Tumor infiltrating lymphocytes (TIL) are undergoing evaluation in the Surgery Branch as a therapy for advanced human malignancy. We are attempting to identify the subset of cells within bulk TIL culture which have specific immune reactivity to the tumor itself. We have developed a method of detecting intracellular calcium in individual TIL after exposure to tumor. The method involves the use of flow cytometry and cell sorting to detect and recover specifically reactive anti-tumor lymphocytes. We showed that in human T cell clones that we could detect an increase intracellular calcium after exposure to specific target cells. We could demonstrate conjugation of the T cell to its target cell as well as signalling with an increase in intracellular calcium in this system using both CD4+ and CD8+ T cells. These cloned populations of T cells, when bound to the target, demonstrated an increase in intracellular calcium and could be sorted. We have begun preliminary experiments in patients TIL and have found that we can also detect tumor T cell conjugates by flow cytometry and that in a subpopulation of these conjugates the signal of an increase in intracellular calcium can be detected. The biologic relevance of such cells is undergoing evaluation with sorting and subculturing experiments. The significance of the project lies in identification of specific anti-tumor reactive T cells in the tumors of patients. We suspect that if these cells are present in the patients tumor and can be enriched and subcultured that such cells may have increased efficacy compared to bulk whole populations of TIL in the therapy of patients with cancer.