CHARACTERIZATION OF NORMAL PERIPHERAL-BLOOD LYMPHOCYTE COLONY-FORMING CELLS - CELL-CYCLE STATUS, SURFACE-MARKERS, AND CELLULAR GROWTH REQUIREMENTS

Abstract

A series of studies was performed to further clarify the nature of lymphocyte colony-forming cells (CFC) from normal peripheral blood. Mononuclear cells were separated into E-rosette-enriched (E+) and E-rosette-depleted (E-) populations and cultured in methylcellulose with conditioned media and irradiated mononuclear cells. Linear plating relationships were obtained with plating efficiencies of 0.26% .+-. 0.02% (mean .+-. SE) for E+ CFC and 0.18% .+-. 0.02% for E- CFC. Cells in E+ colonies were T lymphocytes and in E- colonies were B lymphocytes as determined by cell surface marker analysis. Using the thymidine suicide technique, .apprx. 1/2 of CFC were found to be in cycle at any moment, and plating efficiencies and cell cycle status of E+ CFC were not changed by preincubation with PHA [phytohemagglutinin] in liquid culture for 48 h. Using antibody complement-mediated cytotoxicity, E+ CFC were found to be T101+, OKT3+, and Ia-, while E- CFC were OKT3- and Ia+. Using monocyte-depleted populations obtained by sedimentation at unit gravity, lymphocyte colony growth was absent in monocyte-depleted fractions, and optimal growth occurred with 40% monocytes in culture. In contrast to some previous studies, lymphocyte CFC apparently originate from a small, cycling population of cells bearing mature T or B lymphocyte markers. Entry into cell divison does not confer colony-forming capacity on lymphocytes. Monocytes are critical to growth of E+ CFC, and cultures severely depleted of monocytes would not be expected to form colonies.