Addition of high molecular weight dextran to culture medium prevents the initiation of T lymphocyte-mediated killing by holding the cytolytic T lymphocytes (CTL) and target cells in suspension and preventing intercellular contact. Suspension in 10% dextran was used to interrupt the ongoing formation of adhesions between CTL and target cells already in contact in a centrifuged pellet. The results demonstrate that 1) firm adhesions form between CTL and target cells within 1 min at 37°C; 2) once formed, these adhesions are stable at low temperature and are resistant to mechanical shearing forces; 3) these adhesions can be disrupted by EDTA; 4) immediately after the adhesions form, separation of the CTL from the target cells prevents lysis of the latter; 5) after incubation of targets adhering to CTL for an additional 6 min at 37°C, removal of the CTL no longer prevents target cell lysis. Thus, target cells become “programmed” for subsequent lysis within a few minutes after contact with CTL, after which lysis occurs during the next several hours without further participation of the effector cell.
At 15°C, adhesions form 1/17 as fast as at 37°C. Programming of target cells for lysis occurs 1/76 as fast at 15°C as at 37°C. Thus, the programming for lysis step is about 4-fold more temperature dependent than the adhesion step.
In addition to being detected by subsequent target cell lysis in 10% dextran, the adhering cell clusters can be counted with low power microscopy. This permitted verification that EDTA separates the clusters after programming for lysis is complete. Moreover, the great majority of the clusters seen at 37°C are antigen-specific. Knowledge of the cluster size distribution and the subsequent level of lysis permits the deduction that not less than 6% of the sensitized peritoneal cell populations used were CTL.
This work was supported by National Institutes of Health Research Grant CA-14723-01.