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Several strategies have been used to stimulate the growth of tumorspecific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U /ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 µ,M ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 µM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominately CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.