title: Clinical-scale isolation of 'minimally manipulated' cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease.
abstract: OBJECTIVE
  Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity.
  
  
  METHODS
  Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 10(10) leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting.
  
  
  RESULTS
  A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 10(6) CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection.
  
  
  CONCLUSIONS
  Clinical-scale isolation of "minimally manipulated" CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.
authors.affiliation: Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Odendahl
authors.affiliation: Department of Internal Medicine II, Division of Haematology and Oncology, Julius Maximilian University Medical Centre, Würzburg, Germany.
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Grigoleit
authors.affiliation: Institute for Transfusion Medicine and Immunohaematology, Goethe University Medical Centre, and German Red Cross Blood Donation Service Baden-Württemberg-Hessen, Frankfurt am Main, Germany.
authors.forename: Halvard
authors.initials: H
authors.lastname: Bönig
authors.affiliation: Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München (TUM), Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Neuenhahn
authors.affiliation: Clinical Cooperation Group 'Immune Monitoring, Helmholtz Centre Munich (Neuherberg) and TUM, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Albrecht
authors.affiliation: Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München (TUM), Munich, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Anderl
authors.affiliation: Stage Cell Therapeutics, Goettingen, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Germeroth
authors.affiliation: Institute of Immunology, Medical Faculty, Dresden University of Technology (TUD), Dresden and Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Schmitz
authors.affiliation: Department of Medicine I, University Hospital of Dresden, Dresden, Germany and CRTD, Dresden, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Bornhäuser
authors.affiliation: Department of Internal Medicine II, Division of Haematology and Oncology, Julius Maximilian University Medical Centre, Würzburg, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Einsele
authors.affiliation: Institute of Immunology, Medical Faculty, Dresden University of Technology (TUD), Dresden and Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany.
authors.forename: Erhard
authors.initials: E
authors.lastname: Seifried
authors.affiliation: Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München (TUM), Munich, Germany; Clinical Cooperation Group 'Immune Monitoring, Helmholtz Centre Munich (Neuherberg) and TUM, Germany; Clinical Cooperation Group 'Antigen-specific Immunotherapy, Helmholtz Centre Munich (Neuherberg) and TUM, Germany; German Centre for Infection Research (DZIF), Munich, Germany.
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Busch
authors.affiliation: Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany; Transfusion Medicine, Medical Faculty Carl Gustav Carus, University of Technology Dresden and CRTD, Dresden, Germany. Electronic address: T.Tonn@blutspende.de.
authors.forename: Torsten
authors.initials: T
authors.lastname: Tonn
mesh_terms: D000956:Antigens, Viral / Q000276:immunology; D019496:Cancer Vaccines; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000628:therapy*; D004351:Drug Resistance; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D055503:Protein Multimerization; D012008:Recurrence; D019809:Streptavidin / Q000737:chemistry; D013602:T-Lymphocytes, Cytotoxic / Q000473:pathology* / Q000637:transplantation; D014775:Virus Activation
chemical_list: D000956:Antigens, Viral; D019496:Cancer Vaccines; D015395:Histocompatibility Antigens Class I; D019809:Streptavidin
keywords: CMV-specific T cells; CliniMACS; MHC streptamer technology; adoptive T-cell transfer