Expansion and tumour specific cytokine secretion of bryostatin-activated T-cells from cryopreserved axillary lymph nodes of breast cancer patients

Current adoptive immunotherapy strategies in cancer patients require large numbers of activated T-cells and are limited by the availability of autologous tumour. We describe a novel method of T-cell activation that produced relatively rapid, high-fold expansion of stored, frozen lymphocytes obtained from the lymph nodes of 20 breast cancer patients during axillary dissection but does not require autologous tumour. In vitro exposure of thawed cells to bryostatin-1 (B), a non-tumour promoting protein kinase C activator and ionomycin (I), a calcium ionophore, at day 0 followed by culture in low dose interleukin-2 (IL-2 20 units ml^-1) and restimulation again on day 10 results in 269-28,206 fold (geometric mean = 2254) expansion in cell numbers counted 17 days after initial stimulation. Analysis of cell surface markers revealed that B/I expanded human cells were predominantly T-cells (83-97%) and consisted of a mixture of CD8⁺ (46-74%) and CD4⁺ (4-30%) cells. Bill expanded cells did not lyse autologous tumour cells when tested in a 4-h ⁵¹Cr release assay, but murine studies reported previously have demonstrated specific and curative in vivo efficacy in MCA-105 tumour-bearing mice despite an inability to lyse autologous tumour in vitro. B/I expanded T-cells from five of six patients secreted the cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in response to co-culture with autologous tumour cells but not with irrelevant B/I expanded T-cells mediated specific, curative anti-tumour effects in vivo, and lay the groundwork for a clinical trial of this novel strategy for the adoptive immunotherapy of breast cancer patients.