PROJECT SUMMARY/ABSTRACT Blood transfusions are life-saving for many patients with blood loss or blood production defects, but there are complications that limit their effectiveness and/or lead to medical sequelae. One such sequela is the development of acute lung injury, or transfusion-related acute lung injury (TRALI). TRALI has long been the number one cause of transfusion-related death and a major contributor to in-hospital morbidity. The pathogenesis of TRALI is incompletely understood, but there is consensus that TRALI is a classic example of antibody-mediated disease with a prominent role of HLA/MHC antibodies present in the donor blood product inciting lung injury in susceptible recipients. Indeed, we have developed a mouse model of TRALI based on transfusion of MHC Class I antibody into mice expressing the cognate MHCI antigen. In this application, we will use this model as a platform to investigate three specific aims. In Aim 1, we will determine the critical site of cognate antigen expression required to initiate TRALI by testing mice that are engineered to be deficient or sufficient in antigen expression in specific cellular locations. Novel mouse tools will be used to selectively delete Class I expression (B2mfl/fl) or selectively express allo-Class I protein (Con-Kd) on the endothelium or on specific immune cells. Using these complimentary mouse tools, we will definitively address the critical site of antigen expression required to initiate TRALI. In Aim 2, we will turn our attention to the biochemical characteristics of antibodies associated with the induction of TRALI. A single mouse monoclonal antibody against Class I (34-1- 2S) has been described to produce TRALI, whereas many other antibodies against the same antigen do not cause TRALI; it is unclear why this antibody is unique in its pathogenicity. We will test the hypotheses that antibody affinity, IgG subtype, glycosylation, and cross-reactivity each influence the induction of TRALI. These studies will utilize cutting-edge antibody engineering and binding and affinity assays to determine the properties of antibodies that impart pathogenicity in TRALI. In Aim 3, we will focus on the downstream events resulting from antibody engagement and recognition by immune cells. FcγRs are immune receptors that are the proximal trigger of antibody-mediated immune responses. Using a new mouse model that allows for the conditional deletion of activating FcγRs (Con-Fcγ-KO), we will determine the critical cells responsible for antibody recognition in TRALI. We will further determine if an inhibitory FcγR (FcγRII) influences TRALI severity. Finally, we will humanize the 34-1-2S antibody and infuse it into mice engineered to express human FcγRs in the absence of murine FcγRs. 34-1-2S will be expressed as each of the different human IgG subtypes (IgG1-IgG4) testing the hypothesis that IgG subtype affects TRALI in humans. The results of these experiments will definitively map the cells, antibody ...