Phenotype and cytolytic activity of mouse tumor-bearer splenocytes and tumor-infiltrating lymphocytes from K-1735 melanoma metastases following anti-CD3, interleukin-2, and tumor necrosis factor-α combination lmmunotherapy

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 10⁴ tumor cells and received a single 5 μg i.p. dose of anti-CD3 on day 3, followed by either IL-2 alone or fourfold less IL-2 with TNF (25,000 U/day) given at 3-day intervals. A single dose of anti-CD3 followed by low-dose IL-2 and TNF caused the greatest reduction in metastases compared to anti-CD3 alone, higher doses of IL-2 alone, or IL-2 + TNF. Reduction in metastases (>80%) 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.