Restimulation of cytolytic T-lymphocytes in long-term mixed leukocyte cultures. 1. Physical and proliferative characteristics of cytolytic T-lymphocytes restimulated at low cell density.

Some physical and proliferative characteristics of cytolytic thymus-derived lymphocytes (CTL) have been investigated in long-term mixed leukocyte cultures (MLC). Velocity sedimentation analysis of MLC cells restimulated by homologous alloantigens at low responding cell density indicated that a shift from large cycling CTL to much smaller (probably non-cycling) CTL occurred between the third and sixth day in secondary cultures. This change in physical characteristics as a function of growth phase was accompanied by a parallel change in the responsiveness of secondary MLC cells to a further alloantigenic stimulus; restimulated Day 3 secondary cells gave rise to a transient CTL response (peaking after 2–3 days) whereas the response of restimulated Day 6 secondary cells increased for 4 days and reached much higher peak levels. Repeated stimulation of MLC cells under the latter conditions led to dramatic increases in both CTL activity and viable cell number. In particular, four sequential restimulations at 7-day intervals resulted in a calculated absolute increase of approximately 500,000-fold in both parameters. Cells derived from such extensive proliferation retained their original lytic specificity and were uniquely T cells as determined by surface markers. These results raise interesting questions regarding the extent and regulation of CTL proliferation in MLC.