This results clearly demonstrated that DCs have processed MelanA MART 1 Ag taken up from the tumor cells and presented it to M27 clone in their own HLA A 0201 conte t. As early as 6 hs after DCs loading with Apo Nec, these cells could efficiently induce IFN release, and we were able selleck inhibitor to measure CTL cross presentation even 72 hs post DC Apo Nec co culture. Several authors have identified gp100 as a regression Ag, since the induction of anti gp100 immunity correlated with the regression of documented metastases in melanoma. Besides, anti MelanA MART 1 CD8 T lymphocytes have also been detected in melanoma patients by tetramer staining and ELISPOT, correlating with clinical outcome and regres sions.

Labarriere et al reported that the use of purified melanoma apoptotic bodies to load DCs plus maturation with cytokines, efficiently cross primed CTLs specific for NA 17A Ag but not for MelanA MART 1 Ag. The authors could not detect MelanA MART 1 epitopes in apobodies using a MelanA MART 1 specific mAb. In our case, not only DCs matured after phagocytosis of Apo Nec cells but the induction of IFN secretion by a CTL clone specific for MelanA MART 1 peptide was found. Thus, our results suggest that a vaccine such as DC Apo Nec has the potential to initiate an immune response spe cific for MelanA MART 1 and gp100 Ags and probably for other Ags e pressed by these cells. Recently, Palucka et al. have assayed in a phase I clinical trial a vaccine com posed of DCs loaded with killed allogeneic melanoma cells demonstrating objective clinical responses and MART 1 specific CD8 T cell immunity.

However, in this study the authors used a single HLA A 0201 negative all ogeneic melanoma cell line killed after a combination of TNF treatment, irradiation and culture in serum free medium, plus the addition of CD40L to activate DCs. Our results further support the use of apoptotic necrotic allo geneic tumor cells as a comple source of multiple melanoma native Ags to load DCs and show that a good maturation signal could be obtained with this particular mi ture of melanoma cells, which also allows the cross presentation of melanoma Ags to specific CTLs. As we have demonstrated here, cross presentation for MelanA MART 1 and gp 100 Ags was achieved by DCs that have phagocytosed Apo Nec cells but not by Apo Nec cells themselves, since Apo Nec cells or HLA A 0201 positive Apo Nec cells were not able to induce INF secretion separately. We have also observed that AV-951 Apo Nec cells progressively lost their HLA A 0201 surface e pression after irradiation and that their ability to present MelanA MART 1 and gp100 peptides to CTLs decreased concomitantly.