Project Summary Influenza infections cause substantial morbidity and mortality worldwide. While vaccination is considered to provide protection, the antibody response induced by current seasonal vaccines is generally short-lived. In contrast, natural influenza infection induces durable protective immunity against future influenza virus infections. The immune mechanisms and pathways that are linked with durable protection by natural infection remain unclear particularly in humans. Studies in animal models show that influenza infection, but not immunization with inactivated virus, generates influenza-specific lung tissue-resident memory T (TRM) cells. TRM are retained for a long time locally, and there is evidence that influenza-specific TRM are far more efficient than their splenic counterpart for protection from influenza infection. Infection with influenza virus also induces lung-resident memory B (BRM) cells that rapidly produce specific Abs in the lung following infection challenge. Whereas these observations suggest a key role of influenza-specific lung BRM at the frontline upon re-infection, the mechanism by which lung BRM differentiate into Ab-producing cells remains unknown. We hypothesize that a subset(s) of influenza-specific lung CD4 TRM display properties similar to T follicular helper (Tfh) cells, and that this subset(s) interact with influenza-specific memory B cells in human lung for rapid local Ab response. In this proposal, we will leverage our extensive expertise in human CD4+ T cell subsets to define a ?Tfh-like? subset within human lung CD4 TRM, and determine their mode of action on lung BRM. Aim 1 will define the phenotype of ?Tfh-like? CD4 TRM in human lung. By using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we will comprehensively analyze the transcriptional heterogeneity among human lung CD4+ T cells differentially expressing surface markers, and aim to identify the cell population enriched with the Tfh-gene signature. We will also assess the phenotype of influenza-specific CD4 TRM in human lung by using the IRF4 assay that we have recently established. Aim 2 will comprehensively define the subsets of influenza HA-specific B cells in human lung. By co-culturing ?Tfh-like? CD4 TRM with lung BRM, we will analyze whether the T cells induce B cell differentiation into influenza Ab-producing cells. We will further determine the helper mechanism by blocking several candidate molecules. As such, our study aims to identify the major T and B cell subsets in human lung responsible for rapid local Ab response. We anticipate that our study will increase our knowledge in the local immune protection mechanism of human lung and provide significant insight into the design of efficient influenza vaccines.