Project Summary Influenza remains a continual threat to health of many Americans, resulting in 9.2-35.6 million illnesses, 140,000-710,000 hospitalizations, and 12,000-56,000 deaths annually since 2010 (CDC), as well as the ever-present likelihood of a devastating pandemic that could kill millions. Recent work has also suggested that a universal flu vaccine may be possible, although there is considerable uncertainty about how to achieve this. This renewal application seeks to leverage some of the important methodologies and data that we have developed in this past granting period to understand at a much deeper level what constitutes broad and effective B and T cell responses against influenza so as to better inform next generation vaccine efforts. Specifically, we have developed a unique tonsil organoid system that we can expose to a flu vaccine and produce high affinity antibodies several days to a week later. This gives us an ability to manipulate and test vaccine constructs and adjuvants in a fully human system in order to find the best way to trigger broadly neutralizing antibodies. In addition, we have developed powerful new T and B cell repertoire analysis methods that will allow us to productively analyze large flu- specific TCR and Ig data sets to identify which specificities or other correlates contribute the most to protection or amelioration of diseases in challenge studies. We also plan to further analyze influenza vaccine responses in pregnant women, the elderly and twins, to test various hypotheses that we have developed in our previous work in the CCHI. Lastly we will take advantage of recent advances by the Nolan lab in imaging tissue sections with large numbers of different antibodies to analyze the cellular organization the tonsil organoids with time during a flu vaccine response and after different interventions.