BITE (Bat Immunology Training and Education) - an undergraduate experiential program for developing the next generation of One Health scientists

Project Summary/Abstract Without T cells (e.g., CD4+ helper, CD8+ cytotoxic), mammals cannot have a proper functioning immune system or fight off viral infection. Classically, T cells in mammals develop in the thymus then migrate to peripheral tissues and circulate in the blood. Development of T cells is critical for mounting antiviral response cytokines, immune memory, regulation, pathogen recognition, activation of B-cells to produce antibodies, and cytotoxic response to virus-infected cells. Bats are able to suppress the most highly pathogenic viruses found in mammals (e.g., SARS, Ebola, Rabies, and Nipah) with little to no clinical symptoms or pathology acting as the main reservoir. Yet after 100 years of anatomical study little is known about T cell development, abundance, expression, or T-cell receptors (TCRs) repertoire within bats. Studies of bat T cell development and responses in bats have largely been understudied due to lack of immunological reagents such as antibodies specific for bats. In this resubmission, our research team has provided preliminary data using x-ray computed tomography, histology, and immunohistochemistry that bats have thymus with T cells present. We also provide evidence of age related thymus involution within adult big brown bats (Eptesicus fuscus). In our proposal, we will use museum voucher specimens and collect wild bats to further develop methods for structural and molecular approaches to study lymphoid organs and T cell development. Our team has decades of experience in mammalogy, viral-host interaction, adaptive immune system sequencing (VDJseq), and viral immunology. We will provide ultrastructural determination of the bat’s thymus, spleen, lymph ducts, and bone marrow that will include development of antibodies for flow cytometry/FACS or mRNA based in situ hybridization flow cytometry (Flow-FISH), single molecule fluorescence mRNA in situ hybridization (mRNA-smFISH) and immunohistochemistry (which we have preliminary data) that will elucidate the lymphoid organs of T cell development (Aim 1). In Aim 2, we will assess T cell development in bat primary and secondary lymphoid organs across developmental age (juveniles, subadults, and adults) and examine sexual dimorphism, using bulk organ RNAseq, single cell RNAseq, and RT-ddPCR. Using VDJseq/TCRseq we will measure the TCR repertoire diversity across lymphoid tissues and blood (Aim 2). In Aim 3, we will access and link thymic involution to the susceptibility of bats to pathogenic viruses. Our proposal and team is uniquely posed to provide a new path towards unraveling the ‘black box’ of bat T cell adaptive immune responses. This is foundational work that will enable further immunological study in bats and other small mammals but provide a groundwork to new immunotherapies, vaccines, antivirals, or anti-inflammatory drugs.