Publications by authors named "Jar-How Lee"

27 Publications

  • Page 1 of 1

Development and Validation of a Multiplex, Bead-based Assay to Detect Antibodies Directed Against SARS-CoV-2 Proteins.

Transplantation 2021 01;105(1):79-89

Division of Laboratory Medicine, Department of Pathology, Emory University, Atlanta, GA.

Background: Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining the status of antibodies against SARS-CoV-2 in both candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 in this population. While there are multiple tests to detect antibodies to SARS-CoV-2, their performance is variable. Tests vary according to their platforms and the antigenic targets which make interpretation of the results challenging. Furthermore, for some assays, sensitivity and specificity are less than optimal. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses rather than SARS-CoV-2.

Methods: This study describes the development and validation of a high-throughput multiplex antibody detection assay.

Results: The multiplex assay has the capacity to identify, simultaneously, patient responses to 5 SARS-CoV-2 proteins, namely, the full spike protein, 3 individual domains of the spike protein (S1, S2, and receptor binding domain), and the nucleocapsid protein. The antibody response to the above proteins are SARS-CoV-2-specific, as antibodies against 4 common coronaviruses do not cross-react.

Conclusions: This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting. Test configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients.
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http://dx.doi.org/10.1097/TP.0000000000003524DOI Listing
January 2021

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

Cell 2020 Nov 21;183(5):1264-1281.e20. Epub 2020 Oct 21.

Neuroimmunology and MS Research, Neurology Clinic, University Hospital Zurich, University of Zurich, Zurich 8091, Switzerland. Electronic address:

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4 T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4 T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4 T cells in MS.
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http://dx.doi.org/10.1016/j.cell.2020.09.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707104PMC
November 2020

Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation.

Am J Transplant 2020 05 18;20(5):1365-1374. Epub 2020 Jan 18.

Laboratoire d'Immunologie et Immunogénétique, CHU de Bordeaux, Hôpital Pellegrin, Bordeaux, France.

Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti-HLA-Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti-denatured and anti-native HLA-Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA-Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty-three (31.6%) were anti-denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ = 0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ = 0.289). We studied 52 kidney recipients with preformed anti-HLA-Cw donor-specific antibodies. Those with anti-native HLA antibodies experienced more acute and chronic antibody-mediated rejections (P = .006 and .03, respectively), and displayed a lower graft survival (P = .04). Patients with anti-native HLA-Cw antibodies more frequently had previous sensitizing events (P < .000001) or plausibility of their antibody profile according to known anti-native HLA-Cw eplets (P = .0001). Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.
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http://dx.doi.org/10.1111/ajt.15766DOI Listing
May 2020

A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44.

Nat Immunol 2019 09 29;20(9):1129-1137. Epub 2019 Jul 29.

Research Department Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.

Natural killer (NK) cells can recognize virus-infected and stressed cells using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations. Using NKp44ζ reporter cells and primary human NKp44 NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection, graft-versus-host disease and inflammatory bowel disease.
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http://dx.doi.org/10.1038/s41590-019-0448-4DOI Listing
September 2019

Measuring anti-HLA antibody active concentration and affinity by surface plasmon resonance: Comparison with the luminex single antigen flow beads and T-cell flow cytometry crossmatch results.

Mol Immunol 2019 04 16;108:34-44. Epub 2019 Feb 16.

CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Place Amélie Raba Léon, Bordeaux, France; Immuno ConcEpT, UMR CNRS 5164, 146 rue Léo Saignat, Bordeaux, France; Université de Bordeaux, 146 rue Léo Saignat, Bordeaux, France.

Background: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi-quantitative fluorescence read-out is not closely linked to graft outcome.

Methods: Surface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM).

Results: The five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFB MFI resulted in affinity variations evidenced by SPR.

