Figure 2. In vitro–activated B6/lpr DNTC are cytotoxic to B6 / CD4 and CD8 T cells that are activated by a different antigen. (A) DNTC were purified from the spleens of B6/lpr mice and cultured for 3.5 d in the presence of irradiated BALB/c splenocytes, rIL-2 and rIL-4, and used as effector cells. In vitro–activated H-2d–specific B6 / CD4 (dark bars) and CD8 (light bars) T cells were used as targets. After 18 h of co- culture, cells were harvested and counted, and the percent-specific killing was calculated. The data are expressed as mean percent killing of three replicate cultures. The experiment was repeated three times and represen- tative data is shown. (B) Splenic CD8 T cells from B6 mice or B6 anti– lymphocytic choriomeningitis virus-gp33 were activated in vitro with either irradiated BALB/c spleen cells or gp33 peptide as described previ- ously (21, 23). H-2d–specific B6/lpr DNTC were stimulated indepen- dently by BALB/c spleen cells and cocultured with either H-2d–specific B6 / CD8 T cells (light bars), or p33-specific B6 CD8 T cells (dark bars). The percent-specific killing of each target cell culture was deter- mined. The data are expressed as mean percent killing of three replicate cultures. The experiment was repeated three times and representative data is shown. untreated controls (P 0.04 and P 0.02, respectively). This data indicates that single MHC class I locus–mis- matched DLI can promote the activation and proliferation of lpr DNTC in vivo. Adoptive Transfer of In Vivo–activated B6/lpr DNTC En- hances Specific Skin Allograft Survival. Our finding that in vitro–activated lpr DNTC can kill both CD4 and CD8 T cells suggests that DNTC might be able to inhibit im- mune responses in vivo. To study whether the in vivo–acti- vated lpr DNTC could enhance donor-specific skin graft survival, the spleen and lymph node cells were collected from B6/lpr mice 1 wk after the infusion of bm1 lympho- Figure 3. Increase of DNTC in the lymph nodes and spleen of B6/lpr mice after infusion of bm1 cells. B6/lpr mice were injected intravenously with 4 107 bm1 lymphocytes. 7 d later, (A) lymph node and (B) spleen lymphocytes were harvested from bm1 cell-injected (dark bars) and age- matched control (light bars) mice. The percentage of CD3 CD4 CD8 , CD3 CD4 CD8 , and CD3 CD4 CD8 cell populations were ana- lyzed by flow cytometry. The DNTC population was significantly in- creased in both the spleen (n 5) and lymph nodes (n 4) of DLI- treated mice when compared with age-matched nontreated control mice (n 5; spleen, P 0.02 and lymph node, P 0.04). cytes. Naive B6 / mice were used as skin graft recipients and divided into two groups. One group was infused with a mixture of DLI-treated B6/lpr spleen and lymph node cells, and the other was left untreated as a control. 1 d later, both groups of mice were given tail skin grafts from bm1 (antigen-specific) and BALB/c (third-party) donors. Skin graft survival was monitored as previously described (11). Fig. 4 A shows that the mean survival time (MST) of bm1 skin grafts was significantly enhanced in mice that received B6/lpr lymphocytes that were preactivated by bm1 anti- gens (MST 28.8 d, n 8) when compared with that of the control group (MST 15.7 d, n 6, P 0.046). All third-party BALB/c skin grafts were rejected within the normal time course by both groups, indicating that the en- hancement of skin graft survival was donor specific. To further determine the specific contribution of B6/lpr DNTC in enhancing skin allograft survival, DNTC were isolated from the peripheral lymphocytes of bm1 DLI- treated B6/lpr mice. The in vivo–activated bm1-specific B6/lpr DNTC were adoptively transferred into naive B6 / mice 1 d before transplantation. Each recipient B6 / mouse was then given skin grafts from bm1 (donor-spe- cific) and BALB/c (third-party) mice. Graft survival was compared with the B6 / mice that were given the same skin grafts without pretransplant infusion of B6/lpr DNTC. As shown in Fig. 4 B, the MST of bm1 skin grafts was significantly prolonged in the mice that received in vivo–activated bm1-specific DNTC when compared with the controls (MST 47.5 d, n 10 vs. MST 15.7 d, n 6, P 0.017). In contrast, both treatment groups rejected all third-party BALB/c skin grafts within 2 wk. These data demonstrate that the infusion of in vivo–activated B6/lpr DNTC can significantly enhance donor-specific bm1 skin allograft survival. To test whether B6/lpr DNTC could also enhance the survival of MHC class II–mismatched skin grafts, B6/lpr mice were infused with bm12 (class II–mismatched) spleen 264 The Immune Regulatory Function of lpr DN T Cells