Project Summary/Abstract Influenza (Flu) infection is a leading cause of respiratory disease and associated death in the world. The annual flu epidemics are estimated to result in 3-5 million cases of severe illness and more than 250,000 deaths. A strong effector immunity is desired for viral clearance, it however can lead to immunopathology if not properly controlled. Severe and fatal influenza diseases are accompanied by an aggressive pro-inflammatory response and an insufficient anti-inflammatory immunity. The production of the immunoregulatory cytokine IL- 10 by flu-specific CD8+ T cells is critical in limiting the lung immunopathology during infection, contributing to host survival and recovery. Knowing how to identify these regulatory CD8+ T cells and understanding how they develop and function are essential for learning how to control flu-induced lung immunopathology. This makes our work highly significant. While much is known about the CD4+ regulatory T cells, the molecular signature and mechanisms that regulate the development and function of IL-10+ regulatory CD8+ T cells remain poorly understood, particularly in the context of influenza-induced airway inflammation. We recently generated an IL- 10GFP/Foxp3RFP dual reporter mouse model that enables detection of IL-10 and Foxp3 in live cells, and undertook a screening for the presence of IL-10+ and/or Foxp3+ T cells under normal and pulmonary inflammatory conditions. We found that Foxp3- IL-10+ CD8+ T cells are present in various organs and disease conditions, and are the major contributors to IL-10 production in the airway during influenza infection. These Foxp3- IL-10+ CD8+ T cells exhibit profound immunoregulatory function against pro-inflammatory innate and adaptive immune responses, indicative of a therapeutic potential. There are however no reliable markers for Foxp3- IL-10+ CD8+ regulatory T cells. We hypothesize that molecular signatures and pathways associated with the development and function of IL-10-producing regulatory CD8+ T cells can be exploited to develop therapeutic strategies against influenza-induced lung immunopathology. Our preliminary studies identified a novel surface signature LAG3hiCD25hiBTLAloCD226lo and the critical transcription factor TCF1 in IL-10+ CD8+ T cells during influenza infection. We propose experiments in two Specific Aims to: (1) determine and validate signature surface markers of IL-10-producing regulatory CD8+ T cells; and (2) determine the role of TCF1 in IL-10-producing regulatory CD8+ T cell development and function. This work is highly innovative as we utilize comprehensive biochemical, genetic and genomics approaches with unique transgenic mouse models, and have exciting preliminary data that can be expanded to provide information of surface markers and critical signaling pathways for a better understanding of the development and immunoregulatory function of IL-10-producing CD8+ T cells during Influenza infection. We expect to unravel poten...