The project goals are 1) to develop biocompatible nanoparticles (NP) bearing Pattern Recognition Receptor (PRR) molecules, i.e., agonists for Toll Like Receptors or C-type Lectin Receptors, 2) to determine if these NP- PRR differently affect activation of dendritic cells (DC), 3) to determine if the differently activated DC then influence the generation and functionality of T effector cells, and 4) to evaluate the alloreactive CTL and the autologous tumor associated antigen (TAA)-directed CTL in the GL261 mouse glioma model, where the tumor cell inoculum either is or is not enriched for brain tumor stem cells (BTSC). The latter goal will allow for a determination of BTSC immunosensitivity/immunoresistance. The cancer immunotherapy approach will: """"""""Use various NP-PRR (CpG DNA, imiquimod, LPS and mannosylated BSA) that will be components of a biocompatible PLGA carrier matrix already approved for clinical use to stimulate DC to variable activation states. In some experiments complex NP-PRR will also contain tumor associated antigens (mEphA2, hgp100, mTRP-2 and GARC-1) """""""" Use conventional and Flt3 ligand DC derived from immature bone marrow (BM) as precursor cells """""""" NP-PRR activated DC will then be used to stimulate alloreactive or autologous naive T cells to effector cytotoxic T lymphocytes (CTL) """"""""NP-PRR stimulated DC and subsequently stimulated CTL will be functionally and phenotypically characterized in vitro.""""""""Stimulated alloreactive CTL or TAA-directed CTL will be functionally and phenotypically characterized for their antitumor effects after their adoptive transfer into mice bearing serum-cultured GL261 or the neurosphere-cultured counterpart.
The work proposed here is an interdisciplinary project in translational medicine that brings together several key elements: 1. Cytotoxic T Lymphocytes (CTL)-effector cells for immunotherapy 2. Dendritic Cells (DC)-antigen presenting cells (APC) used to induce CTL effectors in vitro 3. Nanoparticles (NP) and their payload-activation stimuli and antigens delivered to DC 4. Brain Tumor Stem Cells (BTSC)-therapy-resistant tumor cells that should be included as targets of the effector cells The goal of this project is to modulate dendritic cell activation with pathogen associated signaling molecules contained with nanoparticles. These nanoparticles will be used to stimulate dendritic cells to various activation states. The dendritic cells will then be used to stimulate naive T cells into effector cells. The generated CTL will be functionally and phenotypically characterized in vitro and in vivo using mouse glioma cell targets or those enriched for brain tumor stem cells in culture.
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