title: Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-gamma secretion assay.
abstract: OBJECTIVE
  The aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 10(7) cells per m(2) of body surface area to restore protective immunity after stem cell transplantation.
  
  
  METHODS
  A new strategy to generate HCMV-specific CTLs using the interferon-gamma (IFN-gamma) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described.
  
  
  RESULTS
  From 1 to 5 x 10(4) HCMV peptide-specific T lymphocytes (greater than 90% CD3(+)CD8(+)) were isolated from 1 to 2 x 10(8) peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors (n = 14) and expanded ex vivo after a median of 16 days (range 8-28 days; n = 13) to greater than 10(7)/m(2) HCMV peptide-specific CTLs using autologous (n = 2) or allogeneic (n = 11) feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 x 10(9) cells (range 6.1 x 10(8)-3.3 x 10(9)). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2-99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining (n = 14) and 89.4% (range 64.4-99.5%) by peptide-specific IFN-gamma secretion (n = 7). A median of 82.6% (range 76.0-88.0%) showed perforin secretion (n = 3) and 57.5% (range 22.2-80.7%) specific lysis of peptide-pulsed T2 cells (n = 5). A median of 52.2% (range 35.2-7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts (n = 6).
  
  
  CONCLUSIONS
  IFN-gamma secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 10(7)/m(2) HCMV-specific CTLs for adoptive immunotherapy.
authors.affiliation: Medizinische Klinik II, Eberhard-Karls-Universität Tübingen, Tübingen, Germany.
authors.forename: Alfred Lennart
authors.initials: AL
authors.lastname: Bissinger
authors.forename: Georg
authors.initials: G
authors.lastname: Rauser
authors.forename: Holger
authors.initials: H
authors.lastname: Hebart
authors.forename: Friederike
authors.initials: F
authors.lastname: Frank
authors.forename: Gerhard
authors.initials: G
authors.lastname: Jahn
authors.forename: Hermann
authors.initials: H
authors.lastname: Einsele
mesh_terms: D019264:Adoptive Transfer / Q000379:methods; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D007371:Interferon-gamma