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Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3μg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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 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. [ABSTRACT FROM AUTHOR]

Aim Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 μ g/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results As shown, sHLA-A ∗ 02:01 was able to 100% neutralize A2-related responses, leaving other HLA/antibody interactions untouched. In this example, when also subtracting B ∗ 08:01 and B ∗ 49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection. [ABSTRACT FROM AUTHOR]

Aim The measurement of rejection-causing HLA Abs in serological samples of HLA sensitized individuals has clinical relevance in the evaluation of pre- and post-transplant patients. However, due to the increasing demand for more accurate and meaningful data, the determination of MFI values alone is not sufficient anymore to evaluate a patient’s risk potential. In this study, we aimed to demonstrate the enormous complexity of Ab patterns in serological specimen. Methods To visualize variations and different levels of HLA Ab patterns, several investigative tools were applied including HLA Ab-detection assays, neutralization experiments, sera titration analysis and comparative information from human monoclonal Ab profiles derived from single B cells. Results In tier 1, neutralization experiments clearly demonstrated that original response patterns could be deconvoluted into individual sub-patterns and single alleles identified as primary cause of an immunizing event. Depending on exposure, one or several antigens were generally involved. Tier 2 patterns were originated from a single epitope harbored by the immunizing antigen of origin that naturally cross-reacts with related epitope-sharing antigens. Currently, over 100 human monoclonal Ab patterns were collected defining such epitopes and being used to further resolve Tier 1 patterns by a pattern recognition algorithm. Tier 3 involves either individual epitopes shared among related antigens or Abs that recognize a specific HLA/Ab interaction but not necessarily at the same affinity. Tier 4 includes diverse epitope patterns that appear using different isotype-recognizing detection systems. Tier 5 refers to experiments conducted under a time interval, visualizing the appearance/disappearance of different Ab species in time. Conclusion We believe that the knowledge gained about the apparent complexity of specific immune patterns will help us to reach the next chapter of patient care and contribute to a better risk management in Ab-related graft rejection. [ABSTRACT FROM AUTHOR]

Aim: Due to the increasing demand for more accurate and meaningful data in antibody detection assays, which includes quantitative measures of antibody titers, we evaluated a series of serological specimens to demonstrate the benefit of antibody titration analysis. We show that by identifying the linear range of the assay, semi-quantitative observations are possible where titer comparisons can be easily made and saturation issues such as “prozon effects” are eliminated. Methods: To determine linear range and half maximal effective concentrations (EC50), serological specimens from highly sensitized patients were diluted to eight different concentrations and their MFI signal determined using an in-house, 120-allele Luminex-based single specificity solid phase assay platform. Results: Our results show that individual, single-allele titration curves can be generated to establish optimal performance range. We found that titration analysis has two major benefits: (1) eliminating serological interferences at high concentrations, and (2) semi-quantitative observations within the linear range. According to our test series, most sera will cause interferences and high background if tested undiluted. Accurate determinations can only be made by applying serial dilutions to reach highest signal-to-background ratios. Conclusions: Since the physiological responses of the adaptive immune system against an HLA target are highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies has been extremely difficult. Titration analysis offers the possibility to evaluate several of such functional traits of antibodies to any given HLA molecule allowing better interpretation of individual antibody populations in a quantitative matter. This project will likely lead to a better assessment of HLA titers and generate new insights for post-transplant monitoring and early recognition of immunologic rejection. [Copyright &y& Elsevier]

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (k on, 3.63–24.25 × 10 5 per mol per second) and dissociation rates (k off , 0.99–10.93 × 10 −3 per second). Dissociation constants (K D ) ranged from 5.9 × 10 −10  M to 3.0 × 10 −8  M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA. [ABSTRACT FROM AUTHOR]

For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10 6 B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients. [ABSTRACT FROM AUTHOR]

