Deletion of inhibitory genes in post-thymic T cells to study immune tolerance

描述(申请人提供)：使用Cre/lox系统有条件地删除目标基因，为了解特定基因产物在体内选定细胞谱系中的功能作用提供了宝贵的工具。在T细胞生物学研究中，携带靶向等位基因的小鼠通常与由Lck、CD2或CD4启动子驱动的表达Cre的转基因小鼠杂交。在T细胞谱系中，使用这样的系统已经建立了特定信号分子的明确的功能角色。然而，由于这些启动子在胸腺细胞的发育过程中是活跃的，所以最引人注目的表型已经在该隔室中获得。此外，由于这种突变小鼠的胸腺选择经常受到干扰，对外周T细胞的研究结果可能很难解释，甚至不可能解释。利用T细胞系中的柯萨奇和腺病毒受体(CAR)转基因小鼠，我们最近开发了一种使用Cre腺病毒有条件地删除胸腺后T细胞中基因的策略。这一工具现在使我们能够第一次探索在外周T细胞中直接删除特定的感兴趣基因。我们建议应用这项技术来更好地了解脂质磷酸酶PTEN在T细胞生物学中的作用。具有Lck-Cre背景的条件性PTEN缺陷小鼠发生自身免疫和T细胞淋巴瘤。我们推测，这些表型可能是胸腺中PTEN缺失的结果，当胸腺后T细胞中PTEN缺失时，这些表型可能不会发生，在这种情况下，仍然可以看到T细胞免疫力的增强。在第一个特定目标中，将分析对照和PTEN缺失的T细胞在IL-2的产生、增殖和存活方面的增强作用。对分化为效应细胞亚群的影响，以及下游信号事件的调节，也将被调查。在第二个特定目标中，对照和PTEN缺失的T细胞将在体内转移，以研究自身免疫和淋巴瘤发生的可能性。还将检查是否发生了对疫苗接种或针对同基因肿瘤的增强免疫反应。最终，这些实验将允许评估PTEN作为未来潜在的免疫增强干预的药理学靶向是否有进一步发展的兴趣。公共卫生相关性将探索直接在外周T细胞中基因删除负调控分子PTEN的策略。PTEN缺失的T细胞是否能在不引起自身免疫或导致淋巴瘤的情况下，对疫苗或肿瘤产生更好的免疫反应，将进行测试。如果成功，该项目的结果将支持抑制PTEN功能的药物的开发，最终目的是改善宿主对癌症或慢性感染的免疫反应。

DESCRIPTION (provided by applicant): The conditional deletion of targeted genes using the Cre/lox system has provided an invaluable tool for understanding the functional role of specific gene products in selected cell lineages in vivo. For studies of T cell biology, mice bearing targeted alleles are typically intercrossed with transgenic mice expressing Cre driven by the Lck, CD2, or CD4 promoters. Clear functional roles of specific signaling molecules have been established using such systems in the T cell lineage. However, as these promoters are active in developing thymocytes, the most striking phenotypes have been obtained in that compartment. In addition, because thymic selection often becomes perturbed in such mutant mice, the results of studies in peripheral T cells can be difficult, or even impossible, to interpret. Using mice transgenic for the Coxsackie and adenovirus receptor (CAR) in the T cell lineage, we recently have developed a strategy for conditional deletion of genes in post- thymic T cells using a Cre adenovirus. This tool has now allowed us to explore for the first time the deletion of specific genes of interest directly in peripheral T cells. We propose to apply this technique to gain a better understanding of the role of the lipid phosphatase PTEN in T cell biology. Conditional PTEN-deficient mice on an Lck-Cre background develop autoimmunity and T cell lymphoma. We hypothesize that these phenotypes may be a result of PTEN deletion in the thymus and might not occur when PTEN is deleted in post-thymic T cells, in which case heightened T cell immunity may nonetheless be seen. In the first Specific Aim, control and PTEN-deleted T cells will be analyzed for potentiation in IL-2 production, proliferation, and survival. Effects on differentiation into effector cell subsets, and regulation of downstream signaling events, also will be investigated. In the second Specific Aim, control and PTEN-deleted T cells will be transferred in vivo to investigate the potential for autoimmunity and lymphoma development. Whether augmented immune responses to vaccination or against syngeneic tumors occurs also will be examined. Ultimately, these experiments will allow assessment of whether pharmacologic targeting of PTEN as a future potential immunopotentiating intervention is of interest for further development. PUBLIC HEALTH RELEVANCE A strategy will be explored for genetic deletion of the negative regulatory molecule PTEN directly in peripheral T cells. Whether PTEN-deleted T cells generate improved immune responses to vaccines or against tumors, without causing autoimmunity or leading to lymphoma, will be tested. If successful, the results of this project will support development of drugs to inhibit PTEN function with the ultimate aim of improving host immune responses against cancers or chronic infections.