Freshly isolated tumour-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 described methods. Because TIL predominantly are T-lymphocytes, we have applied 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 MHC-restriction and allows for functional testing of all T cells, irrespective of their clonal specificity. In order to assay for induction of DNA fragmentation, we have labelled the target cell nuclei with [3H]thymidine. Upon harvesting fragmented DNA are washed away. Electrophoretic analysis of the fragmented DNA demonstrated the characteristic 'ladder' pattern, consistent with apoptosis. This rapid and simple assay monitors the capacity of different T cells to induce apoptosis in the target cell. It depends on intercellular interactions and clearly discriminates between different T cell subsets. With this assay we demonstrate the functional integrity of the cytotoxic effector arm of freshly isolated TIL.
Ostenstad
B
B
Institute of Immunology and Rheumatology, National Hospital, Oslo, Norway.
Sioud
M
M
Schlichting
E
E
Lea
T
T
Harboe
M
M
D000230:Adenocarcinoma / Q000276:immunology; D017209:Apoptosis; D017252:CD3 Complex / Q000502:physiology; D015179:Colorectal Neoplasms / Q000276:immunology; D016572:Cyclosporine / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004249:DNA Damage; D006801:Humans; D007111:Immunity, Cellular; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*
D017252:CD3 Complex; D016572:Cyclosporine