In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be... Show moreIn kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level. Show less
In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of... Show moreIn kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants. Show less
Kramer, C.S.M.; Bezstarosti, S.; Franke-van Dijk, M.E.I.; Vergunst, M.; Roelen, D.L.; Uyar-Mercankaya, M.; ... ; Heidt, S. 2024
In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation... Show moreIn solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting. Show less
Betriu, S.; Rovira, J.; Arana, C.; García-Busquets, A.; Matilla-Martinez, M.; Ramirez-Bajo, M.J.; ... ; Diekmann, F. 2023
The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal... Show moreThe presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation. Show less
Gille, I.; Hagedoorn, R.S.; Meer-prins, E.M.W. van der; Heemskerk, M.H.M.; Heidt, S. 2023
HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ... Show moreHLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3? domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8(+) T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFN? secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFN? with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the a3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation. Show less
Organs transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the... Show moreOrgans transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA-affinity as well as DSA-concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA-specific monoclonal antibodies and assessed the technology-specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow-induced dispersion analysis (FIDA). While the first three (solid-phase) technologies revealed comparable high binding-strengths, suggesting measurement of avidity, the latter (in-solution) approach revealed slightly lower binding-strengths, presumably indicating measurement of affinity. We believe that our newly developed in-solution FIDA-assay is particularly suitable to provide useful clinical information by not just measuring DSA-affinities in patient serum samples but simultaneously delivering a particular DSA-concentration. Here, we investigated DSA from 20 pre-transplant patients, all of whom showed negative CDC-crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA-concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449-fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre-transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA-concentration and DSA-affinity. Show less
Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in... Show moreLuminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r(2): 0.946, class II r(2): 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r(2): 0.952, class II r(2): 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended. Show less
Recently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The... Show moreRecently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The current sub-study analyzed formation of de novo donor-specific HLA antibodies (dnDSA) in the context of the degree of HLA eplet mismatches. At the time of protocol biopsy at 6 months, 7/29 patients (24%) in the MSC group and 1/27 patient (3.7%) in the control group had developed dnDSA. In the MSC group, all dnDSA were antiHLA-DQ; two patients had anti-DQ alone and five patients combined with anti-class I, HLA-DR or-DP. Despite excess dnDSA formation in the MSC-arm of the study, the evolution of eGFR (CKD-EPI) and proteinuria were comparable 2 years posttransplant. All dnDSA were complement-binding and three patients had antibody-mediated rejection in the protocol biopsy, but overall rejection episodes were not increased. Everolimus had to be discontinued in nine patients because of toxicity, and tacrolimus was reintroduced in six patients because of dnDSA formation. The HLA-DQ eplet mismatch load independently associated with dnDSA (adjusted hazard ratio = 1.07 per eplet mismatch, p = 0.008). A threshold of >= 11 HLA-DQ eplet mismatches predicted subsequent dnDSA in all 11 patients in the MSC group, but specificity was low (44%). Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized maintenance immunosuppression in kidney transplantation. Show less
Matern, B.M.; Niemann, M.; Nemparis, I.; Schimanski, A.; Peereboom, E.T.M.; Kramer, C.S.M.; ... ; Spierings, E. 2023
The International HLA and Immunogenetics Workshop (IHIW) is a recurring gathering of researchers, technologists and clinicians where participants contribute to collaborative projects with a variety... Show moreThe International HLA and Immunogenetics Workshop (IHIW) is a recurring gathering of researchers, technologists and clinicians where participants contribute to collaborative projects with a variety of goals, and come to consensus on definitions and standards for representing HLA and immunogenic determinants. The collaborative and international nature of these workshops, combined with the multifaceted goals of several specific workshop components, necessitates the collection and curation of a wide assortment of data, as well as an adaptable platform for export and analysis. With the aim of ensuring data quality and creation of reusable datasets, specific standards and nomenclature conventions are continuously being developed, and are an integral part of IHIW. Here we present the 18th IHIW Database, a purpose-built and extensible cloud-based file repository and web application for collecting and analyzing project-specific data. This platform is based on open-source software and uses established HLA data standards and web technologies to facilitate de-centralized data repository ownership, reduce duplicated efforts, and promote continuity for future IHIWs. Show less
Meng, T.; Bezstarosti, S.; Singh, U.; Yap, M.; Scott, L.; Petrosyan, N.; ... ; Heidt, S. 2022
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 angstrom radius. Eplet 44KM has been previously... Show moreEplet 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 angstrom 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. Show less
