Bone Marrow Transplantation (2002) 30, 69–74  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt T cell depletion A large-scale method for T cell depletion: towards graft engineering of mobilized peripheral blood stem cells PR Gordon 1 , T Leimig 1 , I Mueller 1 , A Babarin-Dorner 1 , MA Holladay 1 , J Houston 1 , G Kerst 2 , T Geiger 1 and R Handgretinger 1 1 Division of Stem Cell Transplantation, St Jude Children’s Research Hospital, Memphis, TN, USA; and 2 Children’s University Hospital, University of Tuebingen, Germany Summary: We have investigated the feasibility and efficacy of large-scale T cell depletion from granulocyte colony- stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC). The method is based on the use of a CD3 antibody conjugated to magnetic microbeads and magnetic activated cell sorting (Clinimacs). A total of eight large-scale experiments were performed. In four experiments, CD3 + T cells were depleted from PBSC obtained from volunteers mobilized with G-CSF whereas, in four experiments, T cells were depleted from PBSC from stem cell donors, in which the CD34 + stem cells had been removed for allogeneic transplan- tation by positive selection prior to T cell depletion. The mean number of processed mononuclear cells (MNCs) was 3.3  10 10 (range 1.5  10 10 –5.1  10 10 ) with a mean T cell proportion of 35.8% (range 16.7–64.0%). After T cell depletion, the percentage of contaminating T cells was 0.15% (range 0.01–1.01%) with a mean log 10 depletion of 3.4 (range 2.8–4.1). The mean recovery of CD3-negative MNCs after depletion was 76% (range 52–100%). The mean recovery of CD34 + stem cells in the four evaluable experiments was 82% (range 75– 92%). In vitro colony assays and in vivo NOD/SCID repopulation assays showed that this large-scale T cell depletion method has no negative impact on the func- tion of the hematopoietic precursor cells. Therefore, we conclude that this T cell depletion method is a valuable tool for further graft engineering strategies involving mobilized PBSCs. Bone Marrow Transplantation (2002) 30, 69–74. doi:10.1038/sj.bmt.1703619 Keywords: T cell depletion; peripheral blood stem cells; allogeneic transplantation; Clinimacs Correspondence: R Handgretinger, Division of Stem Cell Transplantation, St Jude Children’s Research Hospital, 332 N Lauderdale St, Memphis, TN 38105, USA Received 23 January 2002; accepted 4 April 2002 While bone marrow (BM) has been the source of hemato- poietic stem cells in the past, G-CSF mobilized PBSCs are increasingly used as a stem cell source. Compared to BM, transplantation of allogeneic mobilized PBSC is associated with less regimen-related toxicity 1 and earlier hematopo- ietic recovery. 2 Although the risk of developing acute graft- versus-host disease (GVHD) is not increased after PBSC transplantation compared to bone marrow transplantation, 3,4 the incidence of extensive chronic GVHD is high. 5 The most effective means of prevention of acute and chronic GVHD is in vitro depletion of T lymphocytes from the graft. 6,7 Most of the available T cell depletion strategies have been developed for BM and methods based on antibody/complement 8 or other approaches 9 are not well applicable for PBSC due to the high cell numbers to be processed. Recently, a method for indirect T cell depletion of PBSCs based on the positive selection of CD34 + stem cells has been described. 10 We and others have used this method in matched sibling transplants, 11 MUD transplants 12 and haploidentical transplantation. 6 This indirect approach yields a T cell depletion of 5 log. 10 However, there might be some disadvantages associated with the use of purified CD34 + cells, such as a higher incidence of engraftment fail- ures, 13 delayed immunoreconstitution, 14 the lack of an anti- leukemic effect 15 and the exclusion of CD34-negative stem cells from the graft. In order to overcome the disadvantages of CD34 + positive selection, we have evaluated a strategy to directly deplete T cells from PBSC. In this paper, we describe a rapid and efficient method of T cell depletion based on the Clinimacs technology. This method allows the use of T cell-depleted PBSC and can be the basis for further graft engineering strategies. Materials and methods Mobilization and collection of PBSCs A total of eight large-scale experiments were performed. PBSCs were mobilized from four volunteer donors (experiments 1–4) using G-CSF (480 g/day, Neupogen; Amgen, Thousand Oaks, CA, USA) for 4 days. A single leukapheresis was performed at day 5 using a Cobe Spectra (Cobe, Lakewood, CO, USA).