Project summary: Development of long-lived memory CD8+ T cells is paramount for the control of viral infections, such as influenza. The complete identification of extracellular factors promoting memory CD8+ T cells will favor the better design of CD8+ T cell-inducing immunizations and antiviral therapies. Notably, we have found that one of these factors, extracellular ATP (eATP) – via its receptor P2RX7 - promotes the generation and long-term survival of virus-specific memory CD8+ T cells, both in the circulation (central memory cells – TCM) and in barrier tissues (resident memory cells – TRM) in response to systemic viral infection. These findings highlight an unanticipated role for the “danger signal” eATP for the promotion of antiviral immunity. However, there still are gaps on the understanding of how P2RX7 promotes memory CD8+ T cell long-term survival. This is especially true for the TRM pool, which is relevant both in the context of both systemic and tissue-localized infections. In Aim 1, we will build up on preliminary studies suggesting that P2RX7 upregulation in a subset of memory precursors favor the development of long-lived TRM cells. We are especially interested in understanding how preferential upregulation of P2RX7 at such an early stage of the immune response affects TRM cell survival long after this time window. In Aim 2, we propose to address how P2RX7 expression promotes TRM cell longevity in the context of influenza infection. Our preliminary data suggests that, despite initial seeding of the lung is mostly independent of P2RX7 (and expression of P2RX7 itself is low), influenza-specific lung TRM cells that survive the numerical decay typical of this infection express high levels of P2RX7, and need this receptor to maintain. We will perform experiments to understand (a) the origin of P2RX7-expressing long-lived flu-specific lung TRM cells, and (b) how manipulating P2RX7 expression and its potential upstream regulation promote better survival of lung TRM cells and their protective ability against secondary influenza challenge. Overall, we will test the hypothesis that, in response to multiple viral infections, expression of the eATP sensor P2RX7 selects memory CD8+ T cells for long-term survival in barrier tissues. These studies will establish how dynamic regulation of this eATP sensor promotes the longevity of memory CD8+ T cells in distinct infection and inflammatory contexts. They will also serve as a proof-of-concept for potential future experimental setups aiming to boost the protective ability of memory CD8+ T cells to influenza, a disease of high medical relevance.