PROJECT SUMMARY / ABSTRACT Influenza infection imposes a substantial burden on global health, with several million infections and hundreds of thousands of deaths annually. Current influenza vaccination strategies offer limited seasonal protection due to changing influenza virus epitopes and the appearance of novel and/or pandemic potential strains that may thwart pre-existing influenza immunity mediated by antibodies. CD8+ tissue-resident memory T cells (TRM) afford optimal protection against heterologous influenza strains, as these cells target highly conserved influenza epitopes that remain relatively unchanged year-to-year. CD8+ TRM reside in the upper respiratory tract (URT), comprised of the nasal cavity, larynx, and pharynx, and the lower respiratory tract (LRT), containing the airways and lungs. URT CD8+ TRM are critical for limiting viral replication and preventing severe LRT infection, which leads to significant morbidity and mortality. The requirements for development of CD8+ TRM in the LRT are well understood. Prior work shows that local antigen recognition and specific signals in the lung microenvironment are required for LRT CD8+ TRM formation. However, the signals that drive URT CD8+ TRM development, particularly in the absence of local antigen, have not been extensively characterized. This proposal aims to identify the requirements for URT CD8+ TRM development in the presence or absence of local antigen (ie, after parenteral vaccination). Preliminary data shows that a mouse model of intranasal (i.n.) versus intraperitoneal (i.p.) influenza infection induces URT CD8+ TRM with and without local antigen, respectively. Further, initial studies demonstrate that URT CD8+ TRM cannot form in the absence of local antigen and TGF-b signaling. We will leverage this i.n. versus i.p. infection system to investigate the role that TGF-b, IL-15 and IL-33 cytokine signaling play in URT CD8+ TRM formation. Initial in-vivo mouse studies demonstrate that LRT CD8+ TRM induced in the presence of local antigen decline over time, while URT CD8+ TRM remain stable up to 6 months after i.n. and i.p. priming. We will utilize a tamoxifen-inducible CD103 Cre system and parabiosis model to investigate mechanisms of URT CD8+ TRM maintenance over time with and without local antigen. We will additionally employ an adoptive transfer model to assess URT CD8+ TRM localization and maintenance in the absence or presence of local antigen via immunofluorescent microscopy. This work will define signals required for optimal formation and maintenance URT CD8+ TRM, which will offer critical insights to improve vaccination strategies for preventing severe influenza disease and inducing protective immunity against other respiratory viruses.