates that the expression of functional FasL on DNTC enhances the ability of DNTC to kill CD8 T cell targets. To further determine the specific contribution of FasL expression on lpr DNTC-mediated killing of syngeneic T cells, FasL on lpr DNTC was blocked with various con- centrations of Fas–Fc fusion protein before coincubation with a fixed number of syngeneic Fas CD8 T cells. The Figure 1. Lpr DNTC kill target cells through the Fas/FasL pathway. (A) H-2d–specific B6/lpr DNTC effectors were prepared as described in Materials and Methods and cocultured with primary activated CD8 T cells from either B6 / (light bars), or B6/lpr (dark bars) mice. The per- cent-specific lysis of target cells was calculated. The data are expressed as mean percent killing of three replicate cultures. The experiment was repeated twice and similar results were obtained. (B) Activated B6 Fas CD8 T cells cocultured with DNTC isolated from either B6/lpr (light bars) or B6/gld (dark bars) mice. The percent-specific lysis of target cells was calculated. The data are expressed as mean percent killing of three replicate cultures. (C) 5 104/well of activated B6/lpr DNTC were incubated with Fas–Fc fusion protein in the indicated concentra- tions for 20 min and then cocultured with 104 primary activated B6 / CD8 T cell targets. The mean percent inhibition of specific lysis of B6 / CD8 target cells compared with killing detected in the absence of Fas–Fc is shown. The data represent three replicate cultures. The experiment was repeated twice and representative data are shown. ability of lpr DNTC to kill syngeneic Fas CD8 T cells was significantly inhibited when FasL was blocked on lpr DNTC by increasing concentrations of Fas–Fc fusion pro- tein (Fig. 1 C). Taken together, these data demonstrate that lpr DNTC are able to down-regulate allogeneic immune responses mediated by syngeneic CD8 T cells and the ex- pression of functional FasL on DNTC and Fas on target CD8 T cells is important for lpr DNTC-mediated killing. In Vitro–activated B6/lpr DNTC Are Cytotoxic to B6 / CD4 and CD8 T Cells That Are Activated by a Different Antigen. To further characterize the ability of lpr DNTC to regulate wild-type T cells, we first assessed the ability of H-2d–specific B6/lpr DNTC to kill activated syngeneic H-2d–specific B6 / CD4 , in addition to CD8 , T cells. Fig. 2 A shows that B6/lpr DNTC could dose dependently kill both activated syngeneic H-2d–specific CD8 and CD4 T cells from normal B6 / mice. Next, we addressed the question of whether the killing of syngeneic T cells medi- ated by lpr DNTC was antigen specific. Activated H-2d– specific B6/lpr DNTC were tested for their ability to kill syngeneic T cells that are activated by either the same or a different antigen. Specifically, CD8 T cells from B6 / mice were activated against H-2d as described in Materials and Methods, or B6 transgenic P14 CD8 T cells were stimulated with p33 peptide to create CD8 T cell targets that carried TCR of different specificity from the B6/lpr DNTC. The ability of H-2d–specific B6/lpr DNTC to kill each cell type was compared. Fig. 2 B shows that H-2d– specific B6/lpr DNTC effectively killed both p33- and H-2d–specific CD8 T cells. Together, these data indicate that lpr DNTC that were activated in vitro with allogeneic splenocytes can kill both activated syngeneic CD4 and CD8 T cells, and the cytotoxicity to syngeneic CD8 T cells is not antigen specific in vitro. Infusion of Allogeneic Lymphocytes Leads to Expansion of B6/lpr DNTC. DLI has been shown to enhance organ allograft survival in mice (24) and humans (26). Recently, we have demonstrated that pretransplant DLI induces the activation and expansion of recipient DNTC and the pro- longation of allograft (18) and xenograft survival (unpub- lished data), and that adoptive transfer of activated DNTC clones can enhance donor-specific but not third-party skin graft survival (11). To investigate the function of B6/lpr DNTC in vivo, we first studied whether B6/lpr DNTC can be activated and expand in vivo after the infusion of allogeneic lympho- cytes. B6/lpr mice were given a DLI of bm1 (single class I–mismatched) spleen and lymph node cells. The recipient mice were killed 7 d later and the percentages of CD8 , CD4 , and DNTC in the lymph nodes and spleen were analyzed by flow cytometry. 1 wk after DLI there was no significant change in the percentage of CD8 T cells and a significantly reduced proportion of CD4 T cells in the spleen (P 0.05) but not in the lymph nodes compared with age-matched B6/lpr non-DLI–treated controls. Inter- estingly, the percentage of DNTC in both the lymph nodes (Fig. 3 A) and spleen (Fig. 3 B) of DLI-treated B6/lpr mice was significantly increased compared with age-matched 263 Ford et al. Brief Definitive Report