Aim: Strong humoral responses to allogeneic HLA preclude organ transplantation, promote hyperacute rejection, and contribute to chronic transplant rejection. Therapies exist to prevent B cells from secreting antibodies, and bulk antibody removal is also an option, but there is no means to specifically deplete antibodies that recognize an allogeneic HLA molecule while leaving humoral immunity largely intact. Here, we tested a sHLA Antibody Removal Column (SHARC) for removing anti-HLA antibodies from liter volumes of patient samples. Methods: Approximately 70 milligrams of the soluble class II molecule HLA-DRB1∗11:01/DRA1∗01:01 (DR11) produced in mammalian cells was covalently coupled to a solid support matrix. The HLA-DR11 matrix was packed into a column for the purpose of removing anti-DR11antibodies from patient plasma. 2.5L of patient plasma containing anti-DR11 antibodies was run over the column at a clinically relevant flow rate of 40ml/min. Fractions of the flow through were taken and anti-HLA-DR11 antibodies were measured using a single antigen bead assay. Results: When patient plasma recognizing multiple HLA was passed over the DR11 matrix, antibodies specific for DR11were removed while antibodies for other class II (DQ) passed through the SHARC intact. Additionally there was no change in bulk antibody concentration after the plasma was passed over the column. DR11 specific antibodies that bound to the column were recovered by elution and retained their reactivity for DR11 as well as other specificities with expected shared cross reactivity. Conclusions: Native class II molecules were coupled to an affinity matrix, antibodies specific for class II HLA were removed from patient plasma, and the depleted antibodies were recovered intact from the column. These data represent a proof of concept for selective antibody desensitization as a potential therapeutic. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

Summary: Dendritic cells (DCs) are highly specialized antigen-presenting cells of the immune system that are efficient at presenting peptide-antigen for the activation of T cells and are often the cell type of choice for vaccine targeting by virtue of high expression levels of MHC and costimulatory molecules. Since the level of peptide–MHC complex significantly influences stimulation of T cells, a proof-of-concept potency assay was developed to directly examine the presentation and density of MHC class I peptides derived from the processing of a model tumor antigen, (hCGβ), on the surface of DCs. In this study we first generated antibodies (TCR mimics or TCRm) to two peptide-HLA-A*0201 epitopes derived from hCGβ designated as TMT (40–48) and GVL (47–55). Characterization of each TCRm by ELISA and flow cytometric analysis, demonstrated specific binding to soluble recombinant HLA-A2 protein and HLA-A2.1+ T2 cells loaded with relevant peptide. TCRm reactive against the TMT and GVL epitopes blocked granzyme-B production by peptide-specific cytotoxic T lymphocytes (CTL) lines further supporting their recognition specificity. For the assessment of antigen presentation function, human immature monocyte-derived DCs (iDCs) were treated with the mannose receptor targeting vaccine, B11-hCGβ and matured with Poly I:C. The TMT and GVL epitope reactive CTL lines responded to vaccine-treated but not vehicle-treated mature DCs (mDCs) with TMT and GVL TCRm specifically blocking IFN-γ production. The TCRm were then used to directly confirm specific peptide–MHC complexes on mDCs. TCRm staining of vaccine-treated mDCs showed detection of the TMT and GVL peptide-HLA-A2 complexes. These findings demonstrate that TCRms may be important tools for determining the potency of DC-based vaccines. [Copyright &y& Elsevier]

Aim: Therapies exist for the bulk removal of antibodies in sensitized transplant patients, but there are no means to specifically deplete antibodies that recognize a specific HLA molecule. Current bulk antibody removal procedures may leave a patient compromised immunologically and more susceptible to opportunistic infections. The goal of this project was to create specific HLA-A∗02:01 removal matrices and demonstrate their ability to capture defined anti-HLA antibodies. Methods: Soluble class I HLA-A∗02:01 molecules were produced in a native conformation in mammalian cells, purified by affinity chromatography, coupled to a Sepharose matrix, and loaded into a column enclosure. Various columns were created and tested with an automated chromatography system by initial injection of various HLA antibodies under different loading conditions. After an extensive column wash, the bound antibodies were released by a pH shift and quantitated. Loading and elution profiles were recorded. Results: Initial tests show that HLA columns maintain their structural integrity and function. Multiple passes of the antibody W6/32, recognizing only intact HLA molecules, resulted in consistent and repeatable binding patterns. During the entire performance process, several parameters were identified, determining column saturation, capacity and efficiency values. Specificity was shown by removing a typical A∗02:01 recognition pattern from an A2 sera. In addition, treatment conditions were established to reuse the device. Conclusions: Anti-HLA antibody removal devices are highly efficient in selectively depleting HLA antibodies. Capable of removing up to 1 mg per ml of matrix depending on the avidity of the antibody makes this device very efficient for research studies of HLA antibodies and provides promise for therapeutic applications. Furthermore, this device might also find an application in HLA diagnostics, allowing for the deconvolution of serologic samples and providing a new way to evaluate high PRA patients. [Copyright &y& Elsevier]