Studies on the induction and expression of T cell mediated immunity. VI. Heterogeneity of lytic efficiency exhibited by isolated cytotoxic T lymphocytes prepared from highly enriched populations of effector-target conjugates

The lytic efficiency of isolated alloimmune cytotoxic lymphocytes (CTL) was examined, following the development of a method to highly enrich for cytotoxic T lymphocytes (CTL) by specific binding to allogeneic target cells. Murine effector-target cell conjugates, prepared by mixing populations of immune lymphocytes (generated in vitro and in vivo) with allogeneic tumor target cells, were enriched by rate sedimentation through a discontinuous fetal calf serum (FCS) gradient. The bottom layer contained 80 to 90% conjugated lymphocytes, or an enrichment of 3- to 6-fold and a recovery of approximately 50%. Isolation by a microdilution technique, followed by single-cell, cell-mediated cytotoxicity (CMC) assay proved that an average of 70% of the conjugating lymphocytes from both primary and secondary responses lysed their attached target cell. The absolute frequency of CTL in any immune T cell population was determined by correcting the percentage of conjugates by the factor of those conjugating lymphocytes observed to kill in the single-cell CMC assay. Using these techniques of CTL enrichment and single-cell assay, results obtained that provide evidence for heterogeneity of CTL lytic potential. The intrinsic lytic efficiency of individual CTL was determined by quantitation of the time intervals required for target lysis by 1373 conjugated lymphocytes. Up to 5 hr for primary and 4 hr for secondary CTL was necessary to achieve maximum single-target lysis at 37°C. In addition, the average 1° CTL displayed a slower rate of killing than the average 2° CTL. It was found that the time required for lysis by 50% of the 1° lymphocytes was 2.5 hr and for 2° lymphocytes was 1.2 hr, demonstrating that cytotoxic lymphocytes are heterogeneous in their lytic potential. Heterogeneity of lytic efficiency by CTL was also shown by experiments demonstrating a poor correlation between the frequency of conjugating lymphocytes and the cytotoxicity measured by ⁵¹Cr release.