ELISPOT FOR MEASUREMENT OF ANTIGEN SPECIFIC T CELLS

描述：(申请人提供) 用于癌症疫苗免疫学监测的标准技术 试验包括将氚胸苷掺入作为抗原的一种衡量标准 细胞毒性T细胞(CTL)的特异性增殖和溶血能力 肿瘤，最常通过铬释放来评估。这两种方法是 难以可靠地生产，需要体外扩增，而且不是 量化的。将这些测定系统添加到极限稀释分析中 格式允许对前兆频率进行更准确的评估，但 建立分析所需的免疫效应细胞数量需要 病人要接受白细胞分离术。因此，这些策略不适用于 大范围的免疫监测。 抗原特异性免疫反应在很大程度上由分泌的 调节T细胞生长和分化的细胞因子 人口。T细胞对特定细胞因子的分泌 抗原已成为衡量功能性免疫反应的常用指标。虽然 使用ELISA方法测量T细胞产生细胞因子的方法是 不是定量的，细胞因子的测量提供了详细的特征 T细胞的功能和免疫环境的表型 抗原识别后产生的。最近，一项单细胞测试已经完成。 开发的目的是更准确地评估产生细胞因子的细胞。该方法， 被称为ELISPOT(酶联免疫吸附斑点)可以检测抗原反应 T细胞在这样的细胞能够产生足够数量的蛋白质之前 用酶联免疫吸附试验检测。 虽然ELISPOT已成为免疫监测的“比较器”， 该分析的灵敏度和检出限与 实验室之间的联系。翻译Elispot需要克服的障碍 临床分级监测技术的实验室工具包括最大化 避免任何体外扩增步骤的检测参数，为 在冻存细胞中的应用，确定用于 分析，并定义在一段时间内对 在多个临床样本中。 这项提案的具体目的是：(1)确定Elispot是否可以 确定T细胞对外源蛋白抗原的反应 志愿者捐赠者和癌症患者，(2)确定Elispot是否可以 从志愿者PBL中鉴定T细胞对肿瘤抗原的反应 捐赠者和癌症患者，(3)确定T细胞反应，如 通过ELISPOT检测，预测小鼠转基因肿瘤的肿瘤保护作用 模型，如CEA和neu转基因小鼠，最后，(4)确定 ELISPOT能否预测对癌症抗原的免疫以及水平 所取得的结果与人类临床上的传染病抗原相似 CEA、HER2、GP 100和MAGE3疫苗的试验。

Description: (provided by applicant) Standard techniques used for the immunologic monitoring of cancer vaccine trials have included tritiated thymidine incorporation as a measure of antigen specific proliferation and the ability of cytotoxic T cells (CTL ) to lyse a tumor, most often assessed by chromium release. These two methods are difficult to reliably produce, require in vitro expansion, and are not quantitative. Addition of these assay systems to a limiting dilution analysis format has allowed a more accurate assessment of precursor frequency, but the number of immune effector cells needed to establish the analysis requires patients undergo a leukapheresis. Thus, these strategies are not viable for wide-scale immunologic monitoring. Antigen specific immune responses are largely regulated by secretion of cytokines whose function is to govern the growth and differentiation of T cell populations. Secretion of cytokines by T cells responding to a specific antigen has become a common measure of a functional immune response. Although the measurement of cytokine production by T cells using ELISA methodologies is not quantitative, measurement of cytokines offers a detailed characterization of the function of the T cell and the phenotype of the immune environment generated after antigen recognition. Recently, a single-cell assay has been developed to more accurately assess cytokine-producing cells. The method, termed ELIspot (enzyme-linked immunosorbant spot) can detect antigen reactive T cells before such cells could produce sufficient amount of protein to be detected by ELISA. Although ELIspot has become the "comparator" assay for immunologic monitoring, the sensitivity and limits of detection of the assay vary greatly from laboratory to laboratory . Obstacles to be overcome to translate ELIspot from a laboratory tool to a clinical grade monitoring technique include maximizing assay parameters to avoid any in vitro expansion step, develop the assay for use in cryopreserved cells, determine optimal antigen preparations used in analysis, and define the reliability of the assay to perform over time on the in multiple clinical samples. The specific aims of this proposal are to: (1) determine whether ELIspot can identify the T cell response to foreign protein antigens from the PBL of volunteer donors and cancer patients, (2) determine whether ELIspot can identify the T cell response to tumor antigens from the PBL of volunteer donors and cancer patients, (3) determine whether the T cell response, as measured by ELIspot, predicts tumor protection in murine transgenic tumor models such as the CEA and neu transgenic mice, and, finally, (4) determine whether ELIspot can predict immunization to cancer antigens and whether levels achieved are that similar to infectious disease antigens in human clinical trials of CEA, HER2, gp 100, and MAGE3 vaccines.