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In the recent years, it has been well established that primary respiratory viral infection-induced lung resident memory CD8 T cells (TRM) characterized by the expression of integrins CD49a and CD103, as well as the early-activation marker CD69, constitute the first line of defense against reinfection. On the other hand, viral vector-based respiratory mucosal (RM) vaccination, as well as parenteral vaccination followed by airway luminal manipulation induce lasting and protective lung T cell immunity towards pulmonary tuberculosis (TB). However, it remains poorly understood whether and how these TB vaccination strategies induce TRM in the lung. As such, within this thesis we will investigate generation of lung CD8 TRM upon different TB vaccination strategies and the underlying mechanisms regulating establishment of such cells. Here using distinct models of replication-deficient adenoviral vector-based TB vaccination, we find that RM vaccination leads to generation of lung CD8 TRM identified by the expression of CD69, CD103, and very late activation Ag 1 (VLA-1). These TRM-associated molecules are acquired by CD8 T cells in distinct tissues. In this regard, VLA-1 is acquired during T cell priming in draining mediastinal lymph nodes (dMLNs) and the others acquired after T cells entered the lung. Once in the lung, Ag-specific CD8 TRM continue to express VLA-1 at high levels through the effector/expansion, contraction, and memory phases of T cell responses. We also reveal that VLA-1 is not required for homing of these cells to the lung, but it negatively regulates them in the contraction phase. Furthermore, VLA-1 has a negligible role in the maintenance of such cells in the lung. Separately, we have observed that while parenteral intramuscular vaccination alone does not induce lung CD8 TRM, subsequent RM inoculation of an Ag-dependent, but not a non-specific inflammatory agonist induces lung CD8 TRM. Such generation of lung CD8 TRM needs CD4 T cell help. These findings not only fill the current knowledge gap, but also hold important implications in developing effective vaccination strategies towards mucosal intracellular infectious diseases such as acquired immunodeficiency syndrome (AIDS), TB and herpes virus infection. / Thesis / Doctor of Philosophy (PhD)

There are an estimated 2 billion individuals infected with Mtb, and 37.7 million people living with HIV (PLWH) worldwide. HIV/Mtb co-infection increases the risk of developing active tuberculosis by over 20-fold, and 210,000 of 1.5 million deaths from TB were among co-infected PLWH in 2020. Therefore, development of effective TB vaccination, particularly within the vulnerable PLWH population, is an urgent global issue. With limited in vivo models to study co-infection, humanized NRG (huNRG) mice and humanized DRAG-A2 mice (a next-generation of huNRG mice expressing HLA class I and II transgenes with improved human immune reconstitution, huDRAG-A2) are promising tools for HIV and TB reserach as they develop robust human immune cell populations and recapitulate many aspects of HIV or TB clinical disease. HIV/Mtb co-infection was investigated using huNRG and hu-DRAG-A2 mice in separate experiments where intravaginal (with DMPA pre-treatment) or intraperitoneal HIV-1 infection was administered, respectively, and intranasal infection of Mtb was administered 3.5 weeks later. Both huNRG and huDRAG-A2 mice recapitulated hallmark features of HIV/Mtb co-infection such as severe granuloma pathology, hCD4+ T cell depletion in lung and spleen tissue, and human like lung pathology such as Mtb-infected foamy macrophages in the granuloma. Co-infected huDRAG-A2 mice also displayed significantly higher bacterial burden in the lungs, increased extrapulmonary dissemination into spleen and liver, and significantly lower hCD4+ T cells in the peripheral blood post-Mtb infection when compared to the Mtb-only infected group. To investigate TB vaccine immunogenicity, huNRG and huDRAG-A2 mice were immunized with a novel trivalent vaccine, AdCh68MV. Upon intranasal immunization, both models showed trends of developing higher Mtb antigen-specific hCD4+ T cell responses in the lung and spleen. Overall, this project sets the initial stages of a pre-clinical HIV/Mtb co-infection model in huNRG and huDRAG-A2 mice appropriate for immune investigations, therapeutic and vaccination development. / Thesis / Master of Science in Medical Sciences (MSMS) / There are over 2 billion individuals infected with TB and 37.7 million people living with HIV (PLWH) worldwide. When someone is co-infected with both diseases, the risk of death is greatly increased. Research in co-infection and developing effective TB vaccination for PLWH are urgent global issues. Animal studies are currently limited because studying HIV requires human immune cells. Our lab has established humanized mice (hu-mice) that develop many different human immune cells and are useful for HIV/Mtb co-infection research. When hu-mice were co-infected, they showed more dying lung tissue, immune cell loss, and bacteria in the lungs. Hu-mice were also used to study human immune responses to a novel TB vaccine delivered to the lungs. Trends of higher immune responses towards TB were observed in the lung and spleen of immunized hu-mice. Overall, this project shows the utility of hu-mice as pre-clinical models of HIV/Mtb co-infection and Mtb vaccine studies.