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Background: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings., Study Design and Methods: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry., Results: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity., Conclusion: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine., (© 2020 AABB.)

Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses.Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization.Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG.Conclusion: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency. [ABSTRACT FROM AUTHOR]

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic—very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients. [ABSTRACT FROM AUTHOR]

Background: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT).Study Design and Methods: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages.Results: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients.Conclusion: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn. [ABSTRACT FROM AUTHOR]

Red blood cells (RBCs) have a well-defined lifespan, indicating a mechanism by which senescent cells of a certain age are removed from circulation. However, the specifics by which senescent cells are recognized and removed are poorly understood. There are multiple competing hypotheses for this process, perhaps the most commonly cited is that senescent RBCs expose neoantigens [or senescent antigen(s)] that are then recognized by naturally occurring antibodies, which opsonize the senescent cells and result in clearance from circulation. While there are a large volume of published data to indicate that older RBCs accumulate increased levels of antibody on their surface, to the best of our knowledge, the causal role of such antibodies in clearance has not been rigorously assessed. In the current report, we demonstrate that RBC lifespan and clearance patterns are not altered in mice deficient in antibodies, in C3 protein, or missing both. These data demonstrate that neither antibody nor C3 is required for clearance of senescent RBCs, and questions if they are even involved, in a murine model of RBC lifespan. [ABSTRACT FROM AUTHOR]

Background: Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized.Study Design and Methods: A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing.Results: A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes.Conclusion: These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported. [ABSTRACT FROM AUTHOR]

Background: Generation of antibodies against red blood cell (RBC) antigens can be a clinically significant problem. The underlying mechanisms that regulate the production of RBC antibodies are only partially understood; however, factors such as inflammation significantly increase the rates of RBC antibody generation. Humoral alloimmunization begins with consumption of transfused RBCs by antigen-presenting cells (APCs). Recently, it has become appreciated that there are multiple different types of APCs. The relative contribution of APC subsets to RBC antibodies has not been described in either the quiescent or the inflamed states.Study Design and Methods: To evaluate the types of APCs that consume RBCs, and how inflammation affects this process, C56Bl/6 mice were treated with polyinosinic-polycytidylic acid (poly(I:C)) to induce an inflammatory response and/or were transfused with 3,3'-dihexadecyloxacarbocyanine perchlorate-labeled syngeneic RBCs. Erythrophagocytosis (both at baseline and during inflammation) was analyzed for different subsets of macrophages (MΦ), dendritic cells (DCs), B cells, and monocytes, by a combined approach using flow cytometry and fluorescent microscopy technology.Results: In four independent experiments, erythrophagocytosis at baseline was predominately performed by red pulp MΦ; however, during inflammation both plasmacytoid DCs (pDCs) and monocytes increased RBC consumption. Furthermore, pDCs up regulated MHC-II and activation markers CD80 and CD86. In addition to changing patterns of erythrophagocytosis, inflammation also led to a significant decrease in CD11c+ conventional DC populations and an increase in granulocytes.Conclusions: The nature of APCs that consume transfused RBCs is changed by inflammation. Given that APCs initiate humoral immune responses, these findings provide potential mechanistic insight into how inflammation regulates RBC alloimmunization. [ABSTRACT FROM AUTHOR]

Background: Platelet (PLT) transfusions can be an essential therapy for patients with thrombocytopenia to maintain hemostasis. However, some patients become alloimmunized to antigens on PLTs (typically HLA), which can prevent efficacy of PLT transfusion due to antibody-mediated clearance. In extreme cases, patients with alloimmunization to multiple HLAs can become "refractory" to PLT transfusion, such that insufficient compatible PLT units can be found to meet transfusion needs.Materials and Methods: An in vivo murine model of PLT-induced alloimmunization was refined so as to include both transfusion with allogeneic leukoreduced PLTs and studies of posttransfusion PLT recoveries, allowing assessment of alloimmunization and refractoriness. Basic mechanisms of antibody-mediated PLT clearance were investigated using recipients missing either the C3 complement gene or the common gamma chain for Fc receptors. In addition, the efficacy of using costimulatory blockade as a therapeutic intervention was assessed by testing CTLA4-Ig administration before PLT transfusion.Results: Fcγ receptors (but not complement C3) are required for alloantibody-mediated PLT refractoriness. In addition, levels of anti-MHC predict the extent of refractoriness in a given animal. Finally, costimulatory blockade as a therapeutic modality prevents transfusion-induced PLT refractoriness.Conclusions: Together these findings introduce new experimental methods, basic mechanistic understanding, and a potential therapeutic intervention for alloimmunization to MHC-based antigens on transfused PLTs. [ABSTRACT FROM AUTHOR]

