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Foetal cells are detectable in women decades postpartum, a state termed foetal microchimerism. The interplay between these semi‐allogeneic foetal cells and the mother could be affected by genetic mismatches in the HLA loci. Here, we relate HLA allele and molecular mismatch values to the presence and quantity of foetal microchimerism in the maternal circulation during pregnancy and postpartum. A total of 76 pregnant women were included, of which 59 were followed up 1–8 years postpartum. Maternal and foetal DNA was genotyped for HLA class I and II loci. Foetal cells in maternal buffy coat were detected by qPCR, targeting inherited paternal alleles. Antibody‐verified eplet mismatch and Predicted Indirectly Recognisable HLA Epitopes (PIRCHE) scores were calculated to quantify foetal‐maternal histocompatibility from the mother's perspective. Circulating foetal cells were detected in 50.0% (38/76) of women during pregnancy, and 25.4% (15/59) postpartum. During pregnancy, HLA class II antibody‐verified eplet mismatch load and PIRCHE scores correlated negatively with the presence and quantity of foetal cells in the maternal circulation. Postpartum, HLA class I allele mismatches correlated negatively with foetal microchimerism presence, while HLA class II allele mismatches, HLA class I and II antibody‐verified eplet mismatch load, and PIRCHE‐I and PIRCHE‐II scores correlated negatively with both microchimerism presence and quantity. The correlation between mismatch parameters aimed at evaluating the risk of humoral and T cell‐mediated allorecognition and foetal microchimerism was more evident postpartum than during pregnancy. The observed predictive effect of foetal‐maternal histocompatibility on foetal microchimerism suggests that circulating foetal cells are subject to clearance by the maternal immune system. We propose that allorecognition of foetal cells in the maternal circulation and tissues influences any long‐term effect that foetal microchimerism may have on maternal health. [ABSTRACT FROM AUTHOR]

On 24 January 2023, Eurotransplant has introduced the virtual crossmatch for kidney and pancreas allocation as a better alternative for the physical Complement Dependent Cytotoxicity (CDC) crossmatches at the donor centre, which were associated with a longer cold ischaemia time and false positive reactions. For the time being, the physical CDC crossmatch at the recipient centre will remain in place as the final histocompatibility check. While Eurotransplant is certainly not the first organ allocation organisation to introduce virtual crossmatching, several novel aspects have been introduced, such as calculation of the virtual panel reactive antibody (vPRA) on 11 loci at the second‐field level in addition to the serological broad and split level, electronic HLA typing data transmission using Histoimmunogenetics Markup Language (HML) file format, and the actual virtual crossmatch based on ambiguous, second‐field HLA typing of the donor on all 11 loci. This short communication will focus on these novel aspects of the virtual crossmatch in Eurotransplant. [ABSTRACT FROM AUTHOR]

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology. [ABSTRACT FROM AUTHOR]

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody‐verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody‐induction and binding are limited. In this proof‐of‐concept study, we performed site‐directed mutagenesis to narrow down the antibody‐verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA) antibodies are induced by pregnancy, transfusion, or transplantation. Serum from transplant recipients is regularly screened for IgG HLA antibodies because of their clinical relevance for transplant outcome. While other isotypes of HLA antibodies, such as IgA may also contribute to the alloimmune response, validated detection assays for IgA HLA antibody detection are lacking. Therefore, we modified the commonly used luminex screening assay for IgG HLA antibody detection (IgG‐LMX) into an IgA HLA antibody screening assay (IgA‐LMX). Optimization and validation was performed with IgG, IgA1, and IgA2 isotype variants of HLA‐specific human recombinant monoclonal antibodies (mAbs). Reactivity patterns of IgA1 and IgA2 isotype HLA‐specific mAbs in IgA‐LMX were identical to those of the IgG isotype. Cross‐reactivity with IgG and IgM antibodies and nonspecific binding to the beads were excluded. Further assay validation showed the absence of IgA HLA antibodies in serum from individuals without alloantigen exposure (n = 18). When the IgA‐LMX assay was applied to sera from 289 individuals with known alloantigen exposure through pregnancy (n = 91) or kidney transplantation (n = 198), IgA HLA antibodies were detected in 3.5% of individuals; eight patients on the kidney retransplant waitlist and two women immunized through pregnancy. The majority (90%) of IgA HLA antibodies were directed against HLA class II and were always present in conjunction with IgG HLA antibodies. Results of this study show that this validated IgA‐LMX method can serve as a screening assay for IgA HLA antibodies and that the incidence of IgA HLA antibodies in alloantigen exposed individuals is low. [ABSTRACT FROM AUTHOR]

