title: A peripheral blood-derived monolayer supports long-term cultures of human CD4+ and CD8+ T lymphocytes.
abstract: An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
authors.forename: N
authors.initials: N
authors.lastname: Sutkowski
authors.forename: M L
authors.initials: ML
authors.lastname: Kuo
authors.forename: P S
authors.initials: PS
authors.lastname: Amenta
authors.forename: J P
authors.initials: JP
authors.lastname: Dougherty
authors.forename: Y
authors.initials: Y
authors.lastname: Ron
mesh_terms: D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000032:analysis; D016516:CD4-CD8 Ratio; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology*; D002450:Cell Communication; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D046508:Culture Techniques / Q000379:methods*; D004261:DNA Replication; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012514:Sarcoma, Kaposi / Q000473:pathology; D013997:Time Factors; D014162:Transfection
chemical_list: D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D006133:Growth Substances; D011993:Recombinant Fusion Proteins