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

Marcus Odendahl, G. Ulrich Grigoleit, Halvard Bönig, Michael Neuenhahn, Julia Albrecht, Florian Anderl, Lothar Germeroth, Marc Schmitz, Martin Bornhäuser, Hermann Einsele, Erhard Seifried, Dirk H. Busch, Torsten Tonn

Research output: Contribution to journalArticlepeer-review

48 Scopus citations


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 × 1010 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 × 106 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.

Original languageEnglish
Pages (from-to)1245-1256
Number of pages12
Issue number9
StatePublished - Sep 2014


  • Adoptive T-cell transfer
  • CMV-specific T cells
  • CliniMACS
  • MHC streptamer technology


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