Conclusion: The SAFB MFI and T-FCXM ratio, both semi-quantitative parameters, only partially reflected the subtlety of the anti-HLA antibody/antigen interaction as it can be analyzed by SPR. Future clinical studies using SPR for anti-HLA antibodies characterization could bring novel insights into the understanding of HLA/anti-HLA interaction and therefore anti-HLA antibodies pathogenicity.
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http://dx.doi.org/10.1016/j.molimm.2019.02.006DOI Listing
April 2019

Mapping molecular HLA typing data to UNOS antigen equivalents.

Hum Immunol 2018 Nov 15;79(11):781-789. Epub 2018 Aug 15.

Department of Pathology and Laboratory Medicine, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, USA. Electronic address:

Background: Histocompatibility labs must convert molecular HLA typing data to antigen equivalencies for entry into the United Network for Organ Sharing (UNOS) UNet system. While an Organ Procurement and Transplantation Network (OPTN) policy document provides general guidelines for conversion, the process is complex because no antigen mapping table is available. We present a UNOS antigen equivalency table for all IPD-IMGT/HLA alleles at the A, B, C, DRB1, DRB3/4/5, DQA1, and DQB1 loci.

Methods: An automated script was developed to generate a UNOS antigen equivalency table. Data sources used in the conversion algorithm included the World Marrow Donor Association (WMDA) antigen table, the HLA Dictionary, and UNOS-provided tables. To validate antigen mappings, we converted National Marrow Donor Program (NMDP) high resolution allele frequencies to antigen equivalents and compared with the UNOS Calculated Panel Reactive Antibodies (CPRA) reference panel.

Results: Normalized frequency similarity scores between independent NMDP and UNOS panels for 4 US population categories (Caucasian, Hispanic, African American and Asian/Pacific Islander) ranged from 0.85 to 0.97, indicating correct antigen mapping. An open source web application (ALLele to ANtigen ("ALLAN")) and web services were also developed to map unambiguous and ambiguous HLA typing data to UNOS antigen equivalents based on NMDP population-specific allele frequencies (http://www.transplanttoolbox.org).

Conclusions: Computer-assisted interpretation of molecular HLA data may aid in reducing typing discrepancies in UNet. This work also sets a foundation for molecular typing data to be utilized directly in the UNet match run as well as the virtual crossmatch process at transplant centers.
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http://dx.doi.org/10.1016/j.humimm.2018.08.002DOI Listing
November 2018

Letter to the editor.

Authors:
Jar-How Lee

J Immunol Methods 2019 11 16;474:112449. Epub 2018 May 16.

Transplant Diagnostics, Division of Thermo Fisher Scientific, Canoga Park, CA, United States. Electronic address:

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http://dx.doi.org/10.1016/j.jim.2018.05.001DOI Listing
November 2019

Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.

HLA 2017 02;89(2):90-97

Departments of Oncology and Pediatrics, Georgetown University, Washington, District of Columbia.

Background: Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype.

Materials And Methods: Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations.

Results: Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons.

Conclusion: Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.
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http://dx.doi.org/10.1111/tan.12951DOI Listing
February 2017

Reassessment of T Lymphocytes Crossmatches Results Prediction With Luminex Class I Single Antigen Flow Beads Assay.

Transplantation 2017 03;101(3):624-630

1 Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 2 UMR 5164 CNRS, Université Bordeaux Segalen, Bordeaux, France. 3 One Lambda, Inc., Canoga Park, CA. 4 Service de Néphrologie, Transplantation, Dialyse, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.

Background: In virtual crossmatch (XM) strategies, a correct anticipation of XM results is required for appropriately allocating organs. We reassessed the ability to predict T lymphocyte flow cytometry and complement dependent cytotoxicity XM results with the mean fluorescence intensity (MFI) in Luminex class I single antigen flow beads (SAFB) assay, after correction of complement interference and exclusion of antidenatured HLA antibodies.

