Rat heterotopic heart transplantation: quantification and analysis of cell mediated cytotoxicity

Limiting dilution analysis was used to measure the frequency of PVG-reactive cytotoxic T lymphocytes (CTL) within the peripheral blood mononuclear cell population of Lewis rats before heterotopic transplantation of PVG rat derived cardiac tissue, and in both the blood and graft-infiltrating cell populations at daily time points afterwards. Before surgery, the frequency of PVG-reactive cells within the blood was between 1/31,700 and 1/50,300; however, this value increased rapidly on day 4 after transplantation to reach values of up to 1/1,100 by day 7. The frequency of these cells was first measurable in the graft-infiltrate on day 2 and also showed a rapid increase 4 days after surgery; peak values up to 1/4,800 were recorded on day 5. This time corresponded with that of functional cardiac rejection and maximum infiltration of the graft by mononuclear cells. The similar kinetic changes and absolute values recorded for the frequency of donor-reactive CTL within the blood and graft-derived cell populations was indicative of a rapid bi-directional passage of cells between these pools and provided no evidence for specific sequestration of CTL by the graft. Cells purified from the graft on post-operative day 5 mediated an immediate specific cytotoxicity towards PVG target cells (44% lysis during a 4 h assay at an effector:target ratio of 100:1) which was of a higher activity than would be predicted on the basis of an effector population containing only 1/4,800 PVG-reactive CTL. This finding implies that other mononuclear cell types than CTL present within the graft-infiltrating population were capable of mediating target cells lysis.