PROJECT SUMMARY: Pulmonary T cells are critical for host protection from influenza A virus (IAV) infections. While current understanding of influenza immunity is focused on conventional MHC-restricted T cells that recognize peptide antigens, unconventional innate-like T cell subsets, such as CD1d-restricted invariant natural killer T (NKT) cells, are emerging as integral components of the respiratory immune system where they play both a protective and immunopathological role in lung disease. This is principally through their production of large amounts of cytokines in barrier organs like the lung where NKT cells preferentially accumulate. Although NKT cell activities are thought to make up a substantial portion of influenza immunity, relatively little is known about their impact on human infections due to a lack of suitable animal models. The current proposal seeks to address this knowledge gap using swine, which offer an excellent translational model to determine the role NKT cells play in shaping human influenza immunity. Using our extensive expertise in porcine NKT cells and the swine influenza challenge model, we propose three aims: Aim 1 will examine the significance of NKT cells as an important host factor contributing to IAV susceptibility. We have already created NKT cell-deficient CD1d knockout (KO) pigs and discovered that they shed less virus than NKT cell-intact pigs. Aim 2 will use the same pig stock to determine how NKT cells shape the pre-existing immunity afforded by inactivated and modified live virus vaccines. Addressing this question is important to inform vaccinologists since NKT cells have previously been found to generate immune responses that could stimulate durable protection against viral infections. Aim 3 will use our CD1d-KO pigs to determine whether NKT cells modulate vaccine associated enhanced respiratory disease (VAERD), which is a dangerous condition caused by the use of inactivated IAV vaccines containing a virus of the same hemagglutinin subtype as the subsequent challenge strain, but with substantial antigenic shift. This induces non-neutralizing IgG Abs that enhances virus uptake into the target cells. These independent but interconnected aims strongly align with the goals of this funding opportunity: to develop novel models that will accurately reflect influenza immunity in humans, including to better mimic pre-existing immunity for vaccine responses and to better understand special high-risk human populations. Our findings should be of considerable value for reducing influenza transmission and improving vaccine safety and efficiency as there are several therapeutic options to modulate the frequency and function of NKT cells.