The major goal of this proposal is to test the hypothesis that """"""""a more efficient anti-tumor T cell response can be generated through antigen presentation by specialized antigen presenting cells such as dendritic cells (DC) grown to immunogenic maturity in an environment of danger and/or tissue damage and made to present the relevant tumor associated antigen through the MHC class I and class II pathways"""""""". Using the human melanoma antigen MART-1 system as a prototype, the specific aims are: 1) to undertake a comprehensive analysis of both CD4+ and CD8+ T cell responses to MART-1 presented in vitro by engineered and immunogenically matured DC; 2) to define the role of and the mechanism by which CD4+ T cells facilitate and amplify CTL response; 3) to examine the molecular basis of """"""""help"""""""" (IL-2 message/IL2R expression, signaling through cytokine common receptor gammac, and anti-apoptotic vs pro-apoptotic mechanism (Bcl/Bax); and 4) to examine the in vitro immunogenicity of compartmentalized epitope presentation by DC genetically engineered with VSV pseudotyped retrovector expressing EGFP-TAA fusion protein plus intracellular trafficking signal sequences and bacterial immuno-stimulatory sequences (ISS). Myeloid DC grown in GM-CSF and IL-4 will be transduced with an adenovector to express the MART-1 antigen and matured to """"""""immunogenic competence"""""""" through CD40 signaling, with a variety of bacterial stimulants, or by making the DC capture MART-1 antigen from apoptotic cells. The conditioned DC will be used to generate CTL and helper T cell in vitro. T cell responses will be monitored in CTL assay, Fastimmune assay, and tetrameter binding assay. The role of the CD4+ T cells on the DC as well as on the CTL will be examined in appropriate co-cultures and the robustness of the CTL response will be determined in CTL assay, Fastimmune assay and in tetramer binding assay to obtain a quantitative assessment of CTL expansion. We shall also test a number of avenues to enhance antigen presentation by compartmentalized class I and/or class II loading of antigen via engineered DC. These are: a) endosomal localization with chimeric polypeptide expressing the TAA and intracellular trafficking signals: b) trafficking of TAA and heat shock -TAA fusions; c) TAA trafficking under a polarized Th1 condition engineered internally by expressing chimeric TAA and bacterial ISS sequences; and d) nuclear localization of EGFP:TAA chimeras. These studies will provide a much needed understanding of the rules of engagement of DC and CD4+ T cells with translational implications.
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