PERIPHERAL BLOOD-DERIVED MONOLAYER SUPPORTS LONG-TERM CULTURES OF HUMAN CD4(-LYMPHOCYTES() AND CD8(+) T)

An in vitro culture system has been developed for the long-term mainte nance of primary, human peripheral blood and umbilical cord blood T ly mphocytes, which does not rely on the use of stimulatory cytokines, an tigen, 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 (C D45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Fa ctor 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 cel ls. 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 r etroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retro viral vector. The infection efficiency was similar to that obtained fo r fresh peripheral blood T cells, indicating that the long-term-cultur ed cells might be suitable for certain gene therapy applications. (C) 1995 by The American Society of Hematology.