Kupffer cells are abundant liver macrophages that play an important role in liver tolerance, both in terms of suppressing adaptive immunity and in protecting the liver from injury. We previously identified a subset of sessile Kupffer cells, whih are radio-resistant in a bone marrow chimera and long-lived in a transplanted liver, but non-motile (hence the name). However these cells are very difficult to isolate, so little is known abou their functional relationship with bone marrow-derived Kupffer cells. The significance of this exploratory/developmental proposal is that to understand Kupffer cells and their roles in immunity and tolerance, it is critical to understand the sessile Kupffer cells. If they act differetly from bone marrow-derived Kupffer cells, a substantial knowledge gap will result since they are so difficult to isolate, and have not been studied. The innovation of our approach is to use a novel transgenic tool, the RiboTag mouse, in which it is possible to transfer an HA tag to ribosomes in situ using the Cre recombinase. By crossing the RiboTag mouse with a macrophage-specific Cre transgene, we will document Kupffer cell gene expression in situ, avoiding cell isolation and the problem of highly-selective cell recovery. Coupling this approach with our knowledge of the behavior of sessile Kupffer cells in a radiation bone marrow chimera will allow us independently to assay gene expression in sessile, versus bone marrow-derived Kupffer cells. A collaborator with expertise in bioinformatics will allow us to make the best objective use of this information. To document the response of the two subsets of Kupffer cells in tolerance, we will exploit a model of CD8+ T cell- mediated liver injury in which our preliminary data show that Kupffer cells are suppressing liver injury, and in which we already know that the bone marrow-derived cells show an anti-inflammatory pattern of gene expression in the inflamed liver. In this context, we will use the (Cre x RiboTag) mice to determine whether sessile and bone marrow-derived subsets of Kupffer cells respond in the same way to suppress liver damage. The important knowledge gain will be to establish that these two kinds of Kupffer cells have distinct functions in immune tolerance, or alternatively to reveal that they respond alike. If they are different, it will be mandatory to address their special role in more biologicaly complex models, including liver transplantation tolerance and liver infection. We will need to understand sessile Kupffer cell biology in humans also. Furthermore, sessile Kupffer cells may share ontogeny, and possibly biology with Langerhans cells of the skin and with microglia. Insights into sessile Kupffer cells may raise issues for other non-bone marrow-derived tissue-resident macrophages.