Quantitative Studies on the Behavior of Sensitized Lymphocytes in Vitro

Abstract

Numerous studies have implicated cells of the lymphoid series as agents responsible for the destruction of solid tissue homografts in vivo (1)(2)(3)(4)(5). This view has been substantiated by recent demonstrations that lymphoid cells (6-15) and macrophages (16, 17) procured from specifically immunized animals have a cytocidal effect on appropriate homologous "target" cells in vitro. It was previously reported that by the 6th to 7th day after grafting with homologous skiu, lymphocytes from the regional lymph nodes of mice or rats had acquired the capacity to injure foreign target renal cells in culture, and that neither isoimmune serum nor complement was required for this destructive reactivity (15). The present communication is concerned with the application of a sensitive, reproducible in vitro assay system to elucidate further quantitative aspects of the destruction of homologous target cells by sensitized lymphocytic cells. Particular attention has been paid to the influence of (a) variation in the ratio of attacking lymphoid cells to target ceils (b) the duration of contact with lymphoid cells and (c) the possible influence of the presence or absence of isoimmune serum on the cytocidal activities of sensitized lymphocytes. Materials and MethodsPrinciple of the Test System.--Rats were immunized to the foreign transplantation antigens of a homologous strain. Cell suspensions consisting of known numbers of lymphocytes prepared from the lymph nodes of normal, i.e. non-immune, or sensitized animals were then added to previously "seeded" monolayer cultures of knoWn numbers of target cells of donor strain origin. After a specified period of incubation, the number of target cells remaining in the

Histocompatibility antigen-activated cytotoxic T lymphocytes. I. Estimates of the absolute frequency of killer cells generated in vitro.

“…2). This is consistent with previous conclusions of others (2,(4)(5)(6)) that cell-mediated cytotoxicity is the outcome of a single cell event. It follows from the Poisson distribution that the dose of cells which give 37% negative wells contains, on the average, a single CTL (7).…”

“…This figure was based on limiting dilution of immune lymphocytes and analysis of target cell survival with an electronic particle counter (2). There were certain weaknesses in that study: (a) it involved relatively long assay periods (48 h), making it difficult to rule out that new killer cells were generated in the cultures; (b) the assumption that a killer cell could affect only a single target cell, an assumption which if not valid leads to erroneously high estimates of CTL frequency; and (c) it could not be determined at the time whether killer cells were exclusively of thymic origin.…”

Quantitative Studies on the Behavior of Sensitized Lymphocytes in Vitro

“…The procedures and system used to assay the destructive effect of specifically sensitized lymphoid cells on homologous target cells have been described before (1). To determine the effect of imuran on this destructive reaction, known numbers of lymphoid cells were suspended in growth medium with or without the drug and then added to Leighton tube cultures of Lewis target tumor cells of the Le-1 line.…”

“…On the basis of results presented in Part 1 of this series (1) it was suggested that the destructive effect of sensitized lymphocytes upon homologous cells in tissue culture depends, in part, on an active metabolism of the lymphoid cells. To provide further information on this point, the experiments to be described were conducted to ascertain whether the antimetabolite imuran, an imidazole derivative of 6-mercaptopurine, has any influence on the cytocidal and cytostatic activities of sensitized lymphoid cells against homologous target cells in vitro.…”

Cell-Mediated Cytotoxicity During Rejection and Enhancement of Allogeneic Skin Grafts in Rats

“…Based on the postulate that one aggressor cell kills one target cell, i.e. the one-hit theory (31,32), the number of CL per spleen was calculated. In adult unmanipulated Le recipients of allografts, CL were 0.5 % of lymph node cell suspensions and 0.3 % of suspensions of spleen cell and peripheral blood lymphocytes tested at the height of CMC.…”