Search

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement-mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer "decoy" therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jash, Usaneerungrueng, Howie, Qiu, Luckey, Zimring and Hudson.)

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system., (Copyright © 2020 Shinde, Howie, Stegmann, Hay, Waterman, Szittner, Bentlage, Kapp, Lissenberg-Thunnissen, Dekkers, Schasfoort, Ratcliffe, Smolkin, Vidarsson, Schoot, Hudson and Zimring.)

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: 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., (© 2020 AABB.)

There are four subtypes of human IgG with different effector functions. Quantifying the relative amount of each IgG subtype is important for laboratory diagnosis in multiple settings. However, in an evolving landscape of the appreciation of human variability and the need for precision/personalised laboratory diagnosis, it has also been shown that there are numerous natural variants of IgG subtypes, with at least 29 having been described. It has recently been reported that commercially available polyclonal antisera to IgG3 cross react with variants of other IgG subtypes, while available monoclonal anti-IgG3 have a blind-spot for the IgG3-04 variant. Herein, we report that IgG3-04 contains an epitope in common with all known IgG3 variants and absent in other subtypes. A novel monoclonal anti-IgG3 is described that is specific to IgG3 but without any blind-spots for known IgG3 variants, providing a remedy to the problem of genetic variability of IgG3., Competing Interests: Competing interests: Bloodworks NW has filed intellectual properties around the PUMA1 reagents in this report- HLH, LK, JNL and JCZ and are each employees of Bloodworks NW. XW has no competing interests to declare., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Methods designed to monitor humoral immune responses, in a variety of settings, typically use a broadly reactive detection reagent (e.g. polyclonal anti-Ig (immunoglobulin)) in order to characterize antibody responses. In the context of murine models of immunity, which are widely used, this would typically be antisera to mouse Ig or mouse IgG. However, there are 4 different subtypes of mouse IgG; thus, the validity of the above approach, as a general screen for humoral immune responses, depends upon the assumption that the antisera recognize all IgG subtypes. This seems like a reasonable assumption, since polyclonal antisera recognize multiple epitopes; however, herein we report that two commercial sources of goat anti-mouse Ig are hyporeactive with IgG3. Given that relative IgG3 levels are different in distinct types of immune response, these findings demonstrate a potential for misinterpretation, and suggest a need to modify immunological methods in this context., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2016 AABB.)

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 FcγR, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory FcγR 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 FcγR to AMIS, C57BL/6 versus FcRγ-chain(-/-) or FcγRIIB(-/-) 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 FcγR involvement. These results may have implications for the understanding of RhD prophylaxis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

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-[alpha]/-[beta] 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.sup.+] 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., Introduction Immunoglobulins are most commonly thought of as immune effector molecules. However, it has been known for over a century that Igs can also substantially enhance primary humoral immunity to [...]

BACKGROUND: Alloantibodies are a clinically significant sequelae of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing PLT transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T-cell help; however, PLT-specific CD4+ T cells have not been visualized in vivo, and specifics of their stimulation are not completely understood. STUDY DESIGN AND METHODS: A murine model of alloimmunization to transfused PLTs was developed to allow in vivo assessment and characterization of CD4+ T cells specific for PLT major histocompatibility complex (MHC) alloantigen. PLTs were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. PLT-specific CD4+ T-cell responses were visualized by using a T-cell receptor transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets. RESULTS: C57BL/6 recipients of BALB/c leukoreduced PLT transfusions produced BALB/c antibodies, with proliferation of antigen-specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response. CONCLUSION: We report a novel model to study antigen-specific CD4+ T cells during alloimmunization to PLT transfusion. The presented data support a critical role for CD4+ T-cell help in the humoral response to PLT transfusion and establish the spleen as a required microenvironment for effective CD4+ T-cell priming against donor PLT-derived MHC I. [ABSTRACT FROM AUTHOR]