Marcus Odendahl1, G Ulrich Grigoleit2, Halvard Bönig3, Michael Neuenhahn4, Julia Albrecht5, Florian Anderl4, Lothar Germeroth6, Marc Schmitz7, Martin Bornhäuser8, Hermann Einsele2, Erhard Seifried7, Dirk H Busch9, Torsten Tonn10. 1. Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany. 2. Department of Internal Medicine II, Division of Haematology and Oncology, Julius Maximilian University Medical Centre, Würzburg, Germany. 3. Institute for Transfusion Medicine and Immunohaematology, Goethe University Medical Centre, and German Red Cross Blood Donation Service Baden-Württemberg-Hessen, Frankfurt am Main, Germany. 4. Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München (TUM), Munich, Germany. 5. Clinical Cooperation Group 'Immune Monitoring, Helmholtz Centre Munich (Neuherberg) and TUM, Germany. 6. Stage Cell Therapeutics, Goettingen, Germany. 7. Institute of Immunology, Medical Faculty, Dresden University of Technology (TUD), Dresden and Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. 8. Department of Medicine I, University Hospital of Dresden, Dresden, Germany and CRTD, Dresden, Germany. 9. 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. 10. 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.
Abstract
BACKGROUND AIMS: 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.
BACKGROUND AIMS: 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: Cameron J Turtle; Laïla-Aïcha Hanafi; Carolina Berger; Theodore A Gooley; Sindhu Cherian; Michael Hudecek; Daniel Sommermeyer; Katherine Melville; Barbara Pender; Tanya M Budiarto; Emily Robinson; Natalia N Steevens; Colette Chaney; Lorinda Soma; Xueyan Chen; Cecilia Yeung; Brent Wood; Daniel Li; Jianhong Cao; Shelly Heimfeld; Michael C Jensen; Stanley R Riddell; David G Maloney Journal: J Clin Invest Date: 2016-04-25 Impact factor: 14.808
Authors: M Neuenhahn; J Albrecht; M Odendahl; F Schlott; G Dössinger; M Schiemann; S Lakshmipathi; K Martin; D Bunjes; S Harsdorf; E M Weissinger; H Menzel; M Verbeek; L Uharek; N Kröger; E Wagner; G Kobbe; T Schroeder; M Schmitt; G Held; W Herr; L Germeroth; H Bonig; T Tonn; H Einsele; D H Busch; G U Grigoleit Journal: Leukemia Date: 2017-01-16 Impact factor: 11.528