FRESHLY ISOLATED TUMOR-INFILTRATING T-LYMPHOCYTES HAVE A HIGH CYTOTOXIC POTENTIAL, AS MEASURED BY THEIR ABILITY TO INDUCE APOPTOSIS IN THE TARGET-CELL

To test if freshly isolated tumour-infiltrating lymphocytes (TIL) can induce apoptosis in a target cell, we have combined two previously des cribed methods. Because TIL predominantly are T-lymphocytes, we have a pplied a redirected approach. When the target cells that express anti- human-CD3 monoclonal antibodies in their membranes bind to the T cell receptor-associated CD3-complex, signals are generated, which activate T cell effector mechanisms. This approach circumvents problems with M HC-restriction and allows for functional testing of all T cells, irres pective of their clonal specificity. In order to assay for induction o f DNA fragmentation, we have labelled the target cell nuclei with [H-3 ]thymidine. Upon harvesting fragmented DNA are washed away. Electropho retic analysis of the fragmented DNA demonstrated the characteristic ' ladder' pattern, consistent with apoptosis. This rapid and simple assa y monitors the capacity of different T cells to induce apoptosis in th e target cell. It depends on intercellular interactions and clearly di scriminates between different T cell subsets. With this assay we demon strate the functional integrity of the cytotoxic effector arm of fresh ly isolated TIL.