Summary:We studied the phenotype and antitumor cytolytic activity of splenocytes from mice with K-1735 pulmonary metastases and tumor-infiltrating lymphocytes (TILs) from these metastases following treatment with anti-CD3, IL-2, and TNF combination immunotherapy. Mice were injected with 5 x 10480%) using the three agents was equal to or exceeded that achieved by ninefold higher concentrations of IL-2 alone. Treatment with anti-CD3 + IL-2 and TNF significantly prolonged survival, and resulted in 60% of mice achieving longterm survival <120 days. This was superior to single agents or other combinations with the three agents causing a synergistic rather than additive effect. The anti-CD3-activated splenocytes were a heterogeneous population of T cells, with an increased number of CD8+cells compared to splenocytes from mice treated with high doses of IL-2 alone. Analysis of TILs showed a greater proportion of CD8+cells in anti-CD3-treated mice compared to IL-2 alone, but a lower proportion of CD4+cells. Lymphokine-activated killer (LAK) and natural killer (NK) activities of both splenocytes and TILs in vitro increased following anti-CD3/IL-2 + TNF treatment, and were consistently greater than that generated with four times more IL-2 alone. TNF appeared to potentiate cytolytic activity rather than affect phenotypic changes. These results indicate that the sequential use of anti-CD3, IL-2, and TNF for LAK induction and maintenance potentiates antitumor activity, and suggests novel strategies for combination immunotherapy.