Antagonism of the Platelet-Activating Factor Pathway Mitigates Inflammatory Adverse Events Driven by Anti-erythrocyte Antibody Therapy in Mice.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet counts primarily due to antiplatelet autoantibodies. Anti-D is a donor-derived polyclonal Ab against the rhesus D Ag on erythrocytes used to treat ITP. Unfortunately, adverse inflammatory/hypersensitivity reactions and a Food and Drug Administration-issued black box warning have limited its clinical use.

Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP.

Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions.

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

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.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis.

B cells and antibodies in refractory immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder mediated by pathogenic autoantibodies secreted by plasma cells (PCs) in many patients. In refractory ITP patients, the persistence of splenic and bone marrow autoreactive long-lived PCs (LLPCs) may explain primary failure of rituximab and splenectomy respectively. The reactivation of autoreactive memory B cells generating new autoreactive PCs contributes to relapses after initial response to rituximab.

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.

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.

THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets.

Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA.

Anti-inflammatory activity of CD44 antibodies in murine immune thrombocytopenia is mediated by Fcγ receptor inhibition.

Monoclonal immunoglobulin G (IgG) antibodies to CD44 (anti-CD44) are anti-inflammatory in numerous murine autoimmune models, but the mechanisms are poorly understood. Anti-CD44 anti-inflammatory activity shows complete therapeutic concordance with IV immunoglobulin (IVIg) in treating autoimmune disease models, making anti-CD44 a potential IVIg alternative. In murine immune thrombocytopenia (ITP), there is no mechanistic explanation for anti-CD44 activity, although anti-CD44 ameliorates disease similarly to IVIg.

New insights into IVIg mechanisms and alternatives in autoimmune and inflammatory diseases.

Purpose Of Review: Intravenous immunoglobulin (IVIg) is an effective treatment for an increasing number of autoimmune and inflammatory conditions. However, IVIg continues to be limited by problems of potential shortages and cost. A number of mechanisms have been described for IVIg, which have been captured in newly emergent IVIg mimetic and IVIg alternative therapies.

Inhibition of platelet phagocytosis as an in vitro predictor for therapeutic potential of RBC antibodies in murine ITP.

Polyclonal anti-D is a first-line therapy for immune thrombocytopenia (ITP). Monoclonal antibodies are desirable alternatives, but none have yet proven successful despite their ability to opsonize erythrocytes (or red blood cells, RBCs) and cause anemia. Here, we examined 12 murine erythrocyte-specific antibodies of different specificity and subtypes and found that 8 of these antibodies could induce anemia in antigen-positive mice.

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.

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.

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia.

GPIbα is required for platelet-mediated hepatic thrombopoietin generation.

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients.

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.

Red blood cell antibody-induced anemia causes differential degrees of tissue hypoxia in kidney and brain.

Moderate anemia is associated with increased mortality and morbidity, including acute kidney injury (AKI), in surgical patients. A red blood cell (RBC)-specific antibody model was utilized to determine whether moderate subacute anemia could result in tissue hypoxia as a potential mechanism of injury. Cardiovascular and hypoxic cellular responses were measured in transgenic mice capable of expressing hypoxia-inducible factor-1α (HIF-1α)/luciferase activity in vivo.

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.

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.