Firstly, using a high affinity mAb specific for MART-1, 2A9 that detects MART-1 by IHC, immunofluorescence and European Blot in melanoma cells, the capacity of HLA-A2+ tumor cells to directly stimulate MART-1 specific CD8 T cells could be related to the expression level of MART-1. vaccinate malignancy individuals. We have previously used gamma-irradiated MART-1 expressing melanoma cells like a source of antigens to vaccinate melanoma individuals by injecting irradiated cells with BCG and GM-CSF or to weight immature DC and use them like a vaccine. Additional clinical trials possess used IFN-gamma triggered macrophage killer cells (MAK) to treat cancer individuals. However, the medical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Therefore, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by medical grade DC or MAK as well as the ability of these cells to mix present MART-1 to CD8+ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically staining melanoma tumors, melanoma cell lines and normal melanocytes, the manifestation level of MART-1 in melanoma cell lines could be related to their ability to activate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8+ T cell clone. Confocal microscopy with Alexa Fluor?647-labelled 2A9 also showed that MART-1 could be recognized in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and FMF-04-159-2 DC co-cultured with melanoma gamma-irradiated cells for different time-points. Therefore, naturally happening MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8+ T cell cross-presentation thereafter. Intro Cutaneous melanoma (CM) FMF-04-159-2 accounts for 4% of all neoplasia and it is the tumor with the fastest growing incidence worldwide [1]. Melanoma tumors are highly resistant to chemotherapy, but more responsive to immunological treatments. A large variety of antigens have been connected to CM, such as Melan A/MART-1 [2], [3], gp100 [4], Tyrosinase [5], TRP-2 [6], and NY-ESO-1 [7]. MART-1 is definitely a hydrophobic transmembrane protein without glycosylation sites highly enriched in early melanosomes. MART-1 is necessary for gp100 function, another antigen connected to CM, involved in the rules of melanosome formation [8]. MART-1 is definitely expressed in pores and skin and retinae melanocytes and in the majority of melanoma tumors, but it is definitely absent from additional cells and tumors. It was isolated thanks to the specific acknowledgement by T lymphocytes of MART-1 derived peptides, specially in the context of the Neurod1 HLA-A0201 haplotype, present in tumor infiltrates from melanoma FMF-04-159-2 individuals [2], [3]. Therefore, MART-1 is definitely immunogenic in humans and has been widely exploited to induce anti-melanoma immunity in individuals by means of several vaccination strategies. Among them, the use of MART-1 peptides either injected with adjuvants and/or pulsed on DC has been tested in medical settings, although with very modest outcomes so far [9]C[11]. Also, MART-1 specific immune responses are frequently assessed to monitor the ability of melanoma vaccines to induce immunity in treated individuals [12]. Using whole irradiated FMF-04-159-2 tumor cells to weight DC could be preferable to develop DC-based vaccines since melanoma cells could contribute with known antigens such as MART-1 and probably unknown antigens. We have used this strategy to vaccinate melanoma individuals with a mixture of gamma-irradiated melanoma cell lines and BCG, a potent inflammatory adjuvant [13], and plus GM-CSF to further entice DC to the vaccination site [14]. We while others have shown in murine models [15], and in humans [16], [17] that when DC engulf gamma-irradiated melanoma cells, antigens can be cross-presented for the generation of HLA class I/peptide-complexes, permitting the induction of specific CTLs. However, in the human being, the fate and immunogenic potential of DC that have phagocytosed dying tumor cells or their debris remains an open issue. The use of irradiated allogeneic tumor cells is based on the paradigm FMF-04-159-2 that tumor cells would only result in MHC-restricted tumor-specific immunity after becoming phagocytosed by DC, the main initiators of immune response able to activate na?ve CD8+ T cells [18]. After phagocytosis, DC develop to a mature phenotype, diminish their phagocytic ability, express HLA class II and co-stimulatory molecules on their surface, and acquire the capacity to present antigens in the.