A Peripheral Blood-Derived Monolayer Supports Long-Term Cultures of Human CD4+ and CD8+ T Lymphocytes

• Published in: Blood 1995-06, Vol.85 (11), p.3213-3222
• Main Authors: Sutkowski, Natalie, Kuo, Ming-Ling, Amenta, Peter S., Dougherty, Joseph P., Ron, Yacov
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; AIDS/HIV; Animal cells; Biological and medical sciences; Biomarkers - analysis; Biotechnology; CD4-CD8 Ratio; CD4-Positive T-Lymphocytes - cytology; CD8-Positive T-Lymphocytes - cytology; Cell Communication; Cells, Cultured; Child; Child, Preschool; Culture Techniques - methods; DNA Replication; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Extracellular Matrix Proteins - analysis; Female; Fetal Blood - cytology; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Genetic Therapy - methods; Genetic Vectors; Growth Substances - metabolism; Humans; Infant; Infant, Newborn; Lymphocyte Activation; Male; Methods. Procedures. Technologies; Middle Aged; Recombinant Fusion Proteins - biosynthesis; Sarcoma, Kaposi - pathology; Time Factors; Transfection
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V85.11.3213.bloodjournal85113213

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 I (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.