title: A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus-specific human CD4 and CD8 T lymphocytes.
abstract: BACKGROUND
  Recent clinical trials have demonstrated the efficacy of adoptive cellular therapy with virus-specific lymphocytes in patients with defective cellular immune responses. Immunoreconstitution has become a challenge for cellular immunology and for transfusion medicine. In fact, both expertises are required to provide effective and safe cellular products. Because of in vitro manipulation, T-lymphocyte cultures are at risk of contamination even under good manufacturing procedure (GMP) conditions.
  
  
  METHODS
  To further improve the quality of these GMP cellular products, a procedure was designed for purification, stimulation, and expansion of antigen-specific CD4 and CD8 T-lymphocytes in a sealed, unbreached system. Leukopacks from the blood bank that fulfill the requirements of a GMP product were the starting material. Gradient separation and washing were performed in bags with sterile connecting devices on the bench-top, as well as addition of ingredients (antigen, interleukin-2) or transfer to larger bags.
  
  
  RESULTS
  The method is described in detail, and it is shown that increase in number of cytomegalovirus-specific CD4 or CD8 T-lymphocytes was similar to procedures based on open culture systems. Cell expansion after 4 weeks ranged from 800- to 2400-fold for CD4 lymphocytes and 300- to 900-fold for CD8 lymphocytes. Antigen specificity and loss of alloreactivity were demonstrated on the expanded cells with proliferation, intracytoplasmic interferon gamma-gamma staining, cytolytic activity, and pentamer binding.
  
  
  CONCLUSIONS
  This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.
authors.affiliation: Advanced Biotechnology Center, Genoa, Italy. lipira@cba.unige.it
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Li Pira
authors.forename: Laura
authors.initials: L
authors.lastname: Bottone
authors.forename: Federico
authors.initials: F
authors.lastname: Ivaldi
authors.forename: Roberta
authors.initials: R
authors.lastname: Pelizzoli
authors.forename: Marco
authors.initials: M
authors.lastname: Risso
authors.forename: Gino
authors.initials: G
authors.lastname: Tripodi
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Manca
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media; D003587:Cytomegalovirus / Q000276:immunology*; D006801:Humans; D016219:Immunotherapy, Adoptive; D008213:Lymphocyte Activation
chemical_list: D003470:Culture Media