Red Blood Cell Alloimmunization and Hemolytic Disease of the Fetus and Newborn: New Approaches Using Animal Models
Authors: Hendrickson Jeanne E. 
Published: October 2019

Abstract
Women may become alloimmunized to foreign red blood cell (RBC) antigens after exposure during transfusion or pregnancy/delivery. These alloantibodies can be detrimental to developing fetuses/neonates, with six in every 1000 pregnancies being affected by maternal alloantibodies. Despite being described more than 80 years ago, hemolytic disease of the fetus and newborn (HDFN) has few preventive or therapeutic options. Outside of antigen avoidance, the most effective maternal preventive therapy to date is Rh immune globulin. However, it targets just one of the more than 50 antigens implicated in HDFN, and its mechanism of action remains unknown. Furthermore, treatment options for affected fetuses are limited largely to intrauterine transfusions of antigen negative RBCs. With a decrease in HDFN cases due to Rh(D), KEL has emerged as one of the most widely implicated antigens. We now describe a transgenic murine model in which the human KEL antigen is expressed on murine RBCs, with clinical significance in both pregnancy and transfusion settings. Transgenic animals were generated with expression of the human KEL2 antigen (referred to herein as KEL) on their RBCs using a β-globin promoter. KEL expression was evaluated on fetal liver and bone marrow erythroid precursors, mature RBCs, and organs. C57BL/6 or MuMT females were bred with transgenic heterozygous KEL males. Maternal serum was collected during pregnancy and after delivery, with anti-KEL glycoprotein responses measured by flow cytometric crossmatch. Newborn pups were evaluated for KEL positivity, degree of anemia, and presence of maternal anti-KEL. Additionally, the effect of anti-KEL alloantibodies on fetal RBC precursors was evaluated at multiple stages of gestation. Lastly, females alloimmunized through pregnancy were transfused with incompatible KEL RBCs labeled with a lipophilic dye, and females alloimmunized through transfusion were bred with KEL males. Transgenic mice have RBC specific expression of the KEL glycoprotein, with antigen detected on early and late RBC precursors in the fetal liver and bone marrow but not on any tested organ. Fetal/maternal blood exchange was maximally detected after delivery, with the majority of mothers developing anti-KEL glycoprotein antibodies (IgM and all IgG subclasses) within weeks of delivery. All IgG subclasses of anti-KEL crossed the placenta and bound to RBC precursors and mature RBCs in developing KEL positive pups; KEL negative pups had positive indirect antiglobulin tests. Maternal anti-KEL glycoprotein titers increased with antigen exposure, with fewer KEL positive pups born during successive pregnancies to alloimmunized C57BL/6 but not MuMT females. Affected KEL positive pups were anemic, and some were stillborn and hydropic-appearing. As has been described in humans, maternal anti-KEL alloantibodies suppressed the development of fetal KEL RBC precursors at all levels of maturation. Additionally, newborn pups had a relative reticulocytopenia, with reticulocyte recovery within a week after birth. Multiparous alloimmunized females exhibited rapid clearance of transfused incompatible KEL RBCs, and females alloimmunized through KEL RBC transfusion also had pups affected by HDFN. To the best of our knowledge, this is the first animal model of HDFN in which pregnancy stimulates alloantibodies to a paternally derived RBC antigen, with these alloantibodies being clinically significant in both pregnancy and transfusion settings. The anti-KEL glycoprotein alloantibodies generated in this model have similar effects on murine RBCs as anti-KEL1 alloantibodies have on human RBCs. Such effects include suppression of erythropoiesis, fetal anemia, and even hydrops fetalis. This model thus provides a platform to study not only the induction, placental transfer, and consequences of maternal RBC alloantibodies, but also to investigate potential preventive therapies (including “KEL” immune globulin). Long-term translational goals of this work include minimizing the dangers of RBC alloantibodies to developing fetuses and neonates, through the development of targeted maternal immunomodulatory therapies.

Disclosures:
No relevant conflicts of interest to declare.