Grail-targeting breaks the immune tolerance to melanoma

摘要 抗癌免疫治疗的疗效受到多种肿瘤逃避机制的限制，包括 肿瘤反应性T细胞的耐受诱导。因此，打破T细胞耐受性的策略以及此后 需要增强T细胞对肿瘤细胞的杀伤作用。这样一种创新和理性的方法是 通过抑制耐受相关因子的活性来操纵T细胞的内在特性。 我们已经确认E3泛素连接酶，Grail是T细胞耐受的重要组成部分，并 显示Grail缺乏导致CD4+T细胞高反应性和调节性T细胞缺陷 抑制功能。因此，靶向圣杯可能有助于打破肿瘤免疫耐受；然而， GRAIL在肿瘤发展和细胞毒性CD8+T淋巴细胞(CTL)功能中的作用尚不清楚。 我们的初步数据显示，GRAIL的丧失增强了CTL的细胞溶解功能。此外，利用 对于淋巴瘤肿瘤，我们已经确定缺乏Grail的CTL对于控制已建立的肿瘤是必不可少的。 值得注意的是，在淋巴瘤患者的CTL中，Grail的表达水平显著升高，提示 Grail在淋巴瘤免疫耐受中的新作用，为探索Grail在淋巴瘤免疫耐受中的作用提供了理论基础 黑色素瘤，常见于淋巴瘤环境中。进一步认识圣杯在不同环境中的作用 肿瘤的类型将有利于发展对这些恶性肿瘤的共同治疗方法。我们 有证据表明，小鼠体内缺乏圣杯可以控制移植的B16黑色素瘤。此外，在 在黑色素瘤患者样本中，我们发现来自肿瘤组织的CD8+T细胞中高表达Grail，提示 免疫细胞中的圣杯表达也可能是控制黑色素瘤的一大障碍。 因此，我们建议研究圣杯靶向CTL对黑色素瘤的治疗潜力。 以及它们使用鼠标和人类系统的调节机制。 在目标1中，我们将通过基因敲除来研究Grail KO CTL在黑色素瘤生长控制中的作用 接近了。我们还将评估圣杯控制抗肿瘤功能和持久性的机制(S) CTL的数量。此外，我们将尝试确定GRAIL促进肿瘤反应性CD8+控制的靶点 利用尖端技术，包括双杂交筛选和微阵列分析，实现T细胞功能 关于基因表达的。在目标2中，我们将建立一种有效的针对人和小鼠的圣杯靶向方法 CD8+T细胞，以提高对已确定的恶性肿瘤的过继治疗效率。 总体而言，拟议的研究将有助于验证GRAIL作为免疫耐受的功能性介体。 以及评估Grail缺陷的CD8+T细胞治疗黑色素瘤的潜力。 这项工作的意义是重大的，因为结果将提供对 了解圣杯在黑色素瘤中的作用并将潜在地引导小说的发展 逆转黑色素瘤和其他恶性肿瘤免疫耐受的靶向治疗。

Abstract Efficacy of anti-cancer immunotherapy is limited due to various tumor evasion mechanisms including the tolerance induction of tumor-reactive T cells. Therefore, strategies to break T cell tolerance and thereafter enhance T-cell cytotoxicity towards tumor cells are needed. One such innovative and rational approach is to manipulate the intrinsic properties of T cells by suppressing the activity of tolerance-associated factors. We have acknowledged the E3 ubiquitin ligase, Grail as an essential component of T-cell tolerance and showed that Grail deficiency resulted in CD4+ T cell hyper-responsiveness and defective regulatory T cell suppressive function. Thus, targeting Grail may help to break tumor immune tolerance; however, the role of Grail in tumor development and in the function of cytotoxic CD8+ T lymphocytes (CTLs) remains unknown. Our preliminary data show that loss of Grail enhanced cytolytic functionality of CTLs. Furthermore, utilizing lymphoma tumors, we have determined that Grail-deficient CTLs are essential for control of established tumor. Remarkably, Grail expression level was significantly higher in CTLs from lymphoma patients, suggesting the novel role of Grail in immune tolerance to lymphoma and providing a rationale for exploring the role of Grail in melanoma that is often observed in a lymphoma setting. Further understanding the function of Grail in different cancer types will be beneficial for development of common therapeutic approach for these malignancies. We have evidence that Grail deficiency in mice confers control of implanted B16 melanoma tumor. Moreover, in melanoma patient samples, we found high Grail expression in CD8+ T cells from the tumor tissues, suggesting that Grail expression in the immune cells could be a big obstruction for controlling melanoma as well. Therefore, we propose to investigate the therapeutic potential of Grail-targeted CTLs for melanoma as well as their regulatory mechanisms using both mouse and human systems. In Aim 1, we will investigate the role of Grail KO CTLs in melanoma growth control by utilizing gene knockout approaches. We will also assess the mechanism(s) whereby Grail controls anti-tumor function and persistence of CTLs. Moreover, we will try to identify targets through which Grail facilitates control of tumor-reactive CD8+ T cell function by employing cutting edge technologies, including two-hybrid screening and microarray analysis of gene expression. In Aim 2, we will establish an efficient Grail targeting approach for human and mouse CD8+ T cells in order to improve the efficiency of adoptive therapy for established malignancies. Overall the proposed research will help to validate Grail as a functional mediator of immune tolerance in melanoma as well as evaluate the therapeutic potential of Grail deficient CD8+ T cells for melanoma treatment. The implications from this work are significant since the results will provide mechanistic insights into the understanding of the function of Grail in melanoma and will potentially lead to the development of novel targeted treatments to reverse immune tolerance in melanoma and other malignancies.