We and others have demonstrated that DNTC and clones from transgenic mice can down-regulate immune responses by killing activated alloreactive CD8 T cells that express functional Fas receptors but not CD8 T cells from lpr mice that express nonfunctional Fas (11, 12). Recently, it was found that DNTC from normal mice can also down- regulate allogeneic immune responses in vitro and in vivo (11, 18). These data indicate that peripheral DNTC have a regulatory function in a variety of models. Because DNTC from lpr mice express similar cell surface markers to the regulatory DNTC clones (11, 19) and can kill syngeneic Fas thymocytes in vitro when stimulated by PMA and ionomycin (20), the accumulating DNTC in lpr mice may have a regulatory function. The goal of this study was to characterize the function of lpr DNTC. The results shown here demonstrate that lpr DNTC can down-regulate allo- geneic immune responses mediated by syngeneic wild-type but not lpr CD4 and CD8 T cells both in vitro and in vivo. The inability of DNTC to kill activated lpr CD4 and CD8 T cells may contribute to the development of autoimmune disease in lpr mice. Materials and Methods Mice. C57BL/6 (B6 / ), B6.MRL-Faslpr (B6/lpr), B6Smn. C3H-Faslgld (B6/gld), B6.C-H2bm1/By (bm1), B6.C-H2bm12/By (bm12), BALB/c-H-2-dm2 (dm2, a BALB/c Ld loss mutant), and BALB/c mice were purchased from Jackson Immuno- Research Laboratories. All mice were maintained in the Univer- sity Health Network (UNH; University of Toronto, Toronto, Canada) animal colonies. (B6Xdm2)F1 mice and the B6-P14 (P14) mice possessing a transgenic TCR specific for the lympho- cytic choriomeningitis virus gp33 peptide (21) were bred in UHN animal facilities. Isolation of DNTC. Spleen and lymph node cells of B6/lpr mice were depleted of red blood cells and then passed through a nylon wool column to enrich the T cell population. The cells were then treated with depletion antibodies specific for murine CD4 (RL172, rat IgM; reference 22) and CD8 (3.168.8, rat IgM; reference 22) and rabbit complement (Cedarlane) to deplete CD4 and CD8 T cells. Generation of Effector and Target T Cells for Killing Assays. Al- loreactive H-2d–specific DNTC CD8 and CD4 T cells were generated by incubating each purified cell type with irradiated (20 Gy) BALB/c splenocytes. gp33-specific CD8 T cells were gen- erated by incubating spleen cells from P14 TCR transgenic mice with gp33 (23). The cells were cultured in -MEM media sup- plemented with 10% FCS and 50 U rIL-2. DNTC cultures also received 30 U rIL-4. Killing Assays. Primary activated B6/lpr or B6/gld DNTC were used as effector cells and plated in serial dilutions in a round-bottom 96-well plate. Target cells labeled with 5 Ci/ml [3H]TdR at 37 C for 18 h were washed three times and added in equal portions to each well. Each cell culture was also given 50 U rIL-2, 30 U rIL-4, and irradiated (20 Gy) allogeneic stimulator cells or peptide depending on the experiment. After coculture with effector cells at 37 C for 18 h, the cells were harvested and counted using a TOP COUNT™ cell harvester and plate reader (Packard Instrument Co.). Specific cell killing was calculated us- ing this equation: % Specific killing (SE)/S 100, where E (experimental) is cpm of retained DNA in the presence of effec- tor cells and S (spontaneous) is cpm of retained DNA in the ab- sence of effector cells. Cell Surface Marker Staining and Blocking. B6/lpr spleen and lymph node cells were harvested 7 d after donor lymphocyte infusion (DLI) treatment and stained with fluorescence-conjugated mono- clonal antibodies specifically recognizing CD3, CD4, and CD8 (BD Biosciences). Data were acquired and analyzed on an Epics XL-MCL flow cytometer (Coulter Corp.). Fas ligand (FasL) was blocked using recombinant mouse Fas-Fc chimera/TNFRSF6 (R&D Systems). DLI, Adoptive Transfer of Lymphocytes, and Skin Grafting. B6/ lpr mice were injected with a mixture of 4.0 107 spleen and lymph node cells from either bm1 or bm12 mice. Spleen and lymph node cells from the B6/lpr mice were harvested 7 d after the infusion of allogeneic cells. Naive B6 / mice intravenously received either 4.0 107 full spleen and lymph node cells or DNTC isolated from DLI-treated lpr mice. 1 d later, all B6 / recipient mice were given skin grafts from either bm1 or bm12 mice and a third-party skin graft (BALB/c or [B6Xdm2]F1) as previously reported (24). Survival of the skin grafts was moni- tored and scored until rejection. Statistics. All skin grafting survival data were analyzed using the Student’s t test. Results B6/lpr DNTC Kill Activated Syngeneic CD8 T Cells through the Fas/FasL Pathway. We and others have dem- onstrated that DNTC and clones derived from normal and transgenic mice can kill activated syngeneic CD8 target cells through the Fas/FasL pathway (11, 12). Furthermore, mitogen-activated lpr DNTC can kill syngeneic Fas thy- mocytes (20). These findings suggest that lpr DNTC also have a regulatory role but are unable to execute their function due to the lack of functional Fas expression on their target cells. To test this hypothesis, B6/lpr DNTC were isolated from spleen and lymph nodes, allogeneically stimulated, and used as putative regulatory cells. CD8 T cells from B6 / (Fas / ) and B6/lpr (Fas-mutant) mice were similarly activated and used as targets in a JAM cyto- toxicity assay (25). As shown in Fig. 1 A, B6/lpr DNTC killed B6 / (Fas / ) CD8 T cells much more effectively than the B6/lpr (Fas-mutant) CD8 T cells. This result is consistent with the data obtained from non-lpr DNTC and indicates that allogeneically activated lpr DNTC can kill activated syngeneic CD8 T cells from wild-type but not Fas-mutant lpr mice, which suggests that Fas–FasL in- teraction is an important mechanism in B6/lpr DNTC- mediated killing. Autoimmune-prone gld mice express FasL in a nonfunc- tional conformation that is not able to ligate Fas on target cells (13, 14). As seen in lpr mice, DNTC also accumulate in gld mice concomitant with the development of autoim- mune disease symptoms (13, 14). To determine if the ex- pression of FasL on DNTC is important for the killing of CD8 T cells, DNTC from B6/gld (FasL-mutant) mice were purified and their ability to kill activated syngeneic Fas CD8 T cells was compared with DNTC from B6/lpr (FasL / ) mice. As shown in Fig. 1 B, the cytotoxicity to 262 The Immune Regulatory Function of lpr DN T Cells