Separation of human T-lymphocyte colony-forming cells on Percoll gradients

Formation of T-lymphocyte colonies in semi-solid agar by mitogen-stimulated peripheral blood mononuclear cells is a sensitive indicator of a proliferative response. The exact identity of the T-lymphocyte colony forming cell (T-CFC) is not known, nor is it known if more than one T-CFC exists. It is possible that different subsets of mononuclear cells, each responding to diverse mitogens, give rise to different T-CFC. In this study, we separated mononuclear cells into seven subsets based on their density utilizing Percoll at concentrations of 40% to 55%. Following separation, the cells from each fraction were stimulated by phytohemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (Con A), or staphylococcal protein A (SPA), and cultured in a semi-solid agar system. Each fraction was fully characterized by immunologic and cytochemical cell markers. Monocytes were found in the light density fraction, whereas T-lymphocytes and large granular lymphocytes were predominantly seen in the heavier density fractions. B lymphocytes were concentrated in the middle density fractions. Cells from fraction 1 (the lightest density fraction) formed significantly more T-cell colonies when stimulated by PHA than did fractions 4, 5, 6, or 7. This effect was not observed when other mitogens were used. We conclude that mononuclear cells can be separated into enriched cell subpopulations by Percoll fractionation and that PHA-stimulated T-CFC may also be enriched by Percoll fractionation. In addition, the data suggest that different subsets of T-CFC may exist.