Brief Deﬁnitive Report The Immune Regulatory Function of Lymphoproliferative Double Negative T Cells In Vitro and In Vivo Megan S. Ford,1 Kevin J.Young,1 Zhuxu Zhang,1 Pamela S. Ohashi,2 and Li Zhang1 1Department of Laboratory Medicine and Pathobiology and Department of Immunology, University of Toronto, Multi Organ Transplantation Program,Toronto General Hospital Research Institute, University Health Network,Toronto, Ontario M5G 2C4, Canada 2Department of Medical Biophysics and Immunology, University of Toronto, Ontario Cancer Institute, University Health Network,Toronto, Ontario M5G 2M9, Canada Abstract Lymphoproliferative (lpr) mice, which lack functional Fas receptor expression and develop autoimmune lymphoproliferative disease, have an accumulation of T cell receptor- CD4 CD8 (double negative T cells [DNTC]) in the periphery. The function of the accumulating DNTC is not clear. In this study we demonstrate that B6/lpr DNTC can dose dependently kill syngeneic CD8 and CD4 T cells from wild-type B6 mice through Fas/Fas ligand interactions in vitro. We also demonstrate that B6/lpr DNTC that are activated and ex- pand in vivo are able to specifically down-regulate allogeneic immune responses mediated by syngeneic Fas CD4 and CD8 T cells in vivo. B6/lpr DNTC that have been preactivated in vivo by infusion of either class I– (bm1) or class II– (bm12) mismatched allogeneic lympho- cytes are able to specifically enhance the survival of bm1 or bm12, but not third-party skin allografts when adoptively transferred into naive B6 / mice. These findings clearly demon- strate that B6/lpr DNTC have a potent immune regulatory function in vitro and in vivo. They also provide new insights into the mechanisms involved in the development of autoimmune disease in lpr mice. Key words: autoimmune • transplantation • immune tolerance • suppressor T lymphocytes • Fas ligand Introduction Compelling evidence indicate that regulatory T cells play an important role in the maintenance of immune tolerance to self- and foreign antigens (1–3). Various subsets of T lymphocytes have been isolated from peripheral lymphoid organs in mice and humans that have the ability to down- regulate the proliferation of autoimmune effector cells (4– 6). CD4 CD25 T cells are the most extensively studied regulatory T cells. Eliminating CD4 CD25 T cells from the periphery of mice leads to the development of systemic autoimmune diseases and adding these T cells back amelio- rates experimentally induced autoimmune symptoms (7). Other regulatory T cells, including CD4 CD45Rblow (1), CD4 DX5 T cells (8), CD8 T cells (9), -TCR cells (10), and -TCR CD3 CD4 CD8 double negative T cells (DNTC; references 11 and 12) have also been demon- Address correspondence to Li Zhang, Toronto General Hospital Research Institute, Department of Laboratory Medicine and Pathobiology, CCRW 2-809, 101 College Street, Toronto, Ontario M5G 2C4, Canada. Phone: 416-340-4915; Fax: 416-597-9749; E-mail: lzhang@transplantunit.org strated in various models to have a potent role in down- regulating immune responses. Lymphoproliferative (lpr) mice, which have a mutation in the gene responsible for the production of functional Fas molecules (13), develop systemic autoimmune disease concurrent with an accumulation of mature peripheral DNTC (14, 15). The development of autoimmune dis- ease has been suggested to be caused by autoreactive T cells in the periphery from either incomplete negative se- lection in the thymus (16) or as more recent studies indi- cate, impaired peripheral deletion (17). In particular, T cells that are reactive to mouse endogenous superantigens are deleted in the thymus of Fas-null mice, but peripheral deletion in response to bacterial superantigen is impaired in the same mice (17). These data suggest that Fas muta- tion leads to the breakdown of peripheral tolerance. However, the mechanism leading to the impaired periph- eral deletion remains unknown. Furthermore, the func- tion of the accumulating DNTC in the periphery of lpr mice is still unclear. 261 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/07/261/7 $5.00 Volume 196, Number 2, July 15, 2002 261–267 http://www.jem.org/cgi/doi/10.1084/jem.20020029