Antibody-mediated Immune Suppression Research Articles

Complement C3 and marginal zone B cells promote IgG-mediated enhancement of RBC alloimmunization in mice.

Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-α/-β receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both CD4+ T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon.

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

AbstractRed 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.

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. 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. Antigen copy number affected the antibody dose required for AMIS induction with higher antigen copy numbers requiring larger doses of antibody. For instance, 5μg 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. 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.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in a Preclinical Model By Monoclonal Anti-HPA-1a Antibody Prophylaxis: Structural and Functional Properties of Antibody Variant Isoforms

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans. DisclosuresSkogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.

Antibody-mediated immune suppression by antigen modulation is antigen-specific

AbstractAlloantibodies developing after exposure to red blood cell (RBC) alloantigens can complicate pregnancy and transfusion therapy. The only method currently available to actively inhibit RBC alloantibody formation is administration of antigen-specific antibodies, a phenomenon termed antibody-mediated immune suppression (AMIS). A well-known example of AMIS is RhD immune globulin prophylaxis to prevent anti-D formation in RhD− individuals. However, whether AMIS is specific or impacts alloimmunization to other antigens on the same RBC remains unclear. To evaluate the specificity of AMIS, we passively immunized antigen-negative recipients with anti-KEL or anti-hen egg lysozyme (HEL) antibodies, followed by transfusion of murine RBC expressing both the HEL-ovalbumin-Duffy (HOD) and human KEL antigens (HOD × KEL RBC). Significant immunoglobulin G deposition on transfused HOD × KEL RBC occurred in all passively immunized recipients. Complement deposition and antigen modulation of the KEL antigen occurred on transfused RBC only in anti-KEL–treated recipients, whereas HEL antigen levels decreased only in the presence of anti-HEL antibodies. Western blot analysis confirmed the specificity of antigen loss, which was not attributable to RBC endocytosis and appears distinct for the 2 antigens. Specifically, removal of KEL was attenuated by clodronate treatment, whereas loss of HEL was unaffected by clodronate in vivo but sensitive to protease treatment in vitro. Antigen-specific modulation correlated with antigen-specific AMIS, with anti-KEL treated recipients forming antibodies to the HOD antigen and anti-HEL–treated recipients developing antibodies to the KEL antigen. Together, these results demonstrate that passively administered antibodies can selectively inhibit the immune response to a specific antigen.

ABO incompatibility and RhIG immunoprophylaxis protect against non‐D alloimmunization by pregnancy

Hemolytic disease of the fetus and newborn (HDFN) is caused by maternal antibodies against fetal red blood cell antigens, most often anti-D, -K, or -c. ABO incompatibility between mother and child and anti-D immunoprophylaxis (RhIG) are known to reduce the risk of D immunization and subsequent HDFN. However, no immunoprophylaxis has been developed to prevent non-D immunizations. We evaluated whether ABO incompatibility has a preventive effect on formation of non-D alloantibodies, by performing a case-control study including pregnant women with newly detected non-D antibodies, identified within a nationwide data set, immunized during their first pregnancy and/or delivery. Subsequently, we assessed a possible protective effect of RhIG in a subgroup with non-Rh antibodies only. The proportions of previous ABO incompatibility and of RhIG administrations of these women were compared to the known rate of 19.4% ABO incompatibility and 9.9% RhIG administrations (D- women carrying a D+ child) in the general population of pregnant women. A total of 11.9% of the 232 included immunized women had a possible ABO incompatibility in their first pregnancy (vs. expected 19.4%; 95% confidence interval [CI], 7.3-18.8; p=0.036). Furthermore, 1.0% women with non-Rh antibodies were D-, delivered a D+ child, and had therefore received RhIG, whereas 9.9% was expected (95% CI, 0.18-5.50; p=0.003). We found that ABO incompatibility and RhIG reduce the risks not only for D, but also for non-Rh immunizations, suggesting that antibody-mediated immune suppression in this condition is not antigen specific.

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.

