Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion. Project 3

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Red blood cell (RBC) transfusion is the most common therapeutic modality given to patients in America, with approximately 1 out of every 70 Americans being transfused each year. Although RBC transfusions save lives, some patients develop alloantibodies against donor RBC blood group antigens. Clinically significant alloantibodies have adverse effects in transfusion, pregnancy, and transplant settings. There are very few effective therapeutic interventions to prevent RBC alloimmunization, in general, and/or to eliminate alloantibodies in already sensitized patients. Thus, there is an unmet clinical need to understand the risk factors that can identify (1) which RBC units are immunogenic and (2) which patients are at high risk for alloimmunization; this knowledge will lead to novel strategies to prevent and/or alleviate these adverse events. Alloimmunization risk may be impacted by (1) the donor RBC unit and (2) the transfusion recipient’s immune system. Observationally, higher reticulocyte counts are observed in RBC units from repeat blood donors because compensatory reticulocytosis lasts longer than the interval between donations. Additionally, transfusion recipients at highest risk of alloimmunization also have ongoing reticulocytosis (e.g., patients with hemolytic anemias). Thus, we generated tractable murine models to test the hypothesis that reticulocytes modulate alloimmunization rates. Herein, we provide robust preliminary data that identify reticulocytes as a significant risk factor for RBC alloimmunization at both the donor and recipient levels: higher alloimmunization rates/alloantibody levels are seen upon transfusion of RBC donor units containing high reticulocyte counts as well as upon allogeneic RBC transfusion into recipients exhibiting reticulocytosis. Because reticulocytes differ from mature RBCs (e.g., reticulocytes have mitochondria), we hypothesize that reticulocytes contain and/or express ligands that activate immune cells and initiate alloimmune responses. We use both pre-clinical models and samples from humans to elucidate the mechanisms underlying reticulocyte-mediated enhanced alloimmunization. To that end, we developed 4 murine models to study reticulocytes that reflect human settings (e.g., hemolytic anemia, repetitive phlebotomy) as different types of stress erythropoiesis may result in reticulocyte differences. To elucidate how reticulocytes enhance alloantibodies, we will leverage sophisticated methods to identify, track, and measure immune responses to reticulocytes in an RBC unit and/or upon their induction in a transfusion recipient. To translate our findings to humans, we will also study how reticulocytes modulate the function of B cells collected from patients with reticulocytosis (e.g., hemolytic anemia) as compared to healthy controls. Elucidating how reticulocytes enhance alloimmunization will (1) aid in identifying potentially immunogenic donor RBC units and (2) predict which transfusion recipients are at higher risk of developing alloantibodies; together these will prevent future alloimmunization events. Additionally, this knowledge will provide insights into potential therapeutic targets to prevent and/or treat alloimmunization.