A novel genetic tool to investigate T cell development and function

一种研究 T 细胞发育和功能的新型遗传工具

• 批准号: 9808399
• 负责人: Yongqiang Feng
• 金额: $ 26.93万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-22 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808399
• 关键词: Acute; Adaptive Immune System; Address; Adult; Animals; Area; Autoantigens; Autoimmune Diseases; B cell differentiation; B-Lymphocytes; Biochemical; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell physiology; Cells; Code; Diphtheria Toxin; Disease model; Dose; Elderly; Elements; Engineering; Expression Profiling; Frequencies; Gene Expression; Genetic; Genetic Transcription; Goals; Immune; Immune System Diseases; Immune Tolerance; Immune response; Immunologics; Immunosuppressive Agents; Investigation; Knock-in; Knowledge; Label; Lead; Life; Lymphoid; Malignant Neoplasms; Mediating; Methods; Monitor; Mouse Strains; Multiple Sclerosis; Mus; Nature; Neonatal; Organ; Peripheral; Play; Process; Proteins; Rag1 Mouse; Regulator Genes; Regulatory T-Lymphocyte; Reporter; Reporting; Research; Role; Specific qualifier value; System; T-Cell Development; T-Lymphocyte; Tamoxifen; Technology; Testing; Therapeutic; Time; adaptive immunity; differentiated B cell; diphtheria toxin receptor; dosage; experimental study; genetic element; immune activation; immune function; improved; in vivo; insight; loss of function; mouse model; novel; pathogen; postnatal development; prevent; programs; recombinase; therapeutic target; tool

ABSTRACT The adaptive immune system provides remarkable defense against fast-evolving pathogens. In the meantime, tolerance mechanisms coevolved to prevent deleterious immune responses, among which regulatory T (Treg) cells play a dominant, active role. The adaptive immune system is established during postnatal development through continuous differentiation and egress of T and B cells into the peripheral lymphoid and non-lymphoid organs. It appears that the cells of the adaptive immune system that emerge at the early and late stages of life are programmed differently in their immunological function. Which factors are present at particular stages of life to program the function of immune cells differently is an unsolved mystery. How T and B cells developed at different stages of life respond to specific immunological challenges also remains to be fully explored. Revealing these features of the adaptive immune system will offer us better ways to manipulate immune cells for the treatment of immunological diseases. Genetic tracing tools are needed to address these important questions. However, despite significant progress in the field, tracing and investigating T and B cells emerging at given stages of life remain a technical challenge. To solve this issue, we propose a robust, versatile method to trace T and B cells during their early differentiation in experimental mice. In our preliminary experiments, we have successfully engineered this mouse strain. Specifically, we took advantage of transient Rag1 expression during early T and B cell differentiation and used it to drive the expression of three proteins to trace T and B cells with tamoxifen, to report Rag1 expression with fluorescent protein GFP, and to deplete Rag1-expressing cells with diphtheria toxin when necessary. We have validated all these genetic elements and their functions in the mice. In our proposed research, we will improve our methods to meet various experimental requirements. Furthermore, we will use Treg cells as a proof of concept of our technology in revealing the differential immunosuppressive activity and gene regulatory networks of Treg cells developed at the early and later stages of life in mouse models of autoimmune diseases. In summary, our study generates a robust, versatile genetic toolbox in experimental mice to report, trace, and acutely deplete developing T or B cells when necessary at given stages of life, offering a powerful approach to address many significant questions about adaptive immunity. Our research also produces insights into different Treg functions specified during postnatal development and reveals novel factors and mechanisms controlling Treg-mediated immune tolerance.

抽象的 适应性免疫系统可针对快速进化的病原体提供卓越的防御能力。同时， 耐受机制共同进化以防止有害的免疫反应，其中调节性 T (Treg) 细胞发挥着主导、积极的作用。适应性免疫系统是在出生后发育过程中建立的 通过 T 细胞和 B 细胞的持续分化并进入外周淋巴和非淋巴 器官。似乎在生命早期和晚期出现的适应性免疫系统细胞 其免疫功能的编程不同。哪些因素存在于生命的特定阶段 对免疫细胞的功能进行不同的编程是一个未解之谜。 T 细胞和 B 细胞如何发育 生命的不同阶段对特定免疫挑战的反应也有待充分探索。揭示 适应性免疫系统的这些特征将为我们提供更好的方法来操纵免疫细胞 免疫性疾病的治疗。 需要基因追踪工具来解决这些重要问题。然而，尽管在这方面取得了重大进展 在该领域，追踪和研究在生命特定阶段出现的 T 细胞和 B 细胞仍然是一个技术挑战。 为了解决这个问题，我们提出了一种稳健、通用的方法来追踪 T 细胞和 B 细胞的早期分化过程 在实验小鼠中。在我们的初步实验中，我们成功地改造了这种小鼠品系。 具体来说，我们利用了早期 T 细胞和 B 细胞分化期间的瞬时 Rag1 表达，并使用 它驱动三种蛋白质的表达，用他莫昔芬追踪 T 和 B 细胞，用它报告 Rag1 表达 荧光蛋白 GFP，并在必要时用白喉毒素消除表达 Rag1 的细胞。我们有 在小鼠中验证了所有这些遗传元件及其功能。在我们提出的研究中，我们将改进 我们的方法可以满足各种实验要求。此外，我们将使用Treg细胞作为证据 我们的技术概念揭示了差异免疫抑制活性和基因调控网络 自身免疫性疾病小鼠模型中生命早期和晚期发育的 Treg 细胞。 总之，我们的研究在实验小鼠中生成了一个强大的、多功能的遗传工具箱，用于报告、追踪和 在生命的特定阶段，必要时急剧消耗正在发育的 T 或 B 细胞，从而提供了一种强大的方法 解决了有关适应性免疫的许多重要问题。我们的研究还对不同的领域产生了见解 Treg 在出生后发育期间发挥特定功能，并揭示了控制的新因素和机制 Treg 介导的免疫耐受。