Dendritic Cells Genetically Engineered to Express Fas Ligand Induce Donor-Specific Hyporesponsiveness and Prolong Allograft Survival 1 Wei-Ping Min,* Reginald Gorczynski,* Xu-Yan Huang,* Michelle Kushida, ² Peter Kim, ² Masayuki Obataki, ² Ji Lei,* Rakesh M. Suri,* and Mark S. Cattral 2 * Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas 1 cells. FasL-trans- fected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hypore- sponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our find- ings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses. The Journal of Immunology, 2000, 164: 161–167. D endritic cells (DC) 3 are APC that play a critical role in the initiation of immune responses (1). Although the po- tent immunostimulatory capacity of DC is well recog- nized, recent evidence suggests that DC are also capable of induc- ing donor-specific hyporesponsiveness (2). It is not yet clear whether these apparently opposing functions of DC reflect distinct subpopulations of DC (3, 4), or alternatively, distinct functions expressed at unique stages in the developmental cycle of the same cell (5). Understanding the nature of the heterogeneity of DC func- tion would promote the development of DC-based immunotherapy for the treatment of many diseases, including the induction of tol- erance in transplantation and in autoimmune disorders. Genetic modification of DC with genes encoding immunoregu- latory molecules is an alternative approach for artificial generation of tolerogenic DC. Indeed, recent reports suggest that transfection of DC with IL-10 and TGF-b can increase their tolerogenic potential (6, 7). Several attributes make DC ideal vehicles for the delivery of such molecules. They are potent activators of naive T cells, a function related to their Ag-processing capacity and to high levels of expres- sion of MHC and costimulatory molecules. In addition, they have unique migratory capability, enabling them to move from peripheral tissues to secondary lymphoid organs, where they interact with T and B cells (8, 9). The cognate recognition of DC and T cells provides the theoretic opportunity of these immunomodulatory molecules to influ- ence the immune response in an Ag-specific manner. One molecule that may enhance the tolerance-inducing capacity of DC is Fas ligand (FasL), a type II integral membrane protein that belongs to the TNF superfamily (10). Engagement of Fas by FasL initiates a signaling cascade that leads to apoptotic cell death of Fas- bearing cells. Apoptosis induced by Fas/FasL interactions is thought to play a pivotal role in the immune system, regulating both peripheral T cell homeostasis and lymphocyte-mediated cytotoxicity. FasL is expressed in immunoprivileged organs, including the eye and testis, where it has been proposed to contribute to their tolerogenic milieu and paucity of infiltrating inflammatory cells (11–13). There is evi- dence that FasL constitutively expressed on splenic DC and bone marrow-derived DC may be involved in the killing of activated CD4 1 T cells (14, 15). More recently, tolerance induced by infusion of donor bone marrow cells was shown to be dependent on the expression of FasL on the infused cells (16). In the studies described below, we investigate the immunomodulatory effect of DC transduced to express high levels of FasL in vitro and in vivo. Materials and Methods Mice Male C57BL/6 (H-2 b ), BALB/c (H-2 d ), and C3H (H-2 k ) mice were pur- chased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6-lpr/lpr mice were purchased originally from The Jackson Laboratory and bred in our animal facility. All mice were used at 8 –12 wk of age. Generation of bone marrow-derived DC DC were generated from bone marrow progenitor cells, as described by Inaba et al. (17) and modified by Suri et al. 4 Briefly, bone marrow cells *Department of Surgery and Multiorgan Transplant Program, Toronto Hospital Re- search Institute, University of Toronto, Toronto, Ontario, Canada; and ² Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Received for publication August 10, 1999. Accepted for publication October 19, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Physician Services Foundation, Juvenile Diabetes Foundation International, and the Multiorgan Transplantation Programme. 2 Address correspondence and reprint requests to Dr. Mark S. Cattral, The Toronto Hospital, NU10-145, 621 University Avenue, Toronto, Ontario, Canada, M5G 2C4. E-mail address: mcattral@email.msn.com 3 Abbreviations used in this paper: DC, dendritic cell; DNFB, dinitrofluorobenzene; FasL, Fas ligand. 4 R. M. Suri, M. B. Lutz, A. L. J. Ogilvie, S. Robner, M. Nimi, N. Kukutsch, G. Schuler, and J. M. Austyn. 1999. Stably immature dendritic cells induce T cell un- responsiveness in vitro and prolong allograft survival in vivo. Submitted for publication. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00