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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral / immunology
  • Cancer Vaccines*
  • Cell Separation / methods*
  • Cells, Cultured
  • Cytomegalovirus / immunology*
  • Cytomegalovirus Infections / immunology
  • Cytomegalovirus Infections / therapy*
  • Drug Resistance
  • Histocompatibility Antigens Class I / chemistry
  • Histocompatibility Antigens Class I / metabolism
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Protein Multimerization
  • Recurrence
  • Streptavidin / chemistry
  • T-Lymphocytes, Cytotoxic / pathology*
  • T-Lymphocytes, Cytotoxic / transplantation
  • Virus Activation

Substances

  • Antigens, Viral
  • Cancer Vaccines
  • Histocompatibility Antigens Class I
  • Streptavidin