In vitro studies of cell-bound immunity, cloning assay of the cytotoxic action of sensitized lymphoid cells on allogeneic target cells.

The in vitro reaction between sensitized lymphoid cells and target cells has been studied in an allogeneic transplantation system in mice. Mastocytoma cells of the DBA/2 donor strain were injected into C57BL mice, and the spleens of the recipients harvested 8 days later. The immune lymphoid cells thus obtained were tested for their ability to damage in vitro cultures of target mastocytoma cells. The reaction was followed by two methods: (1) microscopic counts, and (2) ability of target cells to form colonies in a semi-solid medium after various periods of contact with immune lymphoid cells. The results show: (a) that the effect on target cells is an exponential function of the number of immune lymphoid cells employed, and (b) that the reaction becomes noticeable after 3 hours and reaches completion within 12 hours when evaluated by cloning techniques, while it takes 24–48 hours to be microscopically clearly demonstrable. Problems arising from use of both methods are discussed

Quantitative assay of the lytic action of immune lymphoid cells of (51)Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs

The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label ((51)Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of (51)Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis

Studies of allograft immunity in mice: I. Induction, development and in vitro assay of cellular immunity

Cellular immunity induced by tumour allografts in inbred mice was studied with the help of an in vitro assay system measuring the cytotoxic effect of sensitized lymphocytes on (51)Cr-labelled target cells. It is shown that lymphoid cells from spleen, lymph nodes and blood of the allograft recipients reach a peak of cytotoxic activity on days 10–11 after immunization. Incubated with labelled target cells at a ratio of 100:1, the sensitized lymphocytes caused the specific release of up to 70 per cent of the radioactivity within 1 hour. A second population of target cells added to the same cell suspension was destroyed at a slightly accelerated rate, suggesting stimulation of the effector cells by the first interaction. The cytotoxic activity of circulating lymphocytes was found to reach a plateau between days 20 and 60 after immunization, while the activity of spleen cells dropped to low levels in the same time period. The specificity of target cell destruction by sensitized lymphocytes is demonstrated by the lack of lytic activity for syngeneic target cells, and by the selective destruction of target cells carrying a tumour specific antigen. Tumour cells, lymphocytes and embryonic fibroblasts of the donor strain are shown to differ considerably in their sensitivity to lysis by immune lymphocytes

Studies of allograft immunity in mice: II. Mechanism of target cell inactivation in vitro by sensitized lymphocytes

The mechanisms of the in vitro interaction of sensitized lymphocytes and allogeneic target cells has been studied in a tumour allograft system in inbred mice. The cytotoxic effect of sensitized lymphocytes is shown to require the presence of Ca(+ +) and Mg(+ +). Pretreatment of the lymphocytes with trypsin led to inhibition of cytotoxicity, followed by spontaneous reversal after 1–3 hours incubation. Reactivation was found to be blocked by an inhibitor of protein synthesis (cycloheximide). Cortisone was not found to inhibit the lytic interaction significantly; an occasional effect is thought to be due to toxicity of cortisone for lymphocytes as revealed by dye exclusion test. Inhibition of DNA-synthesis with FUdR (an inhibitor of the enzyme thymidine synthetase) did not reduce the lytic activity of sensitized lymphocytes. Isologous anti-target cell sera induced in various strains of inbred mice were found to be ineffective in blocking the cellular immune reaction in vitro when directed against a minor part of the antigenic complex, but strongly inhibitory when reactive against a major part or the whole complex. Similarly, target cells lacking several of the sensitizing H-2 antigens were not lysed. An isologous anti-lymphocytic serum induced in the graft donor strain and directed against the recipient strain (lymphocyte donor) did not inhibit the cytotoxic reaction. In a heterologous system on the other hand, the lytic effect of guinea-pig lymphocytes sensitized against mouse target cells was effectively blocked by an anti-lymphocytic serum induced in mice of the graft donor strain by injection of recipient (guinea-pig) spleen cells