PROJECT SUMMARY/ABSTRACT Influenza virus infections cause significant global morbidity and mortality and pose a serious pandemic risk due to the virus’s propensity for reassortment and mutation. Current influenza vaccines elicit strain-specific responses and are only 10-60% effective depending on the year. There is an urgent need for a universal influenza vaccine that elicits robust, persistent, and broadly cross-reactive B and T cell responses. Designing such a vaccine will require a comprehensive understanding of how features from both the host and the antigen modulate the magnitude, quality, and breadth of the influenza-specific response. Most human influenza studies have been limited to peripheral blood sampling, even though the critical cellular decisions that lead to productive adaptive immune responses occur within lymphoid tissues. Our long-term goal is to define the dynamics of the lymphoid tissue microenvironment, including cell-cell interactions and signaling pathways, that elicit protective immune responses in humans. Our central hypothesis is that immune signatures from mucosal lymphoid tissue are significantly more informative than peripheral blood in developing immunization strategies that elicit robust and broadly cross-reactive influenza responses. To address this question, we propose to leverage a high throughput in vitro organoid platform derived from primary human tonsil tissues. Tonsils are considered both lymphoid and mucosal tissues; they are also accessible from otherwise-healthy patients undergoing tonsillectomy for hypertrophy or obstructive sleep apnea. Participants are demographically diverse and cover the full human age span; males and females are represented at similar proportions. Immune organoids derived from tonsils accurately model human germinal center responses, specific antibody secretion, and T cell activation in response to influenza antigens. They are also able to capture host-mediated inter-individual immune variation related to patient age, sex, and immune history. Furthermore, tonsil organoids can be used to track the kinetics of the adaptive immune response and enable the mechanistic insights needed to rationally design a universal influenza vaccine. The goal of this application is to understand how host features and influenza antigen features contribute to both the magnitude and quality of the influenza immune response in humans. This proposal is supported by strong preliminary data and if successful, will open new areas of investigation for universal influenza vaccine development by identifying correlates and predictors of protection. We will combine comprehensive phenotyping and mechanistic experimental approaches to define the key drivers within human lymphoid tissues that lead to narrow, strain-specific responses. The novelty of this application lies in the systems immunology approach that integrates demographic, serological, phenotypic, functional, and repertoire readouts in a well- controlled imm...