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Cytotherapy. 2014 Sep;16(9):1245-56. doi: 10.1016/j.jcyt.2014.05.023.

Clinical-scale isolation of 'minimally manipulated' cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease.

Author information

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.

KEYWORDS:

CMV-specific T cells; CliniMACS; MHC streptamer technology; adoptive T-cell transfer

PMID:
25108651
DOI:
10.1016/j.jcyt.2014.05.023
[Indexed for MEDLINE]
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