ABSTRACT Invariant natural killer T cells (NKT cells) are a conserved T cell population that behaves like innate cells, rapidly secreting cytokines when stimulated. Different subsets of iNKT cells have been reported, including NKT1, NKT2 and NKT17 cells, similar to TH1, TH2 and TH17 cells. The relationships between the different subsets, their stability, and their functional relevance remain incompletely characterized. We have shown in a mouse model (ET-2) that a small alteration in E protein activity during development results in a dramatic change in the differentiation profile of iNKTs, with a decrease in NKT1s and an increase in other subsets, including a novel type. This model provides a unique opportunity to test the impact of different NKT populations in normal immune responses. We chose to test the possible impact of these changes in NKT cells on the immune response to flu, given the relevance of flu as a public health problem and the fact that activation of NKT cells has been successfully used as a novel adjuvant to increase vaccination efficiency. Preliminary results show that ET-2 mice have better outcomes to influenza challenge, and that this correlates with increased numbers a novel NKT subset in the mediastinal LN. In this proposal we will first characterize functionally and molecularly this novel NKT subset, and then use the information to extend our preliminary studies on influenza responses, testing the impact of these cells on different aspects of the immune response where iNKT cells have been implicated, namely recruitment of inflammatory monocytes, production of IL-22, activation of ILC2 and subsequently recruitment of eosinophils, generation of adaptive CD4 and CD8 T cell responses to viral infections, and decreased epithelial damage in the course of the infection. The comparison of the responses of the iNKT present in WT mice with those in ET-2 mice at these different stages of the immune response will facilitate a mechanistic understanding of the physiological role of NKT cells during influenza infection. The results from these experiments will characterize a novel subset of iNKT cells that could be very relevant for immune responses against flu and other pathogens, and increase our understanding of the physiological role of iNKTs during immune responses to flu. Given the interest in using activation of iNKTs as an adjuvant in flu vaccines or in different cancer therapies, these studies could inform future strategies to selectively activate only those NKT subsets that favor the desired outcome of the treatment.