The Third Domain of T cells: the biology of gamma mu T cells in non-eutherian mammals

T细胞是所有脊椎动物的关键免疫细胞类型。T细胞缺乏导致对病原体的易感性和癌症发病率的增加。T细胞有多种类型和亚型。它们的存在和功能可以在远亲物种之间有很大的不同。这个项目研究了一种叫做γμT细胞的新T细胞，这种细胞是在一种有袋动物模型——灰色短尾负鼠身上发现的。γμT细胞存在于有袋动物和鸭嘴兽等单孔动物中。因此，γμT细胞在所有哺乳动物的祖先中都存在，但由于未知的原因，在胎盘哺乳动物（如人类）中丢失了。要了解它们为什么从胎盘哺乳动物中消失，需要了解它们在仍然具有γμT细胞的物种中的功能。本项目研究γμT细胞的发育、分布和基因表达模式，以深入了解其功能。该项目将研究一种新的受体蛋白的功能，称为TCRμ，它定义了γμ T细胞。TCRμ与一种称为纳米体的抗体类型具有结构相似性。纳米体是有用的诊断和治疗工具。如果TCRμ具有纳米体的特性，那么在标准实验动物设施中容易维持的物种中产生这些工具的可能性就会增加。该项目将为从两年制社区学院转入四年制研究型大学的本科生提供研究经验。该项目的博士生将直接与这些本科生一起工作，以获得指导经验并提高自己的职业技能。本项目研究了一种新型T细胞γμ T细胞的发育、分布和表型。所有的研究都将使用一种有袋动物模型——灰短尾负鼠。γμ T细胞的功能尚不清楚，初步结果支持在出生后发育的有限时间内，它们在胸腺中产生，在成年动物中具有有限的组织分布和有限的T细胞受体（TCR）库。本项目将利用逆转录酶PCR和单细胞RNA测序相结合的方法研究：1)γμT细胞发育阶段的时间和进展；2)在成熟过程中和成虫体内的组织分布以及单细胞水平上的组织特异性表型；3)最后，研究γμ tcr的多样性与组织定位之间是否存在关联。此外，本项目将利用噬菌体展示技术研究γμTCR与抗原结合的性质。具体来说，从免疫负鼠脾脏中分离的mRNA将用于利用噬菌体展示载体建立假定的γμTCR抗原结合域的文库。能够结合免疫抗原的克隆将被筛选，并通过测序进一步鉴定具有抗原特异性的克隆。一旦克隆被鉴定出来，这些将被用来鉴定单细胞，将表型与抗原特异性联系起来。预计这些结果将为关于γμ T细胞功能的可验证假设提供基础。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

T cells are a critical immune cell type in all vertebrate animals. T cell deficiencies lead to susceptibility to pathogens and increased cancer rates. There are a variety of T cell types and subtypes. Their presence and function can vary considerably across between distantly related species. This project investigates a new T cell, called the γμT cell, that was discovered in a model marsupial, the gray short-tailed opossum. γμT cells are found in marsupials and monotremes like the duckbill platypus. γμT cells, therefore, are ancient and present in the ancestors of all mammals, but for unknown reasons was lost in the placental mammals such as humans. Understanding why they were lost from the placental mammals requires understanding their function in species that still have γμT cells. This project investigates the development, distribution, and patterns of gene expression of γμT cells to gain insights into their function. This project will investigate the function of a novel receptor protein, called TCRμ, that defines the γμ T cell. TCRμ shares structural similarity to an antibody type called nanobodies. Nanobodies are useful diagnostic and therapeutic tools. If TCRμ has the properties of nanobodies it raises the potential for generating these tools in a species easily maintained in standard laboratory animal facilities. This project will provide research experiences for undergraduates transferring from two-year Community College to a four-year, Research-intensive University. Doctoral students on the project will work directly with these undergraduates to gain mentoring experience and enhance their own career skills. This project investigates the development, distribution, and phenotype of a novel type of T cells, the γμ T cell. All studies will be performed using a model marsupial, the gray short-tailed opossum. γμ T cell function remains unknown and preliminary results support a limited time during postnatal development when they are generated in the thymus, have a limited tissue distribution, and a limited T cell receptor (TCR) repertoire in the adult animal. Using combinations of reverse-transcriptase PCR and single-cell RNA sequencing this project will investigate: 1) the timing and the progression of the γμ T cell developmental stages, 2) the tissue distribution throughout maturation and in the adults along with tissue specific phenotyping at the single cell level, and 3) lastly, investigate if there is an association between the diversity of γμTCR and tissue localization. In addition, this project will use phage display technology to investigate the nature of antigen binding by the γμTCR. Specifically, mRNA isolated from spleens of immunized opossums will be used to create libraries of the putative antigen binding domain of γμTCR using a phage display vector. Clones capable of binding the immunizing antigens will be selected by rounds of panning and characterized further by sequencing to identify the clones that are antigen specific. Once clones are identified these will be used to identify the single cells to associate phenotype with antigen specificity. It is anticipated that these results will provide the basis for testable hypotheses on the function or functions of γμ T cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.