We demonstrated before that primary operated breast cancer patients contain in their bone marrow (BM) cancer reactive memory T cells (MTC) which have to be re-activated to become tumor infiltrating effector cells. The aim of this study was to optimize an ex vivo stimulation protocol for MTC based on autologous dendritic cells (DC). As source of tumor antigens we used lysates from unmodified tumor cells or from tumor cells infected with Newcastle Disease Virus (NDV) which contain IFN-α inducing viral dsRNA as one danger signal. DC from breast cancer patients were pulsed with lysates from the MCF-7 breast cancer line (Tu-L) or from NDV infected MCF-7 cells (TuN-L, viral oncolysates) and compared for stimulatory capacity in an ELISPOT response of autologous BM derived MTC. To analyze potential further danger signals derived from NDV infection, we employed MALDI mass spectrometry, Western blots, FACS cytometry and ELISA tests. DC pulsed with viral oncolysates showed increased expression of co-stimulatory molecules in comparison to Tu-L pulsed DC and induced significantly higher ELISPOT MTC responses. Supernatants from co-cultures of MTC and TuN-L pulsed DC contained increased titers of IFN-α and IL-15. NDV infection of tumor cells resulted in a number of differences in protein expression including a heat-shock protein (HSP27) which became phosphorylated. The results suggest that a DC preparation pulsed with viral oncolysate includes danger signals (e.g. dsRNA, cytokines, HSP molecules) and is superior for MTC stimulation to a DC preparation pulsed with lysate from non-infected tumor cells.

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