T lymphocyte colonies stimulated by different mitogens require diverse culture conditions

Normal human peripheral blood mononuclear cells form colonies of T lymphocytes in a semi-solid agar matrix when stimulated by a variety of mitogens. In this report, we attempt to determine the optimal conditions for the formation of T lymphocyte colonies by cells stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Concanavalin A (Con A), or Staphylococcal protein A (SPA). We conclude that optimal conditions differ for each mitogen used. Cultures stimulated by PWM or Con A showed a significant requirement for feeder layers composed of human peripheral blood mononuclear cells. Two-mercaptoethanol significantly enhanced the number of T-cell colonies when PWM, Con A, or SPA, but not PHA, were added as mitogens. Fetal calf serum (FCS) was required for optimal conditions when Con A or SPA but not PWM or PHA were used to stimulate mononuclear cells. Cells stimulated by PHA or PWM produced more T-cell colonies in a 2-step assay than a 1-step assay, whereas the reverse was true with Con A or SPA. Optimal cell concentrations, mitogen doses, and culture kinetics also differed for each mitogen used in the T-cell colony assay.