Characterization of OKT3-initiated Lymphokine-activated Effectors Expanded with Interleukin 2 and Tumor Necrosis Factor α

Synergistic and cooperative effects in vitro of OKT3, interleukin 2 (IL-2), and tumor necrosis factor α (TNF) as stimuli in generating effectors with lymphokine-activated killer activity were studied. Activation of human peripheral blood mononuclear cells with OKT3 (10 ng/ml) for 48 h, followed by culture in low concentrations of IL-2 (10 units/ml) and TNF (250 units/ml) resulted in higher cell recovery (50- to 3300-fold) compared to the number of cells in the initial culture and enhanced lytic activity against both Raji and fresh lung tumor targets (mean 100-fold) by day 30 compared to those expanded with higher concentrations of IL-2 (100 units/ml) alone. Immunofluorescence analysis of peripheral blood mononuclear cells initiated with OKT3 and expanded with IL-2 plus TNF revealed a selective increase in CD8⁺ cells and a decrease in CD4⁺ by day 28; the opposite effect was observed when cells were incubated with 100 units/ml of IL-2 alone, resulting in a greater proportion of CD4⁺ cells. Almost all cells were CD3⁺ Studies of cytokine receptor expression indicated that OKT3 plus IL-2 plus TNF caused an earlier up-regulation of the IL-2 receptor β chain (Tac) and higher TNF receptor expression by day 6 compared to 100 units/ml IL-2 alone. Significant TNF levels (>17 units/ml) were measured in culture super-natants from peripheral blood mononuclear cells initiated with OKT3 alone. Collectively, our data demonstrate that induction of lymphokine-activated killer activity with OKT3, followed by culture in low concentrations of IL-2 plus TNF is an alternative to the use of high-dose IL-2 alone and suggest that this combination may provide potential advantages in long-term generation of cytolytic cells.