Over the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses theoretically defined polymorphic amino acid configurations, called eplets, for HLA compatibility analysis. Although consideration of eplet mismatch loads has potential for immunological risk stratification of transplant patients, the use of eplet matching in organ allocation algorithms is hindered by lacking knowledge of the immunogenicity of individual eplets, and the possibility that single mismatched amino acids, rather than complete eplets, are responsible for HLA antibody induction. There are several approaches to define eplet immunogenicity, such as antibody verification of individual eplets, and data-driven approaches using large datasets that correlate specific eplet mismatches to donor specific antibody formation or inferior transplant outcomes. Data-driven approaches can also be used to define whether single amino acid mismatches may be more informative than eplet mismatches for predicting HLA antibody induction. When using epitope knowledge for the assignment of unacceptable antigens, it important to realize that alleles sharing an eplet to which antibodies have formed are not automatically all unacceptable since multiple contact sites determine the binding strength and thus biological function and pathogenicity of an antibody, which may differ between reactive alleles. While the future looks bright for using HLA epitopes in clinical decision making, major steps need to be taken to make this a clinical reality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The impact of HLA-DP mismatches on renal allograft outcome is still poorly understood and is suggested to be less than that of the other HLA loci. The common association of HLA-DP donor-specific antibodies (DSA) with other DSA obviates the evaluation of the actual effect of HLA-DP DSA., Methods: From a large multicenter data collection, we retrospectively evaluated the significance of HLA-DP DSA on transplant outcome and the immunogenicity of HLA-DP eplet mismatches with respect to the induction of HLA-DP DSA. Furthermore, we evaluated the association between the MFI of HLA-DP antibodies detected in Luminex assays and the outcome of flowcytometric/complement-dependent cytotoxicity (CDC) crossmatches., Results: In patients with isolated pretransplant HLA-DP antibodies (N = 13), 6 experienced antibody-mediated rejection (AMR) and 3 patients lost their graft. In HLAMatchmaker analysis of HLA-DP mismatches (N = 72), HLA-DP DSA developed after cessation of immunosuppression in all cases with 84DEAV (N = 14), in 86% of cases with 85GPM (N = 6/7), in 50% of cases with 56E (N = 6/12) and in 40% of cases with 56A mismatch (N = 2/5). Correlation analysis between isolated HLA-DP DSA MFI and crossmatches (N = 90) showed negative crossmatch results with HLA-DP DSA MFI <2000 (N = 14). Below an MFI of 10,000 CDC crossmatches were also negative (N = 33). Above these MFI values both positive (N = 35) and negative (N = 16) crossmatch results were generated., Conclusions: Isolated HLA-DP DSA are rare, yet constitute a significant risk for AMR. We identified high-risk eplet mismatches that can lead to HLA-DP DSA formation. We therefore recommend HLA-DP typing to perform HLA-DP DSA analysis before transplantation. HLA-DP DSA with high MFI were not always correlated with positive crossmatch results., (Copyright © 2020 Elsevier B.V. All rights reserved.)

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors., (Copyright © 2019 Ferrata Storti Foundation.)

Recent data suggest that HLA epitope matching is beneficial for the prevention of de novo donor specific antibody (DSA) formation after transplantation. In this review, different approaches to predict the immunogenicity of an HLA mismatch will be discussed. The parameters used in these models are often called epitopes but the actual antibody epitope is far more complex. Exact knowledge of the antibody epitope is crucial if epitope matching is also used as a tool to select compatible donors for (highly) sensitized patients. Evidence is provided that it is not always possible to give an exact definition of an antibody epitope. We conclude that HLA "epitope" matching is superior over HLA antigen matching with respect to the prevention of de novo DSA formation and will enhance the prediction of acceptable HLA mismatches for sensitized patients. However, epitope matching at our current level of knowledge will not solve all histocompatibility problems as unexpected antibody reactivity still may occur., (© 2018 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis; however, its cause(s) and early pathogenesis are unclear. We hypothesized that a bile acid-induced ductular reaction (DR) drives fibrogenesis. The DR was evaluated by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD, and it demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction, liver progenitor cells (LPCs) were treated with taurocholate, and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation, and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin 19, connexin 43, integrin β4, and γ-glutamyltranspeptidase), whereas the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia + LPCs, and increased active γ-glutamyltranspeptidase enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α, and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7 + DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)