PROJECT SUMMARY/ABSTRACT – PROJECT 3 Tissue resident memory T cells (TRM) are non-recirculating CD8+ T cells that become established in peripheral tissues after an infection. Upon re-encounter with the same or related pathogen(s), these memory T cells rapidly reactivate and provide immediate effector function that can mean the difference between life and death in a lethal challenge model. They tend to be specific for conserved antigens, and in the case of influenza, could be part of the solution to achieving more broadly cross-reactive and universal vaccine. Understanding how they are regulated, how they mediate optimal protection, and how they are established and maintained are critical goals of this project. Besides the markers used to identify the T cell types (CD3, CD8, CD44, CD62L, CD69), several other cell surface markers are used to identify memory T cell subsets in the tissues. CD49a, when paired to integrin beta-1 to form VLA-1, is a receptor for collagen in the extracellular matrix and is the prototypic TRM marker first used to define these cells in the tissue by us in 2004. CD103, when paired with beta-7 integrin, binds to E-cadherin expressed in the junctions between epithelial cells. These “markers' of TRM are critical to their establishment and function, yet little has been done to determine the in vivo functions of CD49a and CD103. We propose to test hypotheses related to how each of these adhesion molecules acts to position memory T cells in different anatomical locations, regulate communication with the epithelium, are associated with genetic programming linked to functional differentiation, thereby regulating CD8+ T cell motility, survival, and optimal immune protection. The Specific Aims are: Aim 1: Determine the mechanisms that determine differentiation, establishment, and maintenance of TRM subsets after influenza infection. Aim 2: Investigate mechanisms of T cell-epithelial cell-matrix interactions required for motility and positioning in the airways. Aim 3: Determine the functions of CD49a and CD103 in optimizing immune protection. To achieve these aims, we use combinations of genomics, flow cytometry, in vitro and in vivo models to study the differentiation and function of these cells. A core technology employed is intravital multiphoton microscopy (IV-MPM) applied to a live animal model of influenza tracheitis that our lab developed. We extend this approach to an innovative in vitro system using primary airway organoid cultures incorporating influenza infection and virus-specific CD8 T cells to study fine aspects of the mechanisms regulating cell motility and T- cell/epithelial cell interactions. The results of our studies will lead to novel ways to optimize immune protection from influenza and reduce the burden of disease.