Heterogeneity of cytolytic and suppressor clones of alloactivated cells generated from soft agar colonies

Long-term cell cultures (or clones) were developed from soft agar colonies of lymphocytes alloactivated in mixed leukocyte culture reaction (MLR). Two types of colonies were identified: upper colonies that grew on the agar surface, and lower colonies found within the agar layer. Virtually all cytolytic clones originated exclusively from the upper colonies. Two groups of cytolytic clones could be distinguished, one with strong and the other with weak proliferation upon restimulation. Upper clones were capable of inhibiting primary MLR proliferation and this appeared to be related to their cytolytic effect on the stimulator. Many noncytolytic lower clones were found to suppress primary MLR cultures. Considerable heterogeneity was apparent from differences in the magnitude of suppression and the ability of the clones themselves to undergo stimulator-induced proliferation. Kinetic studies of MLR suppression were conducted to further analyze this heterogeneity. Two major kinetic patterns were observed. One showed a biphasic proliferation pattern of the MLR + clone culture. The first peak appeared to reflect an enhanced proliferation of the clone. The second phase seemed to represent diminished proliferation of the MLR responder. This type of suppression may be related to T cell growth factor depletion from MLR by the proliferating clone. The other kinetic pattern showed a consistently low proliferation of the MLR + clone culture throughout the 8-day assay period. Subsequent testing of these suppressor clones in third-party MLR cultures suggested that the specificity of suppression was unrelated to HLA-DR, MB, MT, and SB.