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.

Treating murine inflammatory diseases with an anti-erythrocyte antibody.

Treatment of autoimmune and inflammatory diseases typically involves immune suppression. In an opposite strategy, we show that administration of the highly inflammatory erythrocyte-specific antibody Ter119 into mice remodels the monocyte cellular landscape, leading to resolution of inflammatory disease. Ter119 with intact Fc function was unexpectedly therapeutic in the K/BxN serum transfer model of arthritis. Similarly, it rapidly reversed clinical disease progression in collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis and completely corrected CAIA-induced increase in monocyte Fcγ receptor II/III expression. Ter119 dose-dependently induced plasma chemokines CCL2, CCL5, CXCL9, CXCL10, and CCL11 with corresponding alterations in monocyte percentages in the blood and liver within 24 hours. Ter119 attenuated chemokine production from the synovial fluid and prevented the accumulation of inflammatory cells and complement components in the synovium. Ter119 could also accelerate the resolution of hypothermia and pulmonary edema in an acute lung injury model. We conclude that this inflammatory anti-erythrocyte antibody simultaneously triggers a highly efficient anti-inflammatory effect with broad therapeutic potential.

Erythrocyte Saturation with IgG Is Required for Inducing Antibody-Mediated Immune Suppression and Impacts Both Erythrocyte Clearance and Antigen-Modulation Mechanisms.

Anti-D prevents hemolytic disease of the fetus and newborn, and this mechanism has been referred to as Ab-mediated immune suppression (AMIS). Anti-D, as well as other polyclonal AMIS-inducing Abs, most often induce both epitope masking and erythrocyte clearance mechanisms. We have previously observed that some Abs that successfully induce AMIS effects could be split into those that mediate epitope masking versus those that induce erythrocyte clearance, allowing the ability to analyze these mechanisms separately. In addition, AMIS-inducing activity has recently been shown to induce Ag modulation (Ag loss from the erythrocyte surface). To assess these mechanisms, we immunized mice with transgenic murine RBCs expressing a single Ag protein comprising a recombinant Ag composed of hen egg lysozyme, OVA sequences comprising aa 251-349, and the human Duffy transmembrane protein (HOD-Ag) with serial doses of polyclonal anti-OVA IgG as the AMIS-inducing Ab. The anti-OVA Ab induced AMIS in the absence of apparent epitope masking. AMIS occurred only when the erythrocytes appeared saturated with IgG. This Ab was capable of inducing HOD-RBC clearance, as well as loss of the OVA epitope at doses of Ab that caused AMIS effects. HOD-RBCs also lost reactivity with Abs specific for the hen egg lysozyme and Duffy portions of the Ag consistent with the initiation of Ag modulation and/or trogocytosis mechanisms. These data support the concept that an AMIS-inducing Ab that does not cause epitope masking can induce AMIS effects in a manner consistent with RBC clearance and/or Ag modulation.

Prevention of hemolytic disease of the fetus and newborn: what have we learned from animal models?

PURPOSE OF REVIEW: This review aims to highlight recent advances in our understanding of how anti-red blood cell (RBC) antibodies prevent erythrocyte immunization with an emphasis on new murine models. RECENT FINDINGS: New murine models with clinically relevant human erythrocyte antigens have been used to understand the alloimmunization process and its inhibition. The search to elucidate the mechanism of action of IgG-mediated inhibition of erythrocyte alloimmunization has provided new evidence in support of a potential role for epitope masking, immune deviation and/or antigen modulation in this process. In addition, recent evidence suggests that blends of monoclonal antibodies targeting nonoverlapping epitopes on the RBC surface can improve the efficacy of monoclonal antibodies approaching that of polyclonal IgG. SUMMARY: Animal models with defined alloantigens have helped to identify important mechanistic components that lead to alloimmunization and its inhibition by IgG. A better understanding of the underlying mechanisms leading to hemolytic disease of the fetus and newborn is required to develop the most effective prevention strategies for future patients.

Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes.