Trogocytosis drives red blood cell antigen loss in association with antibody-mediated immune suppression.

ABSTRACT: Red blood cell (RBC) alloimmunization to paternal antigens during pregnancy can cause hemolytic disease of the fetus and newborn (HDFN). This severe and potentially fatal neonatal disorder can be prevented by the administration of polyclonal anti-D through a mechanism referred to as antibody-mediated immune suppression (AMIS). Although anti-D prophylaxis effectively prevents HDFN, a lack of mechanistic clarity has hampered its replacement with recombinant agents. The major theories behind AMIS induction in the hematologic literature have classically centered around RBC clearance; however, antigen modulation/loss has recently been proposed as a potential mechanism of AMIS. To explore the primary mechanisms of AMIS, we studied the ability of 11 different antibodies to induce AMIS, RBC clearance, antigen loss, and RBC membrane loss in the HOD (hen egg lysozyme-ovalbumin-human Duffy) murine model. Antibodies targeting different portions of the HOD molecule could induce AMIS independent of their ability to clear RBCs; however, all antibodies capable of inducing a strong AMIS effect also caused significant in vivo loss of the HOD antigen in conjunction with RBC membrane loss. In vitro studies of AMIS-inducing antibodies demonstrated simultaneous RBC antigen and membrane loss, which was mediated by macrophages. Confocal live-cell microscopy revealed that AMIS-inducing antibodies triggered RBC membrane transfer to macrophages, consistent with trogocytosis. Furthermore, anti-D itself can induce trogocytosis even at low concentrations, when phagocytosis is minimal or absent. In view of these findings, we propose trogocytosis as a mechanism of AMIS induction.

Antigen copy number and antibody dose can determine the outcome of erythrocyte alloimmunization inducing either antibody-mediated immune suppression or enhancement in a murine model.

BACKGROUND: The administration of anti-D for the prevention of hemolytic disease of the fetus and newborn is one of the most successful clinical uses of the phenomenon of antibody-mediated immune suppression (AMIS). However, despite adequate prophylaxis, failures can still occur in the clinic and are poorly understood. Recently, the copy number of red blood cell (RBC) antigens has been shown to influence immunogenicity in the context of RBC alloimmunization; however, its influence on AMIS remains unexplored. STUDY DESIGN AND METHODS: RBCs expressing approximately 3,600 and approximately 12,400 copy numbers of surface-bound hen egg lysozyme (HEL), named respectively HELmed -RBCs and HELhi -RBCs, and selected doses of a polyclonal HEL-specific IgG were transfused into mice. Recipient HEL-specific IgM, IgG, and IgG subclass responses were evaluated by ELISA. RESULTS: Antigen copy number affected the antibody dose required for AMIS induction with higher antigen copy numbers requiring larger doses of antibody. For instance, 5 µg of antibody caused AMIS for HELmed -RBCs but not HELhi -RBCs, while 20 µg induced significant suppression for both HEL-RBCs. Overall, increasing amounts of the AMIS-inducing antibody were associated with a more complete AMIS effect. In contrast, the lowest tested doses of the AMIS-inducing IgG led to evidence of enhancement at the IgM and IgG levels. DISCUSSION: The results demonstrate that the relationship between antigen copy number and antibody dose can influence the outcome of AMIS. Further, this work suggests that the same antibody preparation can induce both AMIS and enhancement but that the outcome may depend on the quantitative interrelationship of antigen-antibody binding.

Antigen-specific IgG subclass composition in recipient mice can indicate the degree of red blood cell alloimmunization as well as discern between primary and secondary immunization.

BACKGROUND: Despite the vast antigen disparity between donor and recipient red blood cells (RBCs), only 2%-6% of transfusion patients mount an alloantibody response. Recently, RBC antigen density has been proposed as one of the factors that can influence alloimmunization, however, there has been no characterization of the role of antigen density along with RBC dose in primary and secondary immunization. STUDY DESIGN AND METHODS: To generate RBCs that express distinct antigen copy numbers, different quantities of hen egg lysozyme (HEL) were coupled to murine RBCs. The HEL-RBCs were subsequently transfused into recipient mice at different RBC doses and their HEL-specific IgM, IgG, and IgG subclass response was evaluated. RESULTS: Productive immune responses could be generated through a high copy number antigen transfused at low RBC doses or a low copy number transfused at high RBC doses. Further, primary but submaximal humoral immunization predominantly induced the IgG2b and IgG3 subclasses. In contrast, a maximal primary immunization or a secondary immunization induced all four IgG subclasses. DISCUSSION: Our results confirm the existence of an antigen threshold for productive immune responses but indicate that a high antigen copy number alone might not be enough to induce a response, but rather a combination of both antigen copy number and cell dosage may determine the outcome of immunization. Further, this study provides a proof of concept that the IgG subclass composition can be an indicator of the level of RBC alloimmunization as well as discern between primary and secondary immunization at least in this murine model.

The use of IVIg in fetal and neonatal alloimmune thrombocytopenia- Principles and mechanisms.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare neonatal disorder that is caused by alloimmunization against platelet antigens during pregnancy. Although rare, affecting only 1 in 1000 live births, it can cause intracranial hemorrhage and other bleeding complications that can lead to miscarriage, stillbirth and life-long neurological complications. One of the gold-standard therapies for at risk pregnancies is the administration of IVIg. Although IVIg has been used in a variety of different disorders for over 40 years, its exact mechanism of action is still unknown. In FNAIT, the majority of its therapeutic effect is thought the be mediated through the neonatal Fc receptor, however other mechanisms cannot be excluded. Due to safety, supply and other concerns that are associated with IVIg use, alternative therapies that could replace IVIg are additionally being investigated. This includes the possibility of a prophylaxis regimen for FNAIT, similarly to what has been successfully used in hemolytic disease of the fetus and newborn for over 50 years.