Methods: Among 432 XM with T lymphocytes (T-XM), 407 were analyzed after exclusion of antidenatured HLA antibodies. Only ethylenediaminetetraacetic acid-treated serum MFI was considered. Only 1 cellular target HLA antigen for the serum was expressed in 238 cases, 209 and 29 being heterozygous and homozygous for this antigen, respectively. For the remaining 169 cases, at least 2 antigens were recognized. Single antigen flow bead MFI thresholds allowing XM positivity to be predicted were calculated with receiver operating characteristic curves.

Results: T-XM results were tightly associated with SAFB MFI values. Anti-HLA-A and anti-HLA-B antibodies behaved similarly. Prediction and sensitivity SAFB MFI thresholds were determined, respectively, assessing the highest sensitivity/specificity ratio and at least 95% sensitivity for predicting T-XM positivity. Both were slightly lower for HLA-B than for HLA-A, whereas anti-HLA-Cw antibodies induced random XM results. Both thresholds were only slightly diminished for homozygous and for multiple HLA targets, considering the immunodominant, but not the sum, of antibodies MFI.

Conclusions: Antibodies against HLA-A, -B, or -Cw behave differently. A homozygous HLA target does not trigger a twice higher XM positivity. Multiple antibodies are better evaluated through the immunodominant DSA MFI than through the sum of DSA MFI.
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http://dx.doi.org/10.1097/TP.0000000000001239DOI Listing
March 2017

C1q-binding anti-HLA antibodies do not predict platelet transfusion failure in Trial to Reduce Alloimmunization to Platelets study participants.

Transfusion 2016 06 15;56(6):1442-50. Epub 2016 Apr 15.

Blood Systems Research Institute, San Francisco, California.

Background: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 subjects became clinically refractory (CR) to platelets (PLTs) without lymphocytotoxicity assay (LCA)-detectable anti-HLA antibodies. The LCA only detects complement-binding antibodies and is less sensitive than newer assays. Utilizing a more sensitive bead-based assay that does not distinguish between complement-binding versus non-complement-binding antibodies, we have previously shown that while many LCA-negative (LCA-) patients do have anti-HLA antibodies, these low- to moderate-level antibodies do not predict refractoriness. As complement can contribute to PLT rejection, we assessed if previously undetected complement-binding antibodies account for refractoriness among LCA- patients.

Study Design And Methods: Samples from 169 LCA- (69 CR, 100 non-CR) and 20 LCA-positive (LCA+; 10 CR, 10 non-CR) subjects were selected from the TRAP study serum repository. Anti-Class I HLA immunoglobulin (Ig)G and C1q-binding antibodies were measured in serum or plasma with bead-based detection assays. Levels of C1q-binding antibodies were compared between CR and non-CR subjects and correlated with corrected count increments (CCIs).

Results: While some of the LCA- subjects had detectable C1q-binding anti-Class I HLA antibodies, and some LCA+ subjects did not, levels were significantly higher among LCA+ subjects. C1q-binding anti-Class I HLA antibody levels did not differ significantly between CR and non-CR among either the LCA- or the LCA+ subjects. Furthermore, there was no significant correlation observed between CCIs and either C1q-binding or any anti-HLA IgG antibodies.

Conclusions: This work confirms that low- to moderate-level anti-Class I antibodies do not drive PLT rejection, suggesting a role for antibody-independent mechanisms.
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http://dx.doi.org/10.1111/trf.13598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016838PMC
June 2016

Deciphering IgM interference in IgG anti-HLA antibody detection with flow beads assays.

Hum Immunol 2016 Nov 20;77(11):1048-1054. Epub 2016 Feb 20.

CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Bordeaux, France; Université de Bordeaux, UMR CNRS, 5164 Talence, France. Electronic address:

In flow beads assays, the interference of IgM for IgG anti-HLA antibodies detection is not precisely understood. Using the screening flow beads assay for class I HLA antibodies, we analyzed the binding of two IgG mAbs, the anti-class I HLA W6/32 and an anti-beta-2-microglobulin, in the presence of an anti-beta-2-microglobulin IgM mAb. In neat serum, the IgM mAb impaired the detection of both IgG. In EDTA-treated serum, the interference was stronger for the anti-beta-2-microglobulin IgG than for W6/32, in agreement with the finding in surface plasmon resonance that this IgM competed with the anti-beta-2-microglobulin IgG but not with W6/32. The IgM interference was higher in neat than in EDTA-treated serum for both IgG mAbs. The IgM interference was also analyzed with class II single antigen flow beads and sera from two kidney recipients containing IgG and IgM donor specific antibodies. Anti-HLA IgG detection was partially corrected by EDTA, and restored by IgM inactivation with DTT, confirming the results observed with the mAbs. Therefore, three mechanisms can explain the IgM interference for IgG anti-HLA antibodies in flow beads assays: direct competition for antigen, steric hindrance and complement activation.
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http://dx.doi.org/10.1016/j.humimm.2016.02.008DOI Listing
November 2016

Calibration free concentration analysis by surface plasmon resonance in a capture mode.

Talanta 2016 Feb 10;148:478-85. Epub 2015 Nov 10.

Université de Bordeaux, Laboratoire ARNA, 146 rue Léo Saignat, Bordeaux, France; INSERM, U869, Laboratoire ARNA, IECB, 2 rue Robert Escarpit, Pessac, France. Electronic address:

Surface plasmon resonance (SPR) is the gold standard for determining rate and equilibrium constants of bimolecular complexes. Accuracy of these parameters depends on the correct determination of the concentration of the injected analyte. Calibration free concentration analysis (CFCA) has been developed to overcome the limitation of measuring protein concentrations spectroscopically, which may overestimate the fraction of the protein that really binds to the immobilized ligand, i.e. the active concentration. In this work, we demonstrate that CFCA can also be implemented in a capture format for measuring active concentrations. Capture CFCA (CCFCA) was first validated by measuring the concentration of a HLA-B*44:02 antigen solution. The active concentration of this molecule determined by CCFCA was similar to that obtained by covalent CFCA. CCFCA was then used to determine the concentration of the W6/32 pan class I HLA monoclonal antibody over three different HLA molecules captured by another specific antibody. This could not have been performed by covalent CFCA because immobilized HLA molecules cannot withstand regeneration. By exploring different capture levels we also show that CCFCA gives consistent results even at low capture levels. Knowing the active concentration of W6/32, we then determined the rate and equilibrium constants of W6/32-HLA complexes on the same flow cell. CCFCA is of general use for measuring active concentrations and of great interest for analytes recognizing ligands that cannot be covalently immobilized on sensor chips. The capture mode also allows determining the kinetic constants of multiple analyte-ligand complexes on the same flow cell. This increases experiments throughput and reduces sensor chip consumption.
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http://dx.doi.org/10.1016/j.talanta.2015.11.025DOI Listing
February 2016

Evaluation of the iBeads assay as a tool for identifying class I HLA antibodies.

Hum Immunol 2015 Sep 25;76(9):651-6. Epub 2015 Sep 25.

Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France; UMR CNRS 5164, Université de Bordeaux, Bordeaux, France. Electronic address:

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs.
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http://dx.doi.org/10.1016/j.humimm.2015.09.012DOI Listing
September 2015

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation.

Eur J Immunol 2015 Jul 28;45(7):2111-21. Epub 2015 Apr 28.

Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum.
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http://dx.doi.org/10.1002/eji.201445340DOI Listing
July 2015

Clinical impact of preformed donor-specific denatured class I HLA antibodies after kidney transplantation.

Clin Transplant 2015 May 13;29(5):393-402. Epub 2015 Mar 13.

Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France; UMR CNRS 5164, Université de Bordeaux, Bordeaux, France.

Class I single-antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA-sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor-specific antibodies (DSA) using