ORIGINAL ARTICLE Proliferation-based T-cell selection for immunotherapy and graft-versus-host-disease prophylaxis in the context of bone marrow transplantation APachnio 1 ,SDietrich 1 ,WKlapper 2 ,AHumpe 1 ,MSchwake 3 ,RSedlacek 3 ,MGramatzki 1 andCBeck 1 1 II Medical Department, University of Kiel, Kiel, Germany; 2 Department of Pathology, University of Kiel, Kiel, Germany and 3 Department of Biochemistry, University of Kiel, Kiel, Germany Graft-versus-host disease (GvHD) caused by alloreactive T cells within the graft is a major drawback of allogeneic BMT, but depletion of T cells leads to higher rates of relapse, opportunistic infections and graft failure. There- fore, selective removal of GvHD-inducing alloreactive T cells might be beneficial. We describe here the separation of alloresponsive T cells, based on carboxyfluorescein succimidyl ester labeling, in vitro allostimulation and FACS-sorting. In vivo effects of the separated cell populations were investigated in the context of allogeneic BMT in murine models: in vitro resting T cells were shown to survive in the allogeneic host and retain immunor- eactivity against ‘third-party’ antigens. As demonstrated in two different transplantation models, elimination of proliferating cells significantly reduces GvHD but offers no advantages to using T-cell-depleted bone marrow alone concerning engraftment and tumor control. Transplanting T cells that proliferate in response to tumor antigens in vitro may narrow down the spectrum of antigens recognized by T cells and therefore reduce GvHD while maintaining graft-facilitating function and tumor control. Therefore, selecting tumor-reactive T cells on the basis of their proliferative response in vitro may be beneficial for the recipient, less time consuming than T-cell cloning and still reduce the extent of GvHD. Bone Marrow Transplantation (2006) 38, 157–167. doi:10.1038/sj.bmt.1705411 Keywords: T-cell selection; graft-engineering; GvHD; allogeneic bone marrow transplantation Introduction Allogeneic bone marrow (BM) transplantation is an established procedure for a variety of diseases of the hematopoietic system, including leukemia, lymphoma and autoimmune diseases. 1 The treatment is hampered by the development of acute and chronic graft-versus-host disease (GvHD),whichisinducedbyalloreactiveTcellswithinthe graft or given separately as a donor lymphocyte infusion (DLI) (reviewed by Ferrara et al. 2 ) and has a profound impact on survival and quality of life. T cells, on the other hand, confer a protective effect against relapse of the underlyingmalignantdisease 3,4 andsupportengraftment. 5,6 Therefore, an increasing arsenal of immunosuppressive agents is used for GvHD prophylaxis in the clinical applicationofBMtransplantation. 7,8 The complete elimina- tion of T cells in the graft represents an efficient means to circumventGvHDinduction,butleadstoincreasedrelapse rates, 3 impaired engraftment 9 and profound immunosup- pression, resulting in increased mortality caused by opportunistic infections. 10 Repetitive infusion of increasing amounts of donor T cells (DLI 11 ) at intervals after transplantation of T-cell-depleted (TCD) BM proves to be valuable for the treatment of relapse but still bears the risk of GvHD induction. Therefore, in vitro approaches to selectively eliminate alloreactive T cells have been deve- loped: they include removal of activated CD25 þ T cells, 12 CD69-positive cells, 13 induction of activation-induced cell death by treatment with anti-CD95 antibodies 14 and the depletion of proliferating cells using photodynamic label- ing. 15 In vivo elimination of alloreactive T cells after TK suicide gene modification 16 or the addition of expanded regulatory T cells 17,18 was also evaluated. The strategy of identifying leukemia-specific or lineage-specific minor anti- gens on malignant host cells 19,20 may allow selective induction of leukemia-reactive T cells with low alloreacti- vity but is labor-intensive and time consuming. In this study, we evaluated a carboxyfluorescein succi- midyl ester (CFSE)-based method 21 to deplete alloreactive T cells by ex vivo stimulation and subsequent selection of non-proliferating T cells. The approach is based on the observationthatTcellsproliferateinresponsetoallogeneic stimuli 22 and display a distinct phenotype. 23 CFSE labeling and allostimulation allow the reliable identification and FACS-based separation of resting and proliferating T cells via dye dilution of the latter. Resting T cells have been shown to be tolerant towards their stimulators in vitro and Received 27 July 2005; revised 20 March 2006; accepted 20 April 2006 Correspondence: Dr C Beck, II Department of Medicine, Division of Stemcell and Immunotherapy, Universita¨tsklinikum Schleswig-Holstein, Campus Kiel, Schittenhelmstrasse 12, D-24105 Kiel, Germany. E-mail: Dr.Ch.Beck@freenet.de Bone Marrow Transplantation (2006) 38, 157–167 & 2006 Nature Publishing Group All rights reserved 0268-3369/06 $30.00 www.nature.com/bmt