Gene Therapy (2002) 9, 201–207  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Effect of combined cytostatic cyclosporin A and cytolytic suicide gene therapy on the prevention of experimental graft-versus-host disease S Maury 1 , E Litvinova 1 , O Boyer 1 , L Benard 2 , S Bruel 1 , D Klatzmann 1 and JL Cohen 1 1 Biologie et The ´rapeutique des Pathologies Immunitaires CNRS/UPMC ESA 7087, Ho ˆpital Pitie ´-Salpe ˆtrie `re, Paris, France; and 2 Service d’Anatomie Pathologique, Ho ˆpital Pitie ´-Salpe ˆtrie `re, Paris, France The immunosuppressive drug cyclosporin A (CsA) rep- resents the standard preventive treatment of graft-versus- host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppres- sion based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, gan- ciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of Keywords: hematopoietic stem cell transplantation; graft-versus-host disease; suicide gene therapy; cyclosporin A; herpes simplex type-1 thymidine kinase; CFSE Introduction Graft-versus-host disease (GVHD) represents the main and often lethal complication encountered after allo- geneic hematopoietic stem cell transplantation (HSCT). The immunosuppressive drug cyclosporin A (CsA), administered during the first 3 to 6 months following HSCT, is part of the standard preventive treatment of GVHD. 1 This immunosuppressive regimen is only par- tially efficient, and 15 to 60% of the patients indeed develop GVHD despite CsA. 2 Recent introduction of new immunosuppressive drugs, such as FK506, has not reduced the frequency of severe GVHD. 3 T cell depletion of the graft efficiently prevents GVHD but leads to impaired engraftment, 4 prolonged immune deficiency causing an increased rate of infections, 5 and impaired graft-versus-leukemia effects resulting in an increased frequency of leukemia relapse. 6,7 Ex vivo transduction of donor T cells with a suicide gene encoding Herpes simplex type 1 thymidine kinase (TK) offers a new approach for controlling GVHD by treatment with the synthetic nucleoside analog ganciclo- vir (GCV). TK expression allows the transformation of Correspondence: D Klatzmann, CNRS/UPMC ESA 7087, Ho ˆpital Pitie ´- Salpe ˆtrie `re, 83, bd de l’Ho ˆpital, F-75651 Paris Cedex 13, France Received 13 November 2001; accepted 30 November 2001 CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell prolifer- ation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconsti- tution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT. Gene Therapy (2002) 9, 201–207. DOI: 10.1038/sj/gt/3301637 GCV into a component highly toxic for dividing T cells, a metabolism not occurring in non-transduced cells. When TK T cells divide after alloantigen recognition, they become sensitive to GCV and are consequently killed. 8 Thus, suicide gene therapy should permit the selective elimination of T cells that recognize recipient alloanti- gens, while preserving a large repertoire of T cells which were not dividing during GCV treatment. In preclinical experiments using TK T cells from transgenic mice, we and others have previously demonstrated the feasibility of preventing GVHD by this strategy, 9,10 as well as the possibility of controlling ongoing GVHD, when GCV is initiated at the time of clinical signs of GVHD. 9 In humans, suicide gene therapy of GVHD has been evaluated in two different clinical situations. One study concerned patients with relapsing hematological malig- nancy or EBV-induced lymphoproliferation after T cell- depleted allogeneic HSCT who were treated with ex vivo TK-transduced donor lymphocyte infusions. 11 In three patients who developed GVHD after TK T cell infusion, GCV administration induced two complete and one par- tial remission. The other study concerned patients receiv- ing transduced T cells, together with a T cell-depleted allogeneic marrow graft. 12 GVHD occurred in five patients and treatment with GCV was associated with complete or partial response in four and one patient(s), respectively. Other clinical trials now investigate the ability of the suicide gene system to cure, 13 but also to prevent (our unpublished protocol) GVHD.