Tumor Induced Dysregulation of T Cell Immunity

描述（由申请人提供）：越来越多的证据表明，肿瘤环境可以通过促进T细胞功能障碍来抑制有效免疫应答的发展。我们实验室和其他几个实验室的研究表明，肿瘤及其产物促进T细胞死亡的能力是免疫功能障碍的重要机制。用AnnexinV/7AAD染色的肿瘤浸润性T细胞的流式细胞术分析以及原位TUNEL染色揭示了高百分比的这些细胞显示出凋亡/坏死的迹象，表明肿瘤微环境对肿瘤浸润性T细胞（TIL）具有有害作用，尽管RCC的免疫抑制作用也延伸到外周。另外的研究表明，在患有活动性疾病的患者中，对肾细胞癌表达的法师-6和EphA 2的T细胞应答主要是2型（IL-4、IL-5、IL-10）而不是1型（IFNg），已知1型（IFNg）对于有效的抗肿瘤免疫的发展至关重要。然而，通过手术使疾病消失的患者显示Th-1应答（IFNg，ELISPOT）。HLA-DR 4/肿瘤肽四聚体研究显示，与结合流感肽的T细胞和大量外周血T细胞群相比，法师-6和EphA 2特异性CD 4 +T细胞对细胞凋亡高度敏感。我们的数据还表明，神经节苷脂在肿瘤诱导的免疫功能障碍中起重要作用。神经节苷脂占RCC外植体上清液和RCC细胞系诱导的凋亡活性的50%以上。RCC衍生的神经节苷脂还可以在体外诱导Th 2偏好。HPLC和在线质谱分析表明，有几个神经节苷脂，包括GM 2，介导的免疫抑制。在这里，我们计划确定哪种来自RCC组织、上清液和血浆的常见神经节苷脂在诱导T细胞死亡（2型偏倚）方面最有效，以及该偏倚是否与法师-6和EphA 2特异性Th-1 T细胞的凋亡有关。我们还将确定免疫抑制是否是由选择性神经节苷脂从肿瘤脱落介导的，并与T细胞质膜上的脂筏相关，以及抑制性神经节苷脂的表达是否与Th-2偏倚和患者T细胞对凋亡的敏感性增加（aim 1）相关。将检查由神经节苷脂诱导的T细胞死亡是否是由于降解为神经酰胺或其迁移至线粒体所致（aim 2）。抗氧化剂去铁胺（DFO）可以阻断神经节苷脂介导的T细胞凋亡，这一观察结果将进一步研究DFO如何保护T细胞免于凋亡。我们还将定义导致T细胞死亡的事件顺序，包括铁和溶酶体在这一过程中的作用（目的3）

DESCRIPTION (provided by applicant): Mounting evidence suggests that the tumor environment can inhibit the development of an effective immune response by promoting T cell dysfunction. Studies from our laboratory and several others suggest that the ability of tumors and their products to promote T cell death represents an important mechanism of immune dysfunction. Flow cytometry analysis of tumor infiltrating T cells stained with AnnexinV/7AAD as well as in situ TUNEL staining revealed that a high percentage of these cells showed signs of apoptosis/necrosis, suggesting that the tumor microenvironment has a deleterious effect on tumor infiltrating T cells (TILs), although the immunosuppressive effects of RCC extend into the periphery as well. Additional studies demonstrated that in patients with active disease the T cell response to MAGE-6 and EphA2, expressed by renal cell carcinomas was predominately Type-2 (IL-4, IL-5.IL-10) rather than a type-1(IFNg) which is known to be critical for the development of effective anti-tumor immunity. However, patients rendered disease free by surgery displayed Th-1 responses (IFNg, ELISPOT). HLA-DR4/tumor peptide tetramer studies showed that the MAGE-6 and EphA2 specific CD4+T cells were highly sensitive to apoptosis compared to T cells binding influenza peptides and the bulk peripheral blood T cell population. Our data also suggest that gangliosides play in important role in tumor-induced immune dysfunction. Gangliosides constitute over 50% of the apoptotic activity induced by supernatants from RCC explants and by RCC lines. RCC derived gangliosides can also induce a Th2 bias in vitro. HPLC with on line mass spectrometry analysis suggests that there are several gangliosides, including GM2, which mediate the immunosuppression. Here we plan to determine which of the common gangliosides derived from RCC tissue, supernatant and plasma are the most effective at inducing T cell death, a Type-2 bias, and whether the bias is related to the apoptosis of the MAGE-6 and EphA2 specific Th-1 T cells. We will also determine whether the immune suppression is mediated by select gangliosides shedding from tumor and associating with lipid rafts on the plasma membranes of T cells and whether expression of the inhibitory gangliosides correlates with the Th-2 bias and increased sensitivity of patient T cells to apoptosis (aim1). Whether T cell death induced by ganglosides results from degradation to ceramide or to their migration to mitochondria will be examined (aim2). The observation that the anti-oxidant deferoxamine (DFO) can block ganglioside-mediated apoptosis of T cells will be studied further to define how DFO protects T cells from apoptosis. We will also define the sequence of events leading to T cell death, including the role of iron and lysosomes in this process (aim 3)