Activation of cd8⁺murine t cells from tumor-draining lymph nodes by phorbol dibutyrate plus calcium ionophore

When lymphocytes from the lymph nodes draining the site of a progressively growing MCA-105 sarcoma are stimulated in vitro with autologous tumor and low-dose interleukin-2 (IL-2), they will grow and develop the ability to lyse autologous tumor cells in vitro; these lymphocytes can also eradicate tumor metastases in vivo. Phorbol esters and calcium ionophores activate signal transduction pathways in T cells and mimic the events triggered by antigen binding. We therefore sought to determine whether large numbers of MCA-105 tumor-specific, therapeutically active T cells could be obtained from MCA-105 draining lymph nodes (DLNs) following a brief exposure to phorbol dibutyrate (PDBu) and ionomycin (Io). DLN cells primarily stimulated with autologous tumor, followed by a secondary stimulation with PDBu-Io and cultured in 20 U/ml IL-2, demonstrated marked expansion of cell numbers during 3 weeks in culture, had moderate cytolytic activity [37% at effector: Target ratio (E:T) = 80:1], and were all CD8⁺T cells. In contrast, DLN cells stimulated primarily with PDBu-Io and cultured in 20 U/ml IL-2 demonstrated at least 8–10-fold greater growth than antigen-stimulated DLN cells during 3 weeks, were moderately cytolytic (31% at E:T = 80:1), and were a mixed population of CD8⁺and CD4⁺T lymphocytes. DLN cells that were expanded by either protocol, like cells stimulated repeatedly in vitro with tumor cells, could eliminate MCA-105 pulmonary metastases when given with IL-2 in an adoptive immunotherapy model. DLN cells stimulated primarily with PDBu-Io completely eradicated MCA-105 metastases but had no in vivo antitumor activity against the syngeneic B16 melanoma or MCA-203 sarcoma. Moreover, pharmacologically activated DLN cells induced tumor regression without concomitant IL-2 administration. The therapeutic effect was abrogated when CD8⁺T cells were depleted in vivo. Although lymphocytes obtained from the spleens and lymph nodes of non-tumor-bearing animals could be expanded to large numbers after activation with PDBu-Io and culture in IL-2, these cells had no in vivo antitumor activity. Therefore, these results indicate that phorbol dibutyrate plus ionomycin may substitute for tumor antigen and stimulate the growth of T cells that are capable of mediating specific tumor regression.