PROJECT SUMMARY. Broadly reactive humoral immune responses to flu protect against viral variants. Murine data shows that the flu-specific memory B cells (Bmem) in the lung are cross-protective across a number of influenza strains and are functionally distinct from circulating and lymphoid counterparts. As yet we do not know if flu-specific Bmem in the human lung are cross-protective and bridging this knowledge gap is important to design the appropriate vaccine regimens that are universally protective against flu. Flu-specific Bmem in ex vivo lung tissues are rare and this represents a significant technical hurdle in assaying the flu-specific Bmem response in human lung tissues. In order to address this technical hurdle, this application seeks to establish a model system wherein human lung tissue is challenged with influenza virus and maintained viable after this challenge on an advanced cardiopulmonary modality called Ex Vivo Lung Perfusion or EVLP. We anticipate this model will allow us to enumerate and analyze the flu-specific Bmem response at scale in ex vivo human lung tissues. We also anticipate that this model system will allow us to compare binding reactivity to viral variants between flu- specific Bmem located in lung versus mediastinal lymph node tissues to define where cross-protective immunity exists in the respiratory tract.