Thus, 72 hs after irradiation Apo Nec melanoma cells are no longer able to activate CTLs by themselves but may be used as a source of Ags for efficient cross presentation after DCs phagocytosis and processing. Evaluation of Intracytoplasmatic IL 10 and IL 12 The balance between IL 10 and IL 12 in DC Apo Nec cells was quantitated by FACS at selleck MEK162 different time points after phagocytosis followed by 8 hs treatment with Brefeldin A to accumulate intracytoplasmic cytokines. As shown in Discussion There is now considerable e perimental evidence that dying cells are capable of transferring Ags to the immune system for the induction of T cell immunity. Albert et al first demonstrated in the murine model that apoptotic material could be processed and cross pre sented by DCs to stimulate specific HLA restricted CD8 T cells.

In this study, the use of whole apoptotic necrotic tumor cells to load DCs e ploits both the advan tage of maturation signals delivered by necrotic cells as proposed by Gallucci et al and the optimal Ag processing and presentation in HLA class I and class II molecules by DCs of a vast repertoire of known as well as yet unknown Ags from apoptotic cells for the induction of anti tumor immune responses. Some contro versy has arisen since several authors found that the uptake of pure or early apoptotic tumor cells did not induce proper DCs maturation.

However, these studies differ from the current one in several aspects i we used a mi ture of four apoptotic necrotic melanoma cell lines e pressing several native melanoma associated Ags instead of the single cell line approach Entinostat or virally trans duced tumor cells e pressing e ogenous Ags, ii we used serum free medium instead of FCS or autologous serum to generate monocyte derived DCs, iii the method used for tumor Ag preparation consists in gamma irradiation and 72 hs culture in melanoma medium containing FBS as compared to higher energy radiation and culture in serum free medium, UVB e posure or apoptosis induced by viral infection or by Fas pathway. Previous reports showed that early apoptotic melanoma cells or pure apobodies failed to induce mDCs, and either TNF , Poly I C or cytokine cocktails were nec essary to achieve DCs maturation and Ag presentation. In our case, phagocytosis of the mi ture of melanoma cell lines used to load iDCs was enough to generate mDCs without addition of other stimuli.

According to the results reported by Sauter et at we took advantage of DCs maturation induced by necrotic tumor cells present in the mi ture of Apo Nec cell lines, as well as DCs ability of antigen processing and cross presentation for CTL prim ing due to the apoptotic cells. Using this particular melanoma cell lines mi ture we wanted to address the question if the uptake of Apo Nec cells could allow native TAAs to be processed to peptides by DAPT secretase Sigma iDCs, mDCs and cross present them to specific CTLs.