TUMOR LYMPHOKINES, TCR PEPTIDES AND TUMOR REJECTION

肿瘤淋巴细胞因子、TCR 肽和肿瘤排斥

• 批准号: 2098624
• 负责人: ECKHARD R PODACK
• 金额: $ 26.18万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2098624
• 关键词: T cell receptor; antiidiotype antibody; cell mediated cytotoxicity; cytotoxic T lymphocyte; genetically modified animals; interferon gamma; interleukin 2; interleukin 4; interleukin 6; interleukin 8; laboratory mouse; molecular cloning; natural killer cells; neoplasm /cancer immunology; northern blottings; nucleic acid sequence; pore forming protein; synthetic peptide; tissue /cell culture; transfection

The interrelationship, in tumor rejection, between (1) lymphokine production, (2) T-cell receptor usage, and (3) cytotoxicity, as measured in perforin transgenic mice, will be investigated in this proposal. An immunological response to a normally nonimmunogenic tumor is enforced by lymphokinic transfection of the tumor cell prior to transplantation into mice. IL2 transfection of the non-immunogenic Lewis lung cell carcinoma (LLC) renders the tumor immunogenic in such a way that it is rejected in normal mice but not in immunodeficient mice lacking T-cells (Nude,SCID) or NK-cells (Beige). Hence, both a T-cell and an NK-cell response are required for tumor rejection of this particular tumor producing IL2 (LLC- IL2). Even though IL2 producing LLC can retard the growth of normal LLC, a complete immunity resulting in rejection of the non-lymphokine producing tumor has not been achieved. By transfecting LLC with various lymphokines singly, or in a new double expression vector, with two lymphokine cDNAs simultaneously, the induction of an immune response giving complete tumor immunity is sought. Combination of lymphokines will focus on IL2 with IL4, IL6, I18 or gamma interferon. Studies with singly transfected LLC gave rise only to partial immunity. In rejected or retarded tumors the T- cell response will be determined by measuring the presence of T-cell receptor alpha and beta v-region gene families present in tumor infiltrating T-cells. Using a set of already tested (in NOD mice) v- region gene primers for both TCR-chains and the corresponding c-region primer, the cDNAs of the TCRs, generated by reverse transcription, will be amplified by PCR and cloned. The sequence of the most predominant TCR families, present at the tumor site under the influence of various tumor produced lymphokines, will be established for the v-, D-, and J-segments. To manipulate the T-cell response and uncover rejecting or suppressing T- cell clones, mice will be immunized with synthetic peptides corresponding to the TCR sequence. The ensuing anti idiotype antibody and cellular response will be evaluated with regard to its effect on tumor rejection. These studies will be complemented by the isolation and cloning of T-cells specific for lymphokine producing and non-producing LLC. The role of cytotoxicity, specifically the role of perforin, in tumor rejection in this model will be studied by comparing the tumor response of perforin deficient transgenic mice to that of perforin sufficient mice. Perforin deficiency has been achieved in embryonal stem cells by homologous recombination and perforin deficient chimeras are currently under study. These studies will be complemented by reconstituting perforin deficient mice, as well as normal mice, with human perforin transgenes. Human perforin, detectable by antibody and Northern analysis and distinguishable from mouse perforin, is expressed under the murine perforin promoter and under various promoter deletions, designed to enhance perforin expression in T-cell subsets. Improved perforin expression in transgenic mice will be analyzed with regard to its effect on tumor rejection.

（1）淋巴因子之间的肿瘤排斥相互关系 生产，（2）T细胞受体使用和（3）细胞毒性，如测量 在培养基转基因小鼠中，将在该提案中进行研究。 一个 对通常非免疫原性肿瘤的免疫反应由 在移植到肿瘤细胞中的淋巴因子转染 老鼠。 非免疫原性刘易斯肺细胞癌的IL2转染 （LLC）以这种方式拒绝肿瘤免疫原性 正常小鼠，但在缺乏T细胞（裸，SCID）或 NK细胞（米色）。 因此，T细胞和NK细胞响应都是 肿瘤排斥这种特定肿瘤产生IL2所需的必需（LLC-- IL2）。 即使产生IL2的LLC可以阻碍正常有限责任公司的增长，但 完全免疫力导致拒绝非淋应变的产生 尚未实现肿瘤。 通过用各种淋巴细胞转染LLC 单独或在新的双重表达载体中，有两个淋巴细胞代表cDNA 同时，免疫反应的诱导产生完全肿瘤 寻求免疫力。 淋巴细胞组合的组合将重点放在IL2上 IL4，IL6，I18或伽马干扰素。 单转染LLC的研究 仅引起部分免疫力。 在被拒绝或延迟的肿瘤中 细胞反应将通过测量T细胞的存在来确定 肿瘤中存在的受体α和βV-区域基因家族 浸润T细胞。 使用一组已经测试过的（在NOD小鼠中）V- TCR链和相应C区的区域基因引物 底漆是由逆转录产生的TCR的cDNA，将是 由PCR放大并克隆。 最主要TCR的序列 在各种肿瘤的影响下存在于肿瘤部位的家庭 将为V-，D-和J分析建立产生的淋巴细胞。 操纵T细胞响应并发现拒绝或抑制T- 细胞克隆，小鼠将用相应的合成肽免疫 到TCR序列。 随后的抗白痴型抗体和细胞 将对其对肿瘤排斥的影响进行评估。 这些研究将通过T细胞的隔离和克隆来补充 针对淋巴因子产生和非生产有限责任公司的特异性。 的作用 细胞毒性，特别是穿孔蛋白在肿瘤排斥中的作用 该模型将通过比较穿孔蛋白的肿瘤反应来研究 不足的转基因小鼠对培养蛋白的小鼠足够的小鼠。 perforin 通过同源性，在胚胎干细胞中已实现缺乏症 重组和穿孔蛋白缺乏嵌合体目前正在研究中。 这些研究将通过重建穿孔素不足来补充 小鼠以及正常的小鼠，带有人培养素转基因。 人类 穿孔蛋白，可通过抗体和北方分析检测到可区分 从小鼠穿孔蛋白中，在鼠孔培养蛋白启动子和 在各种启动子缺失下，旨在增强穿孔素表达 在T细胞子集中。转基因小鼠中的穿孔蛋白表达改善将是 分析了其对肿瘤排斥的影响。