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop. [ABSTRACT FROM AUTHOR]

Background Red blood cell ( RBC) alloantibodies to nonself antigens may develop after transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans. Study Design and Methods We recently generated transgenic mice with RBC-specific expression of the human KEL glycoprotein, specifically the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system. Results Transfusion of RBCs from KEL2 donors into wild-type recipients (lacking the human KEL protein but expressing the murine KEL ortholog) resulted in dose-dependent anti- KEL glycoprotein immunoglobulin ( Ig) M and IgG antibody responses, enhanced by recipient inflammation with poly( I: C). Boostable responses were evident upon repeat transfusion, with morbid-appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic after transfusion into wild-type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti- KEL1 or anti- KEL2 responses. Conclusions This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long-term translational goals of these studies include improving transfusion safety for at-risk patients. [ABSTRACT FROM AUTHOR]

Background Alloimmunization to antigens on transfused red blood cells ( RBCs) represents a major barrier to chronic transfusion. In extreme cases of multiple alloimmunization, clinicians may be faced with the decision of transfusing incompatible RBCs or risking death from lack of transfusion. The disastrous results of hemolytic transfusion reactions are well understood, and major pathways of clearance have been described. However, well described but poorly understood is the survival of a subset of incompatible donor RBCs during hemolysis, despite antibody binding. Study Design And Methods We utilize a tractable murine model of incompatible transfusion in which RBCs from transgenic donor mice expressing human glycophorin A ( hGPA) are transfused into recipients passively immunized with anti- hGPA. Results As in humans, the majority of RBCs are cleared but a subset of incompatible donor RBCs persist in circulation, despite being bound by antibodies. Data contained herein reject the hypothesis that lack of clearance is due to insufficient antibody or overwhelming of phagocytic machinery; rather, we establish that surviving RBCs represent a distinct population resistant to clearance. Conclusions These studies demonstrate that surviving RBCs during incompatible transfusion can represent a population that is resistant to clearance. [ABSTRACT FROM AUTHOR]

BACKGROUND: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. STUDY DESIGN AND METHODS: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. RESULTS: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice. CONCLUSION: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs. [ABSTRACT FROM AUTHOR]

BACKGROUND: KEL1, also known as 'K,' is one of the most immunogenic red blood cell (RBC) antigens. KEL2, also known as 'k,' differs from KEL1 by a single amino acid. Anti-Kell system antibodies can lead to significant adverse clinical outcomes in humans, including hemolytic complications in alloimmunized transfusion recipients or in infants of alloimmunized mothers. To provide a platform for in-depth immunologic studies of alloimmunization and subsequent sequelae, we generated transgenic mice expressing the human KEL1 or KEL2 antigens. STUDY DESIGN AND METHODS: Vectors were created in which cDNAs encoding either KEL1 or KEL2 were regulated by an erythroid specific β-globin promoter and enhancer. Pronuclear microinjections were carried out into a C57BL6 background, and founder pups were identified by polymerase chain reaction and screened for expression by flow cytometry. RBC life span and antigen stability were assessed by dye labeling RBCs, transfusing into agammaglobulinemic (µMT) recipients, and tracking by flow cytometry. RESULTS: The expression of either KEL1 or KEL2 is RBC specific and first occurs on early RBC precursors. Both KEL1 and KEL2 RBCs have a normal circulatory life span and stable antigen expression. Expression of KEL1 or KEL2 does not result in altered levels of murine Kell, and resulting RBCs have normal hematologic variables. CONCLUSION: The KEL1 and KEL2 mice represent the first murine system of RBC immunity with antithetical antigens, allowing a more precise modeling of human RBC immunology in general and also a platform for development of novel therapeutics to prevent or minimize the dangers of RBC alloimmunization to the KEL1 and KEL2 antigens in particular. [ABSTRACT FROM AUTHOR]

