Non specific binding of antibodies to dying cells was ruled out in every selleck Erlotinib e periment since anti DCs markers binding to isolated Apo Nec cells was less than 5% or absent. DCs maturation was also evaluated by the decrease of DCs endocytosis after Apo Nec cells phagocytosis. FITC D endocytosis was ma imal in iDCS, 99 5% uptake, decreased to 31 3% in LPS maturated DCs and to 33 5% in DC Apo Nec cells. DCs loaded with melanoma apoptotic necrotic cells e press CCR7 and migrate towards MIP 3 beta in vitro DCs migration to lymph nodes is crucial to trigger T lym phocyte priming. MIP 3 chemokine drives the homing of mDCs to the lymph node and interacts specifically with its receptor CCR7 e pressed on the cell surface. iDCs e pressed low levels of CCR7 but increased its e pression after Apo Nec phagocytosis or LPS maturation.

We observed that iDC migrated to MIP 1 but did not respond to MIP 3, on the contrary DC Apo Nec and DC LPS migrated to MIP 3 but failed to respond to MIP 1. Thus, DC Apo Nec cells e press MIP 3 receptor CCR7 and are able to migrate in response to MIP 3, potentially allowing their homing to lymph nodes. DCs loaded with apoptotic necrotic melanoma cells cross present MelanA MART 1 and gp100 Ags to specific T CD8 cells An important issue in this work was to assess if melanoma associated Ags present in the Apo Nec cells mi ture could be cross presented to specific CTLs after DCs phagocytosis. We analyzed IFN secretion by specific CD8 T clones for MelanA MART 1 and gp100 after overnight stimula tion with DC Apo Nec cells.

As observed in Figure 6A and 6B, DC Apo Nec co cultured with the CTL clones effi ciently induced IFN secretion as soon as 6 hs and up to 48 hs after phagocytosis, evidencing cross presentation for both Ags. For MelanA MART 1 Ag we also observed IFN secretion after incubation with DCs loaded with Apo Nec HLA A 0201 negative MelanA MART 1 cells clearly demonstrating cross presenta tion for this Ag. Controls for this e periment were performed, showing specific HLA A 0201 restricted response using either DCs loaded with the corresponding peptides or CTL stimulation with live HLA A 0201 posi tive gp100 MelanA MART 1 melanoma cells but lack of response using live MEL Y2 cell line or with MelanA MART 1 peptide for G154 clone or gp100 peptide for M27 clone. Mel Y3 was rendered Apo Nec by irra diation and also assayed for CTL priming, however, it only induced IFN secretion by the CTL clones at short times after irradiation and culture. After 48 72 hs of culture, when HLA A 0201 positive Apo Nec cells have completed the AV-951 apoptotic necrotic process, they failed to present both Ags to CTLs.