Project Summary This project addresses a new and emerging area- allospecificity and memory of innate immune cells with a specific focus on macrophages and how such cells impact transplant outcomes. The recent reports that graft loss under broad immunosuppression therapies or after aggressive T cell depletion is dominated by macrophages, plus the strong evidence that macrophages can discriminate self from allogeneic non-self in selected transplant models, place macrophages right under the limelight of graft damage. We believe that induction of transplant tolerance requires comprehensive strategies that target both the innate and adaptive immune cells, and this approach demands a detailed understanding of how innate immune cells respond to alloantigens. We provide the first evidence that macrophages may use a very different mechanism to discriminate self from allogeneic non-self. We showed that macrophages potently rejected allogeneic non-self in an antigen- specific manner. One outstanding feature of our findings is that this allospecificity is induced, and requires stimulation by alloantigens and CD40 engagement. We generated new data that the ligands for allospecific macrophages are donor MHC class I molecules, and macrophages most likely use the paired Ig-like receptors (PIRs) to respond to allogeneic non-self. In this project the central hypothesis is that macrophages use paired Ig-like receptors (PIR) to sense allogeneic cells, and that rejection of target cells requires further acquisition cytolytic M1 features. We put together 3 Aims ito test this hypothesis. Aim 1 addresses the allospecificity PIR-A isoforms and signaling apparatus, and its relationships with PIR-B. Aim 2 examines the molecular pathways leading to the induction of PIR and cytolytic activities in macrophages, investigating whether these two processes are regulated by different mechanisms. Aim 3 determines whether allospecific macrophages could be redirected by modulating either their allospecificity or cytolytic pathways to favor graft survival in vivo. We have all the tools to study the PIR system, including PIR-A/B deficient mice and FcR gamma deficient mice. These models and tools put us in a unique position in resolving the questions proposed in this application. This line of inquiry will lead to major advance in our understanding of innate immune cells in transplant tolerance.