EXPANSION AND TUMOR-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 l arge 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 prom oting protein kinase C activator and ionomycin (1), a calcium ionophor e, at day 0 followed by culture in low dose interleukin-2 (IL-2 20 uni ts 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 consist ed of a mixture of CD8+ (46-74%) and CD4+ (4-30%) cells. B/I expanded cells did not lyse autologous tumour cells when tested in a 4-h Cr-51 release assay, but murine studies reported previously have demonstrate d specific and curative in vivo efficacy in MCA-105 tumour-bearing mic e despite an inability to lyse autologous tumour in vitro. B/I expande d T-cells from five of six patients secreted the cytokines tumour necr osis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in resp onse to co-culture with autologous tumour cells but not with irrelevan t tumour. These results are analogous to findings in a murine model, i n which non-cytolytic B/I expanded T-cells mediated specific, curative anti-tumour effects in vivo, and lay the groundwork for a clinical tr ial of this novel strategy for the adoptive immunotherapy of breast ca ncer patients.