Figure 4. Adoptive transfer of bm1 or bm12 DLI-treated B6/lpr lym- phocytes enhances bm1 or bm12 skin graft survival. (A) The spleen and lymph node cells were collected from B6/lpr mice that were injected in- travenously with bm1 lymphocytes. B6 / mice were either left untreated as a control ( and , n 10) or injected with 4 107 bm1 DLI-treated B6/lpr lymphocytes per mouse ( and , n 8) 1 d before transplanta- tion. All recipient mice were then given tail skin grafts from both bm1 (solid lines) and BALB/c (dashed lines) donors. bm1 skin graft survival was significantly prolonged in mice that received lpr lymphocytes when compared with untreated controls (P 0.05). (B) DNTC were purified from B6/lpr mice that had been infused with bm1 cells. Naive B6 / mice were either left untreated as a control ( and , n 10) or injected intravenously with 1.5 107 bm1 DLI-treated B6/lpr DNTC per mouse ( and , n 10) 1 d before transplantation. Each mouse was trans- planted with bm1 (solid lines) and BALB/c (dashed lines) skin grafts. bm1 skin graft survival was significantly prolonged in mice that received lpr DNTC when compared with untreated controls (P 0.017). (C) DNTC were purified from B6/lpr mice that had been infused with bm12 cells. Naive B6 / mice were injected intravenously with 8.5 106 DNTC ( and , n 7) 1 d before transplantation, or were left untreated as con- trols ( and , n 5). Each mouse was transplanted with bm12 (solid lines) and (B6Xdm2)F1 (dashed lines) skin grafts. bm12 skin graft survival was significantly prolonged in mice that received lpr DNTC when com- pared with untreated controls (P 0.0012). and lymph node cells. The in vivo–activated bm12-specific B6/lpr DNTC were isolated and transferred into naive B6 / mice 1 d before transplantation or left untreated as a control. All B6 / recipient mice then received skin grafts from both bm12 mice (antigen-specific) and (B6xdm2)F1 (third-party) donors. Fig. 4 C shows that the mice that re- ceived bm12-specific B6/lpr DNTC had significant en- hancement of bm12 skin graft survival (MST 29.7, n 7) when compared with control mice that did not receive any B6/lpr DNTC (MST 8.2, n 5, P 0.0012). Third-party allogeneic (B6xdm2)F1 skin grafts were re- jected at the same pace by both groups of recipients. These results demonstrate that in vivo–activated B6/lpr DNTC can also specifically enhance the survival of MHC class II– mismatched skin allograft survival. Together, these find- ings confirm and extend the results obtained from non-lpr mice (11, 24) and demonstrate that in vivo–activated B6/ lpr DNTC can inhibit the immune response of syngeneic T cells toward MHC class I and II alloantigens in vivo, and prolong skin graft survival in an antigen-specific manner. Discussion The accumulation of TCR- DNTC cells in the pe- riphery of autoimmune lpr mice and patients has been ob- served for many years. However, the function and mecha- nism of these DNTC has remained elusive. Previous studies have shown that mature peripheral DNTC from transgenic (11, 12) and normal mice (11, 18) are able to down-regulate alloimmune responses mediated by synge- neic CD4 and CD8 T cells in vitro through the Fas–FasL pathway. Furthermore, several studies indicate that lpr DNTC may have a regulatory function. Watenabe et al. (15) demonstrated that the DNTC population in lpr mice could kill allogeneic tumor target cells through a FasL- dependent mechanism. Benihoud et al. (20) showed that lpr DNTC can kill syngeneic Fas-expressing T cells in vitro af- ter stimulation with PMA and ionomycin. The results pre- sented in this paper demonstrate for the first time that the DNTC that accumulate in autoimmune-prone lpr mice possess a potent ability to down-regulate allogeneic im- mune responses mediated by syngeneic CD4 and CD8 T cells from wild-type mice both in vitro and in vivo. It is well known that regulatory T cells play an important role in preventing autoimmune diseases (4–6). Various studies have shown that autoimmune diseases develop be- cause of a lack or a malfunction of regulatory cells (1, 2, 7, 8). In lpr mice, the number of DNTC is much higher than what is seen in normal mice, yet the animals develop au- toimmune disease. Here we demonstrate that although DNTC from autoimmune-prone gld mice that lack func- tional FasL molecules cannot kill activated syngeneic Fas CD8 T cells, lpr DNTC are able to kill both activated syngeneic CD4 and CD8 T cells that express functional Fas receptors. These results indicate that the regulatory function of DNTC in lpr mice is retained. Thus, we specu- late that DNTC might accumulate partly as a failed attempt to suppress peripheral autoimmune T cells, and partly as a result of their own inability to undergo Fas-mediated acti- vation-induced apoptosis. Because lpr DNTC-mediated suppression depends on interactions between FasL on the DNTC and Fas expressed on target cells, autoimmune dis- ease may develop in lpr mice because the mutation of Fas on autoimmune CD4 and CD8 T cells renders them un- 265 Ford et al. Brief Definitive Report