Localization and Function of iNKT Cells in Neuroblastoma

描述(申请人提供)：我们研究的长期目标是为肿瘤免疫疗法的发展提供基本的基础，以实现Vct24不变的自然杀伤T细胞(INKT)介导有效的抗肿瘤免疫反应的潜力。 我们已经证明iNKT细胞可以被肿瘤细胞衍生的趋化因子MCP-1所吸引，但它们不能渗透到N-MYC癌基因过表达和MCP-1低/缺失的原发神经母细胞瘤中。新的数据表明，N-MYC过表达导致肿瘤细胞MCP-1下调。此外，我们最近还证明了IL-7是人iNKT细胞重要的动态平衡因子。原发神经母细胞瘤中IL-7与iNKT TCR RNA呈显著负相关，在神经母细胞瘤细胞系培养上清中加入IL-7对iNKT细胞具有保护作用。 细胞凋亡。因此，我们假设：1)N-MYC调节神经母细胞瘤细胞MCP-1的表达，其治疗靶向可以实现iNKT细胞在肿瘤部位的有效定位；2)神经母细胞瘤细胞产生IL-7的水平决定了iNKT细胞在肿瘤微环境中的存活和功能。本研究的目的是验证这些假说：1)评估神经母细胞瘤细胞中N-MYC过表达阻止iNKT细胞向肿瘤部位侵袭的机制，并测试干扰这一过程的治疗方法；2)评估IL-7在神经母细胞瘤中iNKT细胞存活和功能中的作用。我们研究N-myc调控作用的实验策略是基于基因工程的“功能获得”或“功能丧失”方法。 神经母细胞瘤细胞分别过表达或抑制N-MYC表达。为了测试神经母细胞瘤细胞IL-7的表达和产生是否足以维持iNKT细胞在肿瘤微环境中的持续和功能，我们将使用四环素调控的表达系统转导带有IL-7基因的神经母细胞瘤细胞。我们的体外和体内实验将利用新鲜分离的人外周血iNKT细胞和具有良好特性的iNKT、NK和神经母细胞瘤细胞系。INKT细胞的定位、存活和功能将在NOD/SCID小鼠的人神经母细胞瘤转移模型中进行测试。下调N-myc和上调N-myc的维甲酸 MCP-1将被测试为增加iNKT在神经母细胞瘤中的渗透。由于iNKT细胞不是直接杀伤细胞，但可以激活强大的NK细胞对神经母细胞瘤细胞的杀伤作用，因此其中一个终点将衡量它们诱导/增强NK细胞介导的抗肿瘤效应的能力。意义重大。这项研究的结果将有助于更好地了解肿瘤免疫逃避和免疫抑制的机制，并为开发神经母细胞瘤和其他类型癌症的有效免疫治疗提供新的分子靶点。

DESCRIPTION (provided by applicant): 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.