Simultaneous protein kinase C stimulation with phorbol 12,13-dibutyrate (PDBU) and calcium mobilization with ionomycin (Io) trigger cellular events leading to expression of proliferation-associated genes in human lymphocytes. The effect of a 16-hr exposure to PDBU and Io on the growth and cytotoxic activity of murine splenocytes and tumor-infiltrating lympho- cytes (TIL) cocultured with interleukin-2 (IL-2) was studied. PDBU + Io increased the number of cytotoxic effector cells that could be generated in lymphokine-activated killer cells (LAK) cultures (40-fold) and to a lesser extent in TIL (10-fold). DNA synthesis of TIL increased significantly when exposed to PDBU + Io. Also, TIL stimulated with PDBU + Io demonstrated in vitro tumor-specific lytic activity significantly greater than that of control TIL (500 lytic units vs 50). The cell-surface phenotype of TIL treated with PDBU + Io was identical to that of control TIL (>95% CD-3+, CD-4-, CD-8+). Results of adoptive immunotherapy using splenocytes stimulated by PDBU + Io and cultured in IL-2 were identical to those achieved when standard LAK cultures were used. However, treatment using TIL stimulated by PDBU + Io led to a significant reduction in the number of pulmonary nodules compared to standard TIL. In addition, PDBU + Io-stimulated TIL maintained significant in vivo activity without the need for systemic IL-2 administration. Pharmacologic manipulation of cytotoxic precursor cells is a useful strategy for improving the generation of murine cytotoxic effector cells. By using PDBU + Io, cytotoxic lymphocytes could be generated with improved in vitro and in vivo activity.
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