Antigen-Mediated Immune Suppression Is Antigen Specific

Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies and result in transfusion-related complications, like hemolytic transfusion reactions, or hemolytic disease of the fetus and newborn. Currently, antibody-mediated immune suppression (AMIS) is the only known methodology to actively inhibit RBC alloantibody formation. The best-known example of AMIS is Rh immune globulin, which is given to Rh(D) negative women to prevent the development of anti-D alloantibodies. Mouse models to elucidate the mechanism by which AMIS occurs have used mice genetically engineered to specifically express the human KEL antigen (KEL RBCs) or the Hel-Ova-Duffy antigen (HOD RBCs) on murine RBCs, and found that AMIS to these two antigens is dependent on antigen loss (also called antigen modulation). As RBCs express many antigens simultaneously, we sought to determine whether AMIS to one antigen can impact the immune response to a completely different antigen on the same RBC by generating mice that express both the KEL and HOD antigens (HOD x KEL RBCs).Methods: WT recipients received polyclonal anti-KEL antibody, a cocktail of two anti-HEL monoclonal antibodies, or PBS, followed by transfusion with WT and HOD x KEL donor RBCs. HOD x KEL RBCs were labeled with the lipophilic dye DiI prior to transfusion, while WT RBCs were labeled with the lipophilic dye DiO and served as a RBC tracer population. RBC survival and levels of detectable IgG, Kel antigen, Hel antigen, Duffy antigen, and complement (C3) on transfused cells were measured by flow cytometry at 10min, 1hr, 2hr, and 4hr, and on days 1, 2, 3, and 5 post-transfusion. Serum was collected on day 5 and analyzed for IgM antibody development by flow-crossmatch using KEL or HOD RBCs.Results: Anti-KEL and anti-HEL treated WT recipients demonstrated significant IgG deposition on transfused HOD x KEL RBCs at 10min post-transfusion. Levels of IgG on transfused RBCs decreased over time but did not correlate with complete RBC clearance. RBC survival was nearly 100% over 5 days in recipients passively immunized with anti-HEL antibodies, while a fraction of RBCs (~20%) were cleared within 4hr post-transfusion but not thereafter (survival plateaued at 80% thru day 5) in anti-KEL immunized recipients. Conversely, decreased bound IgG on transfused RBCs was found to correlate with a decrease in target antigen levels. KEL antigen levels decreased on transfused RBCs in the anti-KEL immunized recipients beginning at 1hr post transfusion, as compared to anti-HEL and PBS recipients. Similarly, HEL antigen expression decreased on transfused RBCs in the anti-HEL immunized recipients, but not in the anti-KEL or PBS recipients, demonstrating that passive immunization causes antigen modulation for the corresponding antigen only. C3 deposition was found to occur on transfused RBCs at 10min post-transfusion in the anti-KEL recipients that remained significantly increased at other time-points, but was not detected on RBCs in the anti-HEL or PBS recipients until day 3, when recipients develop IgM antibodies. Flow crossmatch of serum samples showed that anti-KEL treated recipients did not make anti-KEL IgM but did make anti-HOD IgM, while anti-HEL treated recipients did not make anti-HOD IgM but did make anti-KEL IgM.Conclusions: Although AMIS serves as the basis of a widely successful clinical intervention, the mechanism by which it occurs, including any impact on additional antigens, remains incompletely understood. Here we demonstrate that AMIS is antigen specific - that is, passively acquired antibody can inhibit the immune response only to the corresponding antigen. Specifically, murine RBCs expressing both the human and model RBC antigen KEL and HOD, respectively, demonstrate similar clearance kinetics, antigen modulation, complement deposition, and antibody development after transfusion into WT mice as is observed when KEL-only or HOD-only RBCs are used. Although it is unclear whether these findings apply to all RBC antigens and what relevance this holds for human patients, our study provides insight into the specificity of AMIS that may enlighten development of additional therapeutics based on this mode of immune suppression. DisclosuresNo relevant conflicts of interest to declare.

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

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

AbstractAlthough 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 Duffyb) 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.

