T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cylotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions arc required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unscparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18h at 37°C. Cytokines were measured by cytotoxicity assays using L929(murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatanis derived from unseparaled, CD4⁺ (>95% pure) and CD8⁺ (>90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the eylotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was delected). These same supernatanis were cytoloxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h ¹¹¹In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target celts. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.