Project Summary RBC alloimmunization in patients with sickle cell disease (SCD) can lead to potentially fatal hemolytic events. Patients with SCD have the highest incidence of RBC alloimmunization, compared to all other disease populations including those with similar transfusion burdens. Factors explaining this high incidence are poorly understood. Identifying such factors would allow for identification of patients with SCD who may benefit from personalized transfusion protocols and rare antigen-matched units. Inflammation in the transfusion recipient has been shown to promote RBC alloantibody responses. We previously reported that proinflammatory type 1 interferons (IFNα/β) induced by viral infection or autoimmunity promote RBC alloimmunization in pre-clinical transfusion models. Recent studies, including preliminary data included in this application, have revealed that many patients with SCD have an IFNα/β gene signature, defined by leukocyte expression of IFNα/β stimulated genes. However, mechanisms leading to IFNα/β activation, including signaling pathways, receptors, and ligands, are poorly understood. Nearly all IFNα/β-inducing receptors are intracellular receptors for nucleic acids. Thus, phagocytosis of nucleic acid containing cells, including reticulocytes which are elevated in SCD, has the potential to activate IFNα/β pathways. Anti-RBC autoantibodies facilitate erythrophagocytosis, with alloimmunized patients with SCD having an increased incidence of such autoantibodies. While the RBC alloantibody/RBC autoantibody association is poorly understood, our preliminary data indicate that reticulocyte counts correlate with IFNα/β gene scores and erythrophagocytosis of autoantibody-opsonized reticulocytes from patients with SCD induces IFNα/β activation in human macrophages. Preliminary data also show that anti-RBC autoantibodies induce IFNα/β activation and RBC alloimmunization in a pre-clinical transfusion model. In this proposal, we aim to test the hypothesis that RBC autoantibodies promote IFNα/β-mediated RBC alloimmunization in SCD by identifying inflammatory pathways that induce IFNα/β (Aim 1) and determining the effects of autoantibody-induced IFNα/β inflammation on RBC alloimmunization in SCD mice (Aim 2). In Aim 1, IFNα/β pathways induced by autoantibodies will be identified in co-cultures of human macrophages and reticulocytes from patients with and without SCD, and the extension of findings will be tested in pre-clinical murine models. Identified pathways and receptors will inform ligands that induce IFNα/β in SCD. Aim 2 represents the first investigation of IFNα/β-regulated RBC alloimmunization in pre-clinical models of SCD and has the potential to reveal a mechanism that may contribute to the elevated incidence of RBC alloimmunization. Long term goals of this project include improved understanding of proinflammatory interferons in SCD, with the possibility that targeted IFNα/β-modulated therapy may be beneficial. Findings may als...