Fetal/Neonatal Alloimmune Thrombocytopenia: Pathogenesis, Diagnostics and Prevention.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a relatively rare condition (1/1000–1/2000) that was granted orphan status by the European Medicines Agency in 2011. Clinical consequences of FNAIT, however, may be severe. A thrombocytopenic fetus or new-born is at risk of intracranial hemorrhage that may result in lifelong disability or death. Preventing such bleeding is thus vital and requires a solution. Anti-HPA1a antibodies are the most frequent cause of FNAIT in Caucasians. Its pathogenesis is similar to hemolytic disease of the newborn (HDN) due to anti-RhD antibodies, but is characterized by platelet destruction and is more often observed in the first pregnancy. In 75 % of these women, alloimmunization by HPA-1a antigens, however, occurs at delivery, which enables development of antibody-mediated immune suppression to prevent maternal immunization. As for HDN, the recurrence rate of FNAIT is high. For advancing diagnostic efforts and treatment, it is thereby crucial to understand the pathogenesis of FNAIT, including cellular immunity involvement. This review presents the current knowledge on FNAIT. Also described is a program for HPA-1a screening in identifying HPA-1a negative pregnant women at risk of immunization. This program is now performed at the Institute of Hematology and Transfusion Medicine in cooperation with the Department of Obstetrics and Gynecology of the Medical Centre of Postgraduate Education in Warsaw as well as the UiT The Arctic University of Norway.

Antibody-mediated immune suppression of erythrocyte alloimmunization can occur independently from red cell clearance or epitope masking in a murine model.

Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

Fetal and Neonatal Alloimmune Thrombocytopenia: Lessons Learned from Animal Models

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe alloimmune disorder that results from fetal/neonatal platelet opsonization by maternal antibodies, which cross the placenta and result in fetal/neonatal platelet destruction. The frequency of FNAIT has been estimated at 0.5-1.5/1,000 liveborn neonates. However, this number does not include fetuses that die from this disease, since the incidence of FNAIT miscarriage has not been adequately studied. Analogous to autoimmune thrombocytopenia (ITP), the major target antigens in FNAIT are the platelet GPIIIa (β3 integrin) and GPIbα. However, severe bleeding is much more frequent in FNAIT, particularly the occurrence of intracranial hemorrhage (ICH). It is not known why the reported incidence of FNAIT mediated by anti-GPIbα antibodies is at a much lower frequency when compared to ITP, and whether the severe bleeding tendency in FNAIT is due to β3 integrin expression on angiogenic vessels in the developing fetus, which are targeted by cross-reacting maternal anti-β3 integrin antibodies. To study the pathogenesis and to develop new strategies for prevention and treatment, we have established murine models of FNAIT using β3 integrin and GPIbα deficient (-/-) mice. We first transfused these deficient mice with wild-type (WT) platelets to induce anti-β3 or anti-GPIbα antibody responses; we then bred these immunized female mice with WT males, causing FNAIT in the offspring. We found that maternal antiplatelet antibody titer correlated with the severity of FNAIT. These two murine models have revealed fundamental differences between the pathogenesis of anti-β3 and anti-GPIbα-mediated FNAIT. In anti-β3-mediated FNAIT, we found severe thrombocytopenia, ICH, and miscarriage. We also found the impairment of angiogenesis, which may contribute to ICH and intrauterine growth retardation. In contrast, the anti-GPIbα-mediated model revealed a nonclassical form of FNAIT (e.g., miscarriage but not bleeding disorders in neonates). We found that anti-GPIbα antibodies caused thrombosis in the placentas and miscarriage in most pregnant mice, which may partially explain the rarity of anti-GPIbα-mediated FNAIT reported in humans. Despite these substantial differences, there are also similarities between anti-β3 and anti-GPIbα-mediated FNAIT, such as the role of the neonatal Fc receptor (FcRn). FcRn is important for serum IgG homeostasis and for IgG transportation across the placenta. Using FcRn-/- mice, we demonstrated that fetal (but not maternal) FcRn is required to transport maternal antibodies to the fetal circulation and is indispensable for FNAIT. Blocking FcRn with an anti-FcRn antibody markedly reduced the severity of both anti-β3 and anti-GPIbα-mediated FNAIT. This important finding may lead to the development of new therapies (e.g., anti-FcRn antibody) against FNAIT and other maternal pathogenic antibody-mediated fetal/neonatal diseases. These models of FNAIT have allowed us to investigate the efficacy and mechanism of action of several therapies, including the aforementioned anti-FcRn antibody as well as antibody-mediated immune suppression (AMIS) and intravenous IgG (IVIG). In β3-/- mice, prophylactic administration of anti-HPA-1a antibody or murine β3 antisera induced AMIS against human HPA-1a-positive or murine WT platelets, respectively. Importantly, AMIS induced by β3 antisera suppressed the antibody response, thrombocytopenia, and miscarriage in FNAIT mice. These findings support the hypothesis that anti-HPA-1a antibody administration to HPA-1a-negative women after delivery of an HPA-1a-positive child may prevent FNAIT in subsequent pregnancies. The efficacy of IVIG is inconsistent, sources are limited, and the mechanism of action is not fully understood. We demonstrated that IVIG ameliorated both anti-β3 and anti-GPIbα-mediated FNAIT. IVIG decreased antiplatelet antibodies in both maternal and neonatal circulations, fetal platelet clearance, bleeding, and fetal mortality. In summary, we have uncovered fundamental differences in the pathogenesis of anti-β3 and anti-GPIbα-mediated FNAIT and have greatly enhanced our understanding of emerging and existing therapies. We will continue to investigate the pathogenesis of FNAIT so that we may develop more tailored and accessible therapeutic strategies for patients. Disclosures:No relevant conflicts of interest to declare.

