DAN Synthesis and Proliferation of Human Lymphocytes in Vitro

Abstract

Abstract The response of human lymphocytes to phytohemagglutinin (PHA) stimulation was assessed in detailed kinetic studies, in order to define conditions permitting and regulating proliferation. By utilizing live and dead cell counts, cell cycle characterizations, and calculations of rates of entry into S phase and mitosis, it was demonstrated that dilute culture conditions (2 × 105 cells/ml) enable lymphocytes to proliferate for a period of 5 or 6 days. Although cell division occurs in concentrated cultures (2 × 106 cells/ml), net proliferation is not demonstrable due to extensive cell death and earlier decay in the proliferative response. It is projected that without cell death the cell count would rise more than 6-fold in a dilute culture and 2-fold in a concentrated culture under these growth conditions. The kinetic analyses did not demonstrate separate PHA dose thresholds for blastogenesis and entry into S phase. High doses of PHA stimulated a suboptimal response by causing excessive cell death without reducing initial entry into the cell cycle. These results provide a kinetic explanation for the frequent observation of little net proliferation despite substantial 3H-thymidine incorporation in PHA-stimulated lymphocyte cultures. Cell death is identified as a parameter that must be considered in the interpretation of in vitro lymphocyte stimulation studies, and methods for quantifying the role of cell death are demonstrated.