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

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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.