Mechanisms and Prevention of Alloimmunization in Pregnancy

Transfusion only occasionally gives rise to antibody production, because blood cells per se are not markedly immunogenic. However, the immunological changes that occur during pregnancy increase the risk of alloimmunization against red blood cells, platelets, and/or leukocytes. Fetal-maternal bleeding during pregnancy or in relation to delivery is the antigenic stimuli for immunization against red blood cells, whereas other mechanisms, such as trophoblast-derived microparticles, may also play a role in the production of antibodies against platelets. Antibody-mediated immune suppression has for 4 decades successfully been used for prevention of RhD immunization. Result from a mouse model of fetal and neonatal alloimmune thrombocytopenia (FNAIT) suggests that the same principle may be applied for the prevention of FNAIT. A European Union-funded consortium is presently in the process of developing a hyperimmune anti-human platelet antigen 1a (HPA-1a) immunoglobulin G. The idea is to prevent HPA-1a immunization by administering the drug to nonimmunized HPA-1a-negative women after delivery of an HPA-1a-positive child. The anti-HPA-1a will be purified from plasma collected from women who previously have given birth to a child with FNAIT caused by anti-HPA-1a. If the results of the planned phase III trial are favorable, it is possible that a product for prevention of FNAIT will be available within this decade.

Activating and Inhibitory Fcγ Receptors Are Not Required for Successful Antibody-Mediated Immune Suppression to Foreign Erythrocytes

Activating and Inhibitory Fcγ Receptors Are Not Required for Successful Antibody-Mediated Immune Suppression to Foreign Erythrocytes

Toward a prophylaxis against fetal and neonatal alloimmune thrombocytopenia: induction of antibody‐mediated immune suppression and prevention of severe clinical complications in a murine model

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal or neonatal platelets (PLTs). Results from our recent large screening study suggest that the pathophysiology of FNAIT is more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA-1a might therefore be preventable by a prophylactic regimen of inducing antibody-mediated immune suppression (AMIS), which has been documented to be a useful prophylaxis against HDFN. This preclinical proof-of-concept study investigated whether passive administration of anti-β3 integrin could induce AMIS and thereby prevent clinical complications of FNAIT. A murine model of FNAIT using β3 integrin (GPIIIa)-deficient (β3-/-) mice was employed for this study. AMIS in β3-/- mice was induced by intravenous administration of human anti-HPA-1a immunoglobulin G or murine anti-β3 antisera given as prophylaxis after transfusion of HPA-1a-positive human PLTs or murine wild-type PLTs, respectively. AMIS against both human and murine PLT antigens was induced using this prophylactic approach, reducing the amount of maternal PLT antibodies by up to 90%. Neonatal PLT counts were significantly increased and pregnancy outcome was improved in a dose-dependent manner. The incidence of intracranial hemorrhage, miscarriage, and dead-born pups in mice receiving high-dose prophylaxis was reduced to that of normal controls. We also observed that the severity of thrombocytopenia inversely correlated with birth weight. This work conceptually proves that prophylactic administration of PLT antibodies induces AMIS and prevents poor pregnancy outcome in FNAIT.

