The long-term goal of our research is to provide the fundamental basis for the development of tumor immunotherapy that would realize the potential of Vct24-invariant natural killer T (iNKT) cells to mediate effective anti-tumor immune responses. ? We have demonstrated that iNKT cells can be chemoattracted by the tumor cell-derived chemokine MCP-1 but they fail to infiltrate primary neuroblastomas that have N-MYC oncogene overexpression and low/absent MCP-1 New data reveal that N-MYC overexpression causes MCP-1 downregulation in tumor cells. In addition, we have recently demonstrated that IL-7 is an important homeostatic factor for human iNKT cells. There is a strong negative correlation between IL-7 and iNKT TCR RNA in primary neuroblastomas, and addition of IL-7 to neuroblastoma cell line supernatant protects iNKT cells from ? apoptosis. Therefore, we hypothesize: 1) N-MYC regulates MCP-1 expression in neuroblastoma cells and its therapeutic targeting could achieve effective localization of iNKT cells to the tumor sites; 2) the level of IL-7 production by neuroblastoma cells determines iNKT cell survival and function in the tumor microenvironment. The following specific aims are designed to test these hypotheses: 1) to evaluate the mechanisms by which N-MYC overexpression in neuroblastoma cells prevents iNKT cell infiltration to the tumor sites and to test treatments interfering with this process; 2) to evaluate the role of IL-7 in iNKT cell survival and function in neuroblastoma. Our experimental strategy to study the regulatory role of N-MYC is based on """"""""gain of function"""""""" or """"""""loss of function"""""""" approaches by genetically engineering ? neuroblastoma cells to over express or to repress expression of N-MYC, respectively. To test whether expression and production of IL-7 by neuroblastoma cells is sufficient to sustain iNKT cell persistence and function in the tumor microenvironment, we will transduce neuroblastoma cells with IL-7 cDNA using a tetracycline-regulated expression system. Our in vitro and in vivo experiments will utilize freshly isolated human peripheral blood iNKT cells and well characterized iNKT, NK, and neuroblastoma cell lines. Localization, survival, and function of iNKT cells will be tested in a metastatic model of human neuroblastoma in NOD/SCID mice. Retinoic acid that downregulates N-MYC and upregulates ? MCP-1 will be tested to increase iNKT infiltration in neuroblastoma. Since iNKT cells are not directly cytotoxic but can activate potent NK cell cytotoxicity against neuroblastoma cells, one of the endpoints will measure their ability to induce/enhance NK cell-mediated anti-tumor effects. Significance. The results of the proposed investigation will lead to better understanding of the mechanisms of immune evasion and immunosuppression by tumors and to identification of novel molecular targets for development of effective immunotherapy of neuroblastoma and other types of cancer. ? ?
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