THE INITIATION AND REGULATION OF INFLAMMATION IN NEUROBLASTOMA

 描述(申请人提供)：我们研究的长期目标是利用V24不变自然杀伤T细胞(INKT)的自然和工程特性，为开发有效的癌症免疫疗法提供机制基础。在课程中 在这项资助支持的机制研究中，我们发现iNKTS通过对肿瘤来源的趋化因子的反应而向NB转运，并通过靶向肿瘤相关巨噬细胞(TAMs)间接介导抗肿瘤活性。我们还证明了TAMs在一组NB患者中浸润性原发肿瘤，M2样TAMs的存在与一种新的炎性征象有关，这是预后不良的独立预后因素。然而，在NB中负责启动和调节肿瘤支持炎症的机制尚不清楚，将在更新应用中解决。在寻找NB炎症反应的分子触发因素时，我们发现，在所有受检的人和小鼠细胞系以及原发的人类和转基因小鼠肿瘤中，NB细胞亚群表达跨膜形式的肿瘤坏死因子α(tmTNFα)。最近的报道表明，与可溶性(S)肿瘤坏死因子α不同，α优先激活单核细胞中的肿瘤坏死因子受体2，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，将自发发展成NB的NBL-Tag转基因小鼠与iNKT细胞缺陷动物杂交，可以加速肿瘤的生长，增加的频率。然而，TAMS最终逃避了iNKT细胞的控制，而这种逃避与肿瘤的进展有关。因此，我们假设：1)表达肿瘤坏死因子α的NB细胞通过激活M2样TAMs来启动肿瘤支持炎症；ii)iNKT通过杀伤或重编M2样TAMs来调节肿瘤诱导的炎症反应，间接抑制肿瘤生长；iii)TAMs中和iNKT并使肿瘤逃逸。以下特定目的将检验这些假说：1)检测和治疗探索NB细胞中的肿瘤坏死因子α引发肿瘤支持性炎症的机制；2)检测和治疗探索肿瘤微环境中iNKT和TAMS之间相互抑制的机制。我们将使用遗传功能丧失和功能获得的方法来研究Nb来源的肿瘤坏死因子α亚型在巨噬细胞激活和功能分化中的作用，使用人类细胞体外系统、小鼠同基因Nb模型和Nb患者的原发肿瘤标本。为了研究iNKT和TAMS之间的相互作用，我们最近研究了具有和不存在iNKT或ALL NKT遗传缺陷的转基因NB模型。最后，我们将测试TAMS的治疗靶向是否最大限度地发挥iNKT细胞免疫疗法对NB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，并有助于确定针对NB和其他类型癌症的有效免疫治疗的细胞和分子靶点。

 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.