BACKGROUND: Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs. STUDY DESIGN AND METHODS: Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match. RESULTS: Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice. DISCUSSION: Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Patients may present with an antibody against a blood group antigen, a negative direct antiglobulin test (DAT), and a null phenotype. Typically, this represents an alloantibody in a null individual. However, on occasion, the antibody disappears coincident with conversion to a positive red blood cell (RBC) phenotype. This has been called antigen loss, antigen suppression, or weakened antigenicity. Herein, a unique serologic profile that mimics this pattern, when in fact antigen loss did not occur, is described. STUDY DESIGN AND METHODS: RBCs and serum were analyzed using a gel microtyping system and flow cytometry. Genomic DNA was amplified by polymerase chain reaction and sequenced. RESULTS: Initially, an anti-Kpb was detected in MTS gel, RBCs typed K−k−Kp(b−), and the DAT was negative for immunoglobulin G (IgG). Later, the anti-Kpb disappeared and RBCs phenotyped as K−k+Kp(b+). Analysis of initial specimens by flow cytometry identified an immunoglobulin M (IgM) anti-Kpb with a positive IgM-specific DAT; eluates contained an anti-Kpb at immediate spin. Supporting the presence of the Kell glycoprotein, RBCs agglutinated with anti-Jsb. Sequencing showed homozygosity for Kpb with no mutations surrounding the Kpb polymorphism. CONCLUSION: In antigen loss, antibody masking is excluded by a negative DAT. However, because typical DAT reagent does not detect IgM, such reasoning was inaccurate in the current case. In addition, an anti-Kpb resulted in RBCs typing k−, even though no anti-k was detected. Overall, this case suggests that an IgM may mask adjacent epitopes and illustrates the potential to mistake a non-IgG autoantibody as antigen loss. [ABSTRACT FROM AUTHOR]

Blood bank serology has identified hundreds of red blood cell (RBC) antigens contained within numerous blood group systems. Although most blood group antigens are defined by amino acid polymorphisms in the extracellular domain of membrane proteins, it is also possible that additional nonexofacial polymorphisms (NEPs) may exist within cytoplasmic or transmembrane domains. To assess this possibility, we analyzed several blood group molecules by searching the SNPper database for nonsynonymous single-nucleotide polymorphisms. We report the identification of a number of NEPs in the Kell, Kidd, and Duffy molecules. Because the identified NEPs are not exposed on the surface of intact RBCs and are, thus, not accessible to recipient antibodies, they would neither be detected by blood bank serology in vitro, nor would they be recognized targets in hemolytic transfusion reactions in vivo. The presentation of peptides containing NEPs by recipient MHC Class II molecules, however, would nevertheless produce helper T-cell epitopes. In addition to identifying NEPs in human blood group molecules, we explore a novel hypothesis that the presence of NEPs contributes to the immunogenicity of blood group antigens. We further hypothesize that NEPs provide a mechanism by which transfusion can lead to anti-RBC autoantibodies, which are known to occur in humans after transfusion. The scientific basis, existing evidence, approaches to testing, and predicted biology of this hypothesis are presented. [ABSTRACT FROM AUTHOR]

In this article the author discusses the role of suicidal deaths in hemolytic transfusion reactions (HTRs). He says that it has been believed that HTRs can be prevented by cross-matching recipient serum with donor red blood cells (RBCs). However, he states that the question of assessing the undetectable antibody hypothesis still exits. He concludes that eryptosis may be a common pathway of RBC destruction in different pathophysiologic settings which involve perturbations of normal homeostasis.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses. [ABSTRACT FROM AUTHOR]

Polyclonal anti-D has been used to prevent RhD-negative mothers from becoming immunized against RhD positive fetal erythrocytes, and this mechanism has been referred as Ab or IgG-mediated immune suppression (AMIS). Although anti-D has been highly successful, the inhibitory mechanisms remain poorly understood. Two major theories behind AMIS involve the binding of IgG to activating or inhibitory FC7R, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory Fc"R in the AMIS mechanism using the HOD mouse model of RBC immunization. HOD mice contain a RBC-specific recombinant protein composed of hen egg lysozyme (HEL), OVA and human transmembrane Duffy Ag, and erythrocytes from HOD mice can stimulate an immune response to HEL. To assess the contribution of activating and inhibitory Fc-yR to AMIS, C57BL/6 versus Fc"R-chain-/- or Fc"RIIB-/-- mice were used as recipients of HOD-RBC alone or together with anti-HEL Abs (i.e., AMIS) and the resulting immune response to HEL evaluated. We show that anti-HEL polyclonal Abs induce the same degree of AMIS effect in mice lacking these IgG binding receptors as compared with wild-type mice. In agreement with this, F(ab')2 fragments of the AMIS Ab also significantly reduced the Ab response to the HOD cells. In conclusion, successful inhibition of in vivo Ab responses to HOD-RBC by polyclonal IgG can occur independently of activating or inhibitory FC7R involvement. These results may have implications for the understanding of RhD prophylaxis. [ABSTRACT FROM AUTHOR]

