Anti-GD3 NKT cells as effector cells against melanoma

描述（由申请方提供）：GD 3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。GD 3抗体可以缩小啮齿动物和患者的黑色素瘤。在设计用于在辅助环境中免疫患者以对抗GD 3的临床试验中，我们看到了令人惊讶的低复发率，其与抗GD 3抗体的诱导无关。由于GD 3不是由MHC I类或II类呈递的，因此我们考虑了免疫可能诱导针对GD 3的NKT反应的另一种可能性。我们已经在小鼠中诱导了针对GD 3的CD 1限制性NKT细胞，在本项目中，我们建议进一步表征抗GD 3小鼠NKT细胞反应。具体目标1 -确定T细胞受体的使用，细胞因子谱和小鼠CD 1限制性NKT细胞对GD 3的特异性。迄今为止描述的大多数鼠CD 1限制性NKT细胞识别来自细菌或无脊椎动物的糖脂。关于识别哺乳动物细胞上表达的自身糖脂的CD 1限制性NKT细胞，我们知之甚少。具体目标2 -表征GD 3如何加载到小鼠CD 1上。在人类系统中，一些CD 1限制性糖脂抗原需要内化和加载在酸化的晚期内体中;其他CD 1限制性糖脂加载在不需要酸化的高尔基体后囊泡中。很少有人知道如何自我神经节苷脂加载到CD 1，虽然在人类系统中有一些证据表明，GM 1神经节苷脂甚至不需要内化。使用小鼠系统，我们将表征GD 3如何加载到小鼠CD 1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来绘制细胞内加工途径。特定目的3 -检验小鼠NKT细胞对GD 3的作用可以以抗原特异性方式介导抗肿瘤作用的假设。由α-半乳糖神经酰胺（来自海洋无脊椎动物）激活的CD 1限制性NKT细胞可以以似乎不具有抗原特异性的方式介导抗肿瘤作用。目前尚不清楚对肿瘤细胞上表达的抗原具有特异性的CD 1限制性NKT细胞是否具有抗肿瘤作用。我们将测试针对GD 3的CD 1限制性NKT细胞在体外裂解GD 3+小鼠黑色素瘤和在体内排斥GD 3+小鼠肿瘤的能力。这将为基于抗原特异性NKT细胞的免疫学方法提供初步支持。

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.