The major goal of this proposal is to test the hypothesis that """"""""CTL response against human tumor associated antigen (TAA) induced by dendritic cell (DC)-based stimulation is subject to regulation by DC-T helper (Th) cell crosstalks. A better understanding of the rules of the engagement of DC- Th crosstalks that govern the generation and the control of anti-TAA CTL response will have a major impact in DC-based vaccine design."""""""" Aim 1 will undertake a critical and comprehensive re-examination of the human myeloid DC maturation process, in vitro.
Aim 2 will study the role of DC/Th-based crosstalks in the process of CTL generation, in vitro, against Mart-1 as a prototype human TAA.
Aim 3 will examine the mechanism(s) underlying DC/Th-based regulation of CTL priming versus inhibition of priming (tolerance induction), in vitro, in the Mart-1 system. Myeloid DCs grown in GM-CSF and IL-4 will be further polarized to immunogenic DCs (DC1) or to tolerogenic DCs (DC2) and CD4+ T cells will be polarized to Th1 and Th2 phenotypes in an antigen independent (i.e., polyclonally activated) and antigen dependent (i.e., generating antigen specific CD4+ Th1 lines) manner. The Th cells and the DCs, engineered by transduction with an Adeno/Mart-1 vector to express Mart-1 or pulsed with Mart-1 encoded peptides, will be assembled with CD8+ T cells or with Mart-1 tetramer sorted CTL precursors in an in vitro CTL generation system to examine if the DC1/Th1 crosstalk could generate a robust and sustained Mart-1 specific CTL response and if the DC2/Th2 duo could inhibit and/or terminate CTL responses. The rules of the DC/Th engagements and the molecular mechanisms of regulation will be defined by exposing the CTLp or CTLs to the regulatory crosstalk (i.e., by DC2/Th2) and by determining the activation/inactivation status (IL-2/IL-2R) or the death vs survival machinery (Fas/FasL, TNFR, Bcl/Bax genes and proteins) of the CTL at population level and at single cell level. The role of the DC/Th cells on the CTL precursors 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. These studies will provide a much needed understanding of the rules of engagement of DC and CD4+ T cells and will help design a more effective DC and antigen-based immunotherapy for cancer.
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