Transfusion of antibody‐opsonized red blood cells results in a shift in the immune response from the red blood cell to the antibody in a murine model

It is well known that infusion of immunoglobulin (Ig)G-coated cells results in an inhibited antigen-specific humoral immune response compared to the cells themselves, a phenomenon termed antibody-mediated immune suppression (AMIS). Although this AMIS effect has been well described with many different types of cells as well as vaccines and insoluble antigens, the mechanisms behind this effect remain unresolved. To study AMIS in a broad context, three different models of AMIS were studied. In the first, mice were transfused with sheep red blood cells (SRBCs) versus IgG-coated SRBCs. In the second, SRBCs expressing the antigen hen egg white lysozyme (HEL) were studied, and the third model consisted of the diphtheria/tetanus vaccine in the absence versus presence of anti-tetanus IgG. The antibody responses to the SRBCs and HEL-SRBCs, as well as the vaccine, were analyzed for up to 4 weeks after challenge. In these mouse models of immunization, the IgG-coated RBCs or HEL-RBCs induced an antibody response against the IgG, rather than against the RBCs. The decreased response to the RBCs was directly related to the increase of the response against the IgG. The inhibitory AMIS effect using the vaccine strategy again showed an immune response against the IgG, concurrent with a decrease in the immune response against the specific vaccine component targeted. This work demonstrates that, under AMIS conditions, the IgG itself becomes the focus of B cells in the immune system, suggesting a potential mechanism of B-cell regulation.

Administration of Anti-Platelet Antibodies Prevents the Anti-Platelet Immune Response and Bleeding Complications of Neonatal Immune Thrombocytopenia in a Murine Model.

Abstract 223 Background:The human platelet antigen (HPA) 1a is a potent immunogen located on the β3 integrin. Ten % of pregnant HPA1a negative women produce antibodies against the HPA1a antigen if the foetus is HPA1a positive. Fetal/neonatal immune thrombocytopenia (FNIT) can occur if the mother develops alloantibodies against fetal platelets, with intracranial haemorrhage as the most severe complication. The current opinion has been that immunization against the HPA1a antigen takes place during the first HPA1 non-compatible pregnancy. However, results from a large and recent screening study in Norway found that the majority (75%) of women were immunized around time of delivery, and not so often during pregnancy. This indicates that FNIT could be more similar to haemolytic disease of the newborn (HDN) than previously thought. To prevent HDN, antibody mediated immune suppression (AMIS) is induced by administration of anti-D antibodies in connection with RhD-negative pregnancies. The same principle could be used to prevent FNIT by administration of anti-HPA1a antibodies in HPA1a-negative pregnancies. We have previously established a murine model of FNIT using β3 integrin-deficient (β3−/−) mice. The first aim of the current project was to test whether administration of human anti-HPA1a IgG could suppress the anti-human platelet immune response in β3−/− mice after transfusion of human HPA1a positive platelets. For the second part of the project, we used a pure murine model to test whether administration of murine anti-β3 antibodies transfused after delivery could induce AMIS and prevent bleeding complications of FNIT in the subsequent pregnancies. Methods:Human/murine model: Human IgG from 5 donors with high levels of anti-HPA1a antibodies was purified by Protein G affinity chromatography. Purified IgG from one male donor without detectable anti-platelet specific antibodies was used as control IgG. Human platelets were isolated from an HPA1a positive donor. β3−/− mice were immunized by one tail vein transfusion with 2 × 106 human HPA1a positive platelets, with or without subsequent transfusion of 900ug human IgG (100% saturation). After 7 days, the mice were bled and sera collected. The anti-human platelet immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG as detection antibody. Six mice were injected with anti-HPA1a containing IgG. Control IgG (n=6) or no IgG (n=4) were used as negative controls. Pure murine model: High-titer anti-β3 sera were produced by 4 weekly transfusions of 108 wild type (WT) platelets to β3−/− mice. Naïve β3−/− female mice were bred with naïve β3−/− male mice. Within 24 hours of delivery, the mother was transfused with 108 WT platelets with or without immediate transfusion of anti-β3 sera. The transfusions were repeated one week after delivery and the same females were bred again with WT male BALB/c mice. The anti-β3 immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG. The FNIT phenotype was monitored and all live pups were bled from the carotid vein to determine platelet count. Results:Administration of purified anti-HPA1a IgG significantly suppressed the anti-human platelet immune response in β3−/− mice after transfusion of HPA1a positive platelets as compared with control IgG (p < 0.05). In the pure murine model of FNIT, the anti-β3 immune response was markedly suppressed during the subsequent pregnancy in the mice treated with anti-β3 sera. Two out of three mice receiving anti-β3 sera treatment delivered live pups with moderate thrombocytopenia without signs of haemorrhage (mean platelet count 217 ×106/mL). The third mouse receiving anti-β3 sera delivered dead pups. In contrast, all female mice (n = 3) without anti-β3 sera treatment miscarried. Conclusions:We have demonstrated in vivo that AMIS can be induced by administration of anti-platelet antibodies using a murine model of FNIT. Preliminary data indicates that bleeding complications of FNIT can be prevented with this prophylactic approach. Disclosures:Skogen:Prophylix Pharma a/s: Employment, Equity Ownership. Killie:Prophylix Pharma a/s: Equity Ownership. Husebekk:Prophylix Pharma a/s: Equity Ownership. Kjeldsen-Kragh:Prophylix Pharma a/s: Equity Ownership.

Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn: what can we learn from rodent models?

Hemolytic disease of the fetus and newborn can be effectively prevented by administration of anti-D to the mother. In this setting, the IgG purified from the plasma of D-alloimmunized donors prevents the maternal immune response to D-positive red blood cells (RBC). Several monoclonal anti-D antibodies have recently been developed for potential use in the setting of hemolytic disease of the fetus and newborn; the functional assays used to assess the potential success of these antibodies have often assumed antigen clearance as the predominant mechanism of anti-D. Unfortunately, the in-vivo success of these monoclonal antibodies has thus far been limited. A similar inhibitory effect of IgG has been observed in animal models with a vast array of different antigens, referred to as antibody-mediated immune suppression (AMIS). Here, studies of AMIS are reviewed and the relevance of these findings for anti-D-mediated immunoprophylaxis is discussed. In animal models of AMIS, IgG-mediated antigen clearance was not sufficient for prevention of the antibody response to RBC. Furthermore, anti-RBC IgG inhibited B-cell priming to foreign RBC, but failed to prevent a T-cell response and immunological memory. The applicability of AMIS models for determining the true mechanism of anti-D, though uncertain, may nevertheless provide knowledge as to potential mechanisms of action of anti-RBC antibodies.

Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn

Anti-D is routinely and effectively used to prevent hemolytic disease of the fetus and newborn (HDFN) caused by the antibody response to the D antigen on fetal RBCs. Anti-D is a polyclonal IgG product purified from the plasma of D-alloimmunized individuals. The mechanism of anti-D has not been fully elucidated. Antigenic epitopes are not fully masked by anti-D and are available for immune system recognition. However, a correlation has frequently been observed between anti-D-mediated RBC clearance and prevention of the antibody response, suggesting that anti-D may be able to destroy RBCs without triggering the adaptive immune response. Anti-D-opsonized RBCs may also elicit inhibitory FcgammaRIIB signaling in B cells and prevent B cell activation. The ability of antigen-specific IgG to inhibit antibody responses has also been observed in a variety of animal models immunized with a vast array of different antigens, such as sheep RBCs (SRBC). This effect has been referred to as antibody-mediated immune suppression (AMIS). In animal models, IgG inhibits the antibody response, but the T-cell response and memory may still be intact. IgG does not mask all epitopes, and IgG-mediated RBC clearance or FcgammaRIIB-mediated B-cell inhibition do not appear to mediate the AMIS effect. Instead, IgG appears to selectively disrupt B cell priming, although the exact mechanism remains obscure. While the applicability of animal models of AMIS to understanding the true mechanism of anti-D remains uncertain, the models have nevertheless provided us with insights into the possible IgG effects on the immune response.