HLA serological specificities were defined by the reactivity of HLA molecules with sets of sera and monoclonal antibodies. Many recently identified alleles defined by molecular typing lack their... Show moreHLA serological specificities were defined by the reactivity of HLA molecules with sets of sera and monoclonal antibodies. Many recently identified alleles defined by molecular typing lack their serotype assignment. We surveyed the literature describing the correlation of the reactivity of serologic reagents with AA residues. 20 - 25 AA residues determining epitopes (DEP) that correlated with 82 WHO serologic specificities were identified for HLA class I loci. Thirteen DEP each located in the beta-1 domains that correlated with 24 WHO serologic specificities were identified for HLA-DRB1 and -DQB1 loci. The designation of possible HLA-DPB1, -DQA1, -DPA1, and additional serological specificities that result from epitopes defined by residues located at both -DQA1 and -DQB1 subunits were also examined. HATS software was developed for automated serotype assignments to HLA alleles in one of the three hierarchical matching criteria: (1) all DEP (FULL); (2) selected DEP specific to each serological specificity (SEROTYPE); (3) one AA mismatch with one or more SEROTYPES (INCOMPLETE). Results were validated by evaluating the alleles whose serotypes do not correspond to the first field of the allele name listed in the HLA dictionary. Additional 85 and 21 DEP patterns that do not correspond to any WHO serologic specificities for common HLA class I and DRB1 alleles were identified, respectively. A comprehensive antibody identification panel would allow for accurate unacceptable antigen listing and compatibility predictions in solid organ transplantation. We propose that antibody-screening panels should include all serologic specificities identified in this study. Show less
The International human leukocyte antigen (HLA) and Immunogenetics Workshops (IHIWs) have fostered international collaborations of researchers and experts in the fields of HLA, histocompatibility... Show moreThe International human leukocyte antigen (HLA) and Immunogenetics Workshops (IHIWs) have fostered international collaborations of researchers and experts in the fields of HLA, histocompatibility and immunology. These IHIW collaborations have comprised many projects focused on achieving a variety of specific goals. The international and collaborative nature of these projects necessitates the collection and analysis of complex data generated in multiple laboratories, often using multiple methods of acquisition. Collection and storage of these data in a consistent way adds value to IHIW projects, which can be extended to future work. DNA-based genotyping data, especially HLA genotyping data, can be transmitted in the form of a Histoimmunogenetics Markup Language (HML) document. HML facilitates clear communication of a genotype and supporting metadata, such as, sequencing platform, laboratory assays, consensus sequence, and interpretation. Sequence information can be reported relative to known reference sequences, which add meaning and context to genotypes. Selecting the correct reference sequence for a given allele sequence is nuanced, and guidelines have emerged through collaborative community efforts such as Data Standards Hackathons. Here, we describe the guidelines established for the selection of reference sequences to be used in transmission of HLA (and MICA/MICB) genotyping data for the 18th IHIW. Show less
Car, H.; Karahan, G.E.; Dreyer, G.J.; Brand-Schaaf, S.H.; Vries, A.P.J. de; Kooten, C. van; ... ; Heidt, S. 2020
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... Show moreHuman 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. Show less
Drabbels, J.J.M.; Welleweerd, R.; Rooy, I. van; Johnsen, G.M.; Staff, A.C.; Haasnoot, G.W.; ... ; Eikmans, M. 2020
Polymorphic sites in the HLA-G gene may influence expression and function of the protein. Knowledge of the association between high-resolution HLA-G alleles and 3-prime untranslated (3 ' UTR)... Show morePolymorphic sites in the HLA-G gene may influence expression and function of the protein. Knowledge of the association between high-resolution HLA-G alleles and 3-prime untranslated (3 ' UTR) haplotypes is useful for studies on the role of HLA-G in transplantation, pregnancy, and cancer. We developed a next generation sequencing (NGS)-based typing assay enabling full phasing over the whole HLA-G gene sequence with inclusion of the 3 ' UTR region. DNA from 171 mother-child pairs (342 samples) was studied for: (a) HLA-G allele information by the NGSgo-AmpX HLA-G assay, (b) 3 ' UTR haplotype information by an in-house developed sequence-based typing method of a 699/713 base pair region in the 3 ' UTR, and (c) the full phase HLA-G gene sequence, by combining primers from both assays. The mother to child inheritance allowed internal verification of newly identified alleles and of association between coding and UTR regions. The NGSgo workflow compatible with Illumina platforms was employed. Data was interpreted using NGSengine software. In 99.4% of all alleles analyzed, the extended typing was consistent with the separate allele and 3 ' UTR typing methods. After repeated analysis of four samples that showed discrepancy, consistency reached 100%. A high-linkage disequilibrium between IPD-IMGT/HLA Database-defined HLA-G alleles and the extended 3 ' UTR region was identified (D ' = 0.994, P < .0001). Strong associations were found particularly between HLA-G*01:04 and UTR-3, between HLA-G*01:01:03 and UTR-7, and between HLA-G*01:03:01 and UTR-5 (for all: r = 1). Six novel HLA-G alleles and three novel 3 ' UTR haplotype variants were identified, of which three and one, respectively, were verified in the offspring. Show less
In renal transplantation, polymorphic amino acids on mismatched donor HLA molecules can lead to the induction of de novo donor-specific antibodies (DSA), which are associated with inferior graft... Show moreIn renal transplantation, polymorphic amino acids on mismatched donor HLA molecules can lead to the induction of de novo donor-specific antibodies (DSA), which are associated with inferior graft survival. To ultimately prevent de novo DSA formation without unnecessarily precluding transplants it is essential to define which polymorphic amino acid mismatches can actually induce an antibody response. To facilitate this, we developed a user-friendly software program that establishes HLA class I and class II compatibility between donor and recipient on the amino acid level. HLA epitope mismatch algorithm (HLA-EMMA) is a software program that compares simultaneously the HLA class I and class II amino acid sequences of the donor with the HLA amino acid sequences of the recipient and determines the polymorphic solvent accessible amino acid mismatches that are likely to be accessible to B cell receptors. Analysis can be performed for a large number of donor-recipient pairs at once. As proof of principle, a previously described study cohort of 191 lymphocyte immunotherapy recipients was analysed with HLA-EMMA and showed a higher frequency of DSA formation with higher number of solvent accessible amino acids mismatches. Overall, HLA-EMMA can be used to analyse compatibility on amino acid level between donor and recipient HLA class I and class II simultaneously for large cohorts to ultimately determine the most immunogenic amino acid mismatches. Show less
