PROJECT SUMMARY/ABSTRACT Influenza is a respiratory virus that infects an estimated one billion people around the world annually. While there is an influenza vaccine, it is typically only 40-60% effective and people must receive it every year. Additionally, the vaccine does not protect against future, and possibly pandemic, strains of influenza and thus there is a major need for new vaccination strategies. CD8+ T cells can provide cross protection against different strains of influenza because they recognize internal epitopes that are conserved. Specifically, due to their position in the lung, CD8+ tissue resident memory T cells (TRM) can rapidly respond to infection and mediate protection by reducing viral loads and immunopathology. In mice, studies have shown that influenza-specific CD8+ lung TRM have limited durability, but it is unknown how long lung TRM survive in humans. Additionally, the mechanisms by which lung TRM cytokines reduce viral loads and immunopathology is unknown in mice, and even less is known about the function of lung TRM in humans. Here, we propose a series of experiments to fill these critical gaps in knowledge. In Aim 1, we will use longitudinal bronchoalveolar lavage samples from lung transplant patients and antibodies specific for mismatched HLA alleles between recipient and donor to track the duration of donor lung TRM and the development of recipient lung TRM by flow cytometry. Additionally, we will determine the dynamics of flu-specific human lung TRM by performing immunophenotyping on longitudinal bronchoalveolar lavage, blood, and nasal lavage samples from lung transplant patients that become infected with influenza. In Aim 2, we will obtain healthy lungs that are unable to be transplanted in order to examine the effector functions of human lung TRM. Lung cells will be stimulated with influenza peptide pools to determine the cytokines produced by influenza- specific lung TRM, and the impact those cytokines have on neighboring cells in the lung tissue. Additionally, we will then use mice to mechanistically determine the effect of individual lung TRM cytokines to reduce viral loads and activate neighboring innate cells. Through this proposed work we hope to better understand the role that human lung TRM play in the immune response to influenza. Lastly, as lung TRM are generated in response to respiratory viruses, and not just influenza, results of this study could provide insights into the immune system that could improve vaccine design for all respiratory viruses and aid in the prevention of future global pandemics.