Humoral immunization to alloantigens on red blood cells (RBC alloimmunization) remains a major complication, and in some cases barrier to transfusion therapy for myriad diseases. However, the tendency to become alloimmunized is not evenly distributed; rather, patients tend to become alloimmunized to multiple alloantigens (responders) or none at all despite chronic transfusion (nonresponders). However, the factors that regulate responder vs. nonresponder status remain poorly defined. Rates of RBC alloimmunization is increased in patients with autoimmune pathologies, such as systemic lupus erythematosus (SLE). We have recently discovered that mice with SLE-like biology (i.e. develop anti-nuclear antibodies (ANAs)) have an increased rate of RBC alloimmunization. These mice develop ANAs due to a specific cluster of variant genes (NBA2) that contains multiple orthologues to human genes associated with SLE. Importantly, the NBA2 mice do not display organ pathology associated with SLE, leading to the hypothesis that ANA are an isolated component of SLE that affects RBC alloimmunization. Moreover, in NBA2 mice, toll like receptor 7 (TLR7) and TLR9 are known to regulate ANA formation through mutual antagonism (TLR7 promotes and TLR9 suppresses). These findings lead to the central hypothesis of this project: Central Hypothesis: Anti-nuclear antibodies cause increased RBC alloimmunization through activation of B cells as a result of dysregulated TLR7 and TLR9 signaling. This hypothesis is tested using a novel B cell receptor transgenic mouse that recognizes an authentic human blood group antigen (Kpb epitope) on the human KEL glycoprotein. The anti-Kpb mouse is combined with NBA2 mice that are then transfused with transgenic RBCs expressing the KEL-K2 variant of human KEL, which contains the Kpb antigen. This approach allows an in vivo mechanistic study of early activation and differentiation of naïve B cells specific for an RBC antigen on transfused RBCs. Specific aims 1 and 2 test the role of ANAs on RBC alloimmunization, including the manipulation of TLR7 and TLR9 signaling to rigorously test the central hypothesis. Specific aim 3 uses the human cohort of SCD patients studied in project 4. Patients with SCD have high rates of ANAs. The presence or absence of ANAs in patient plasma will be correlated with patterns of alloimmunization. Patient leukocytes will be stimulated in vitro, in the presence or absence of ANA containing plasma and/or TLR7 and TLR9 agonism or antagonism, using an assay that measures B cell activation and differentiation in response to CD4+ follicular T cell help. Synergy between projects comes from crosstalk between purinergic signaling (project 2) and TLR7 and TLR9, that reticulocytes (project 3) provide agonists for TLR7 and TLR9, and the prediction that genetic variations known to predispose humans to ANA formation will emerge as associations in the prospective GWAS study on RBC alloimmunization in patients with SCD (project 4). T...