Autoantibodies and alloantibodies can damage self-tissue or transplanted tissues through either fixation of complement or ligation of FcγRs. Several pathways have been described that imbue self-tissues with resistance to damage from complement fixation, as a protective measure against damage from these Abs. However, it has been unclear whether parallel pathways exist to provide protection from FcγR ligation by bound Abs. In this article, we describe a novel pathway by which cell surface Ag is specifically decreased as a result of Ab binding (Ag modulation) to the extent of conferring protection to recognized cells from Fcγ-dependent clearance. Moreover, the Ag modulation in this system requires FcγR ligation. Together, these findings provide unique evidence of self-protective pathways for FcγR-mediated Ab damage. [ABSTRACT FROM AUTHOR]

Hemolytic transfusion reactions (HTRs) due to incompatible red blood cell (RBC) transfusions are a leading cause of transfusion associated death. Although many transfused incompatible RBCs are cleared, some remain in circulation despite the presence of RBC-specific antibodies, potentially due to "antigen modulation." With a goal of better understanding incompatible RBC clearance, we generated a murine model with RBC-specific expression of a clinically significant human antigen (KEL2) known to be involved in antigen modulation as well as in HTRs. Wild-type (WT) recipients transfused with transgenic KEL2 RBCs generated anti-KEL glycoprotein alloantibodies, which fixed complement, led to intravascular hemolysis, and resulted in decreased levels of KEL2 antigen detectable on cells remaining in circulation. Antigen modulation did not appear to solely reflect removal of RBCs with higher antigen expression, because cells continued to display antigen modulation in the absence of significant clearance. Recipients genetically lacking complement exhibited lesser degrees of incompatible RBC clearance and antigen modulation in comparison with WT or FcγR knock-out (KO) animals, suggesting a role for complement in RBC clearance. In summary, this HTR model may serve as a platform to test strategies to downmodulate antigen and inhibit incompatible RBC clearance, thus potentially mitigating transfusion dangers. [ABSTRACT FROM AUTHOR]

Exposure to nonself red blood cell (RBC) antigens, either from transfusion or pregnancy, may result in alloimmunization and incompatible RBC clearance. First described as a pregnancy complication 80 years ago, hemolytic disease of the fetus and newborn (HDFN) is caused by alloimmunization to paternally derived RBC antigens. Despite the morbidity/mortality of HDFN, women at risk for RBC alloimmunization have few therapeutic options. Given that alloantibodies to antigens in the KEL family are among the most clinically significant, we developed a murine model with RBC-specific expression of the human KEL antigen to evaluate the impact of maternal/fetal KEL incompatibility. After exposure to fetal KEL RBCs during successive pregnancies with KEL-positive males, 21 of 21 wild-type female mice developed antl-KEL alloantibodies; intrauterine fetal anemia and/or demise occurred in a subset of KEL-positive pups born to wild type, but not agammaglobulinemic mothers. Similar to previous observations in humans, pregnancy-associated alloantibodies were detrimental in a transfusion setting, and transfusion-associated alloantibodies were detrimental in a pregnancy setting. This is the first pregnancy-associated HDFN model described to date, which will serve as a platform to develop targeted therapies to prevent and/or mitigate the dangers of RBC alloantibodies to fetuses and newborns. [ABSTRACT FROM AUTHOR]

Most human transfusion recipients fail to make detectable alloantibodies to foreign ABC antigens ("nonresponders"). Herein, we use a murine model to test the hypothesis that nonresponders may be immunologically tolerant. FVB mice transfused with RBCs expressing transgenic human glycophorin A (hGPA) antigen in the absence of inflammation produced undetectable levels of anti-hGPA immunoglobulins, unlike those transfused in the presence of polyinosinic:polycytidylic acid-induced inflammation. Mice in the nonresponder group failed to produce anti-hGPA after subsequent transfusions in the presence of polyinosinic:polycytidylic acid, whereas anti-hGPA levels increased in the responder group. This tolerance was antigen specific, because nonresponders to hGPA produced alloantibodies to ABCs that expressed a different transgenic antigen. This tolerance was not an idiosyncrasy of the hGPA antigen nor of the recipient strain, because B10.BR mice transfused with membrane-bound hen egg lysozyme antigen-transgenic RBCs also demonstrated induced nonresponsiveness. These data demonstrate that RBCs transfused in the absence of inflammation can induce tolerance. [ABSTRACT FROM AUTHOR]