Anti-GD3 NKT cells as effector cells against melanoma

描述（由申请人提供）：GD3神经节苷脂在神经外胚层组织和肿瘤如黑色素瘤、肉瘤和小细胞肺癌上表达。针对GD3的抗体可以缩小啮齿动物和患者的黑色素瘤。在临床试验中，我们设计了对患者进行GD3免疫的辅助设置，我们发现令人惊讶的低复发率与抗GD3抗体的诱导无关。由于GD3不存在于MHC I类或II类中，我们考虑了另一种可能性，即免疫可能诱导NKT对GD3的反应。我们已经在小鼠和本项目中诱导了抗GD3的cd1限制性NKT细胞；我们建议进一步表征抗gd3小鼠NKT细胞反应。特异性目的1-确定T细胞受体的使用，细胞因子谱，以及小鼠cd1限制性NKT细胞对GD3的特异性。迄今为止描述的大多数小鼠cd1限制性NKT细胞识别来自细菌或无脊椎动物的糖脂。对于识别在哺乳动物细胞上表达的自身糖脂的cd1限制性NKT细胞，我们所知甚少。特异性目标2 -描述GD3如何加载到小鼠CD1上。在人体系统中，一些cd1限制性糖脂抗原需要内化并在酸化的晚期核内体中装载；其他限制cd1的糖脂则装载在不需要酸化的后高尔基囊泡中。尽管在人体系统中有一些证据表明GM1神经节苷脂甚至不需要内化，但对于自身神经节苷脂是如何装载到CD1上的知之甚少。利用小鼠系统，我们将描述GD3如何装载到小鼠CD1上，评估内化、内体酸化和高尔基体功能的要求。我们计划使用共聚焦免疫显微镜绘制细胞内加工途径。特异性目的3 -验证抗GD3小鼠NKT细胞以抗原特异性方式介导抗肿瘤作用的假设。被α -半乳糖神经酰胺（来自海洋无脊椎动物）激活的cd1限制性NKT细胞可以以一种似乎不是抗原特异性的方式介导抗肿瘤作用。目前尚不清楚cd1限制性NKT细胞对肿瘤细胞上表达的抗原是否具有抗肿瘤作用。我们将测试cd1限制性NKT细胞抗GD3在体外裂解GD3+小鼠黑色素瘤和体内排斥GD3+小鼠肿瘤的能力。这将为基于抗原特异性NKT细胞的免疫治疗方法提供初步支持。

DESCRIPTION (provided by applicant): GD3 ganglioside is expressed on neuroectodermal tissue and on tumors such as melanoma, sarcoma, and small-cell lung cancer. Antibodies against GD3 can shrink melanoma in rodents and patients. In clinical trials designed to immunize patients against GD3 in the adjuvant setting, we saw surprisingly low relapse rates that did not correlate with induction of anti-GD3 antibodies. Since GD3 is not presented by MHC class I or class II, we considered the alternative possibility that immunization might induce a NKT response against GD3. We have induced CD1-restricted NKT cells against GD3 in mice and in this project; we propose to characterize further the anti-GD3 murine NKT cell response. Specific Aim 1 - Define the T cell receptor usage, cytokine profile, and specificity of mouse CD1-restricted NKT cells against GD3. Most murine CD1-restricted NKT cells described to date recognize glycolipids derived from bacteria or invertebrates. Much less is known about CD1-restricted NKT cells that recognize self-glycolipids expressed on mammalian cells. Specific Aim 2 - Characterize how GD3 is loaded on to mouse CD1. In the human system, some CD1-restricted glycolipid antigens require internalization and loading in acidified late endosomes; other CD1-restricted glycolipids are loaded in post-Golgi vesicles not requiring acidification. Little is known about how self gangliosides are loaded on to CD1 although in the human system there is some evidence that GM1 ganglioside does not even require internalization to be presented. Using the mouse system, we will characterize how GD3 is loaded on to mouse CD1 assessing requirements for internalization, acidification of endosomes and Golgi function. We plan to map the intracellular processing pathway using confocal immunomicroscopy. Specific Aim 3 -Test the hypothesis that mouse NKT cells against GD3 can mediate anti-tumor effects in an antigen-specific manner. CD1-restricted NKT cells activated by alpha-galactosylceramide (from marine invertebrates) can mediate antitumor effects in a manner that does not appear to be antigen-specific. It is not known if CD1-restricted NKT cells specific for an antigen expressed on tumor cells can have anti-tumor effects. We will test the ability of CD1-restricted NKT cells against GD3 to lyse GD3+ mouse melanoma in vitro and to reject GD3+ mouse tumors in vivo. This will provide initial support for an immunotherapeutic approach based on antigen-specific NKT cells.