THE INITIATION AND REGULATION OF INFLAMMATION IN NEUROBLASTOMA

 描述（由申请人提供）：我们研究的长期目标是为开发一种有效的癌症免疫疗法提供机制基础，这种免疫疗法使用V β 24不变的自然杀伤T细胞（iNKT）的天然和工程特性。过程中 在该基金支持的机制研究中，我们发现iNKT响应于肿瘤衍生的趋化因子而向NB运输，并通过靶向肿瘤相关巨噬细胞（TAM）间接介导抗肿瘤活性。我们还证明了TAM浸润NB患者亚组中的原发性肿瘤，并且M2样TAM的存在与新的炎症特征相关，该炎症特征作为预后不良的独立预后因素。然而，负责NB中肿瘤支持性炎症的启动和调节的机制尚不清楚，将在更新申请中解决。在寻找NB中炎症反应的分子触发因子时，我们发现所有检查的人和鼠细胞系以及原发性人和转基因鼠肿瘤中的NB细胞亚群表达TNF α（tmTNF α）的跨膜形式。最近的报道表明，与可溶性TNF α不同，tmTNF α优先激活单核细胞中的TNFR 2，并促进其M2样分化和抑制活性。我们的初步数据表明，iNKT可能通过选择性识别M2极化的巨噬细胞来抵消这一过程。此外，将自发发展NB的NBL-Tag转基因小鼠与iNKT细胞缺陷动物杂交导致肿瘤生长加速和TAM频率增加。然而，TAM最终逃避iNKT细胞控制，并且这种逃避与肿瘤进展相关。因此，我们假设i）表达tmTNF α的NB细胞通过激活M2样TAM引发肿瘤支持性炎症; ii）iNKT通过杀死或重编程M2样TAM调节肿瘤诱导的炎症并间接抑制肿瘤生长; iii）TAM中和iNKT并使肿瘤逃逸。以下具体目标将检验这些假设：1）检查和治疗性探索NB细胞中tmTNF α触发肿瘤支持性炎症的机制; 2）检查和治疗性探索肿瘤微环境中iNKT和TAM之间相互抑制的机制。我们将使用人细胞体外系统、小鼠同基因NB模型和NB患者原发性肿瘤标本，采用遗传功能丧失和功能获得方法研究NB衍生的TNF α亚型在巨噬细胞活化和功能分化中的作用。为了研究iNKT和TAM之间的相互作用，我们最近表征了具有和不具有iNKT或所有NKT的遗传缺陷的转基因NB模型。最后，我们将测试TAM的治疗靶向是否最大化NB的iNKT细胞免疫疗法的抗肿瘤潜力。这些结果有望揭示控制NB炎症启动和调节的新机制，并有助于确定用于开发NB和其他类型癌症有效免疫疗法的细胞和分子靶点。

 DESCRIPTION (provided by applicant): The long-term goal of our research is to provide the mechanistic basis for the development of an effective immunotherapy for cancer using the natural and engineered properties of V24-invariant Natural Killer T cells (iNKTs). In the course of the mechanistic studies supported by this grant we have found that iNKTs traffic toward NB in response to tumor-derived chemokines and mediate anti-tumor activity indirectly via targeting of tumor-associated macrophages (TAMs). We also demonstrated that TAMs infiltrate primary tumors in a subset of NB patients and the presence of M2-like TAMs is associated with a novel inflammatory signature that serves as an independent prognostic factor of poor outcome. However, the mechanisms responsible for the initiation and regulation of tumor-supporting inflammation in NB are unknown and will be addressed in the renewal application. In the search for the molecular triggers of an inflammatory response in NB, we found that a subset of NB cells in all examined human and murine cell lines, as well as primary human and transgenic murine tumors, expresses a transmembrane form of TNFα (tmTNFα). Recent reports suggest that unlike soluble (s)TNFα, tmTNFα preferentially activates TNFR2 in monocytic cells and promotes their M2-like differentiation and suppressive activity. Our preliminary data suggest that iNKTs may counteract this process via selective recognition of M2-polarized macrophages. Moreover, crossing NBL-Tag transgenic mice, which spontaneously develop NB, with iNKT-cell deficient animals resulted in an acceleration of tumor growth and an increase of TAM frequency. However, TAMs eventually evade iNKT-cell control, and the evasion is associated with tumor progression. Therefore, we hypothesize that i) tmTNFα-expressing NB cells initiate tumor-supportive inflammation via activation of M2-like TAMs; ii) iNKTs regulate tumor-induced inflammation and indirectly inhibit tumor growth via killing or reprograming of M2-like TAMs; iii) TAMs neutralize iNKTs and enable tumor escape. The following specific aims will test these hypotheses: 1) to examine and therapeutically explore the mechanism by which tmTNFα in NB cells trigger tumor-supportive inflammation; 2) to examine and therapeutically explore the mechanism of reciprocal inhibition between iNKTs and TAMs in the tumor microenvironment. We will use genetic loss of function and gain of function approaches to study the role of NB-derived TNFα isoforms in the activation and functional differentiation of macrophages using in vitro systems with human cells, a syngeneic NB model in mice, and primary tumor specimens from NB patients. To study reciprocal interactions between iNKT and TAMs we recently characterized transgenic NB models with and without genetic deficiency of iNKTs or all NKTs. Finally, we will test whether therapeutic targeting of TAMs maximizes the anti-tumor potential of iNKT-cell immunotherapy for NB. The results are expected to reveal novel mechanisms that govern the initiation and regulation of inflammation in NB and to help identify cellular and molecular targets for the development of effective immunotherapy for NB and other types of cancer.