QUANTITATIVE STUDIES ON THE BEHAVIOR OF SENSITIZED LYMPHOCYTES IN VITRO
Authors: Wilson Darcy B. 
Published: June 2004

When lymphoid cells, derived from rats immunized with respect to homologous skin, were cultured with target cells originally of donor origin, cytocidal and cytostatic activities of the attacking lymphocytes became evident. By application of a sensitive and reproducible quantitative assay system, various aspects of the mechanism of this destructive interaction between target cells and lymphocytes were examined with the following results.

1. The degree of survival of target cells was inversely related to the number of sensitized lymphocytes. Graphic plots of the data indicated that this relationship was an exponential one similar to "single-hit" inactivation phenomena. One interpretation which could be placed on these results is that a single lymphocyte, if immunologically active, was sufficient to destroy or at least have a detectably adverse effect on one target cell. Furthermore, from such a model it could be computed that, of the lymphocytes derived from an immunized animal, approximately 1 to 2 per cent of the cells were immunologically active; i.e., capable of demonstrable destructive activities against homologous target cells in vitro.

2. Morphological studies on the effect of sensitized lymphoid cells on homologous target cells, aftervarious lengths of time in culture, showed that by 7 hours of incubation, the attacking lymphocytes firmly adhered to the target cells. The cytotoxic effect of these lymphocytes generally occurred after the 20th hour. Quantitative studies supported this conclusion; the latent period, i.e., the time required for detectable degrees of target cell destruction to occur, was approximately 20 hours.

3. A consequence of the incubation of target cells with normal lymphoid cells or even with small numbers of sensitized lymphoid cells was an increase in the rate of division of the target cells. As might be expected, this was reflected in a shorter doubling time of these cells.

4. Extracts prepared from sonically disrupted sensitized lymphocytes proved to be no more deleterious to target cells than similar preparations from normal lymphoid cells. Furthermore, no evidence could be obtained that sensitized lymphoid cells, separated from target cells by a Millipore membrane, were cytocidally effective. These data indicated that if a cell-bound substance is involved in the destruction of homologous cells, either it is not toxic by itself, or it cannot be detached from the sensitized cells. In any case, close apposition of the lymphocytes to the target cells is apparently required for the destruction of the latter in vitro.

5. Serum obtained from immunized animals, if heat-inactivated, did not adversely affect homologous target cells; if employed fresh, slight degrees of toxicity resulted. Specific isoimmune sera did not impart any detectable degrees of immunological reactivity upon otherwise normal lymphoid cells. Immune sera, even in high concentrations, did not augment the effect of sensitized lymphoid cells upon homologous target cells; rather a slight inhibitory effect of these sera was detected.

6. Attempts to detect the presence of complement activity, which might have been provided by the lymphoid cells in culture, were unsuccessful.

On the basis of these results, it was suggested that the destruction of homologous target cells by sensitized lymphoid cells occurs as a two step process. First, the attacking lymphocytes attach to their targets via a non-toxic cell-bound substance having an immunologic specificity, and then, destruction of the target cells follows the result of some process dependent on the metabolic activity of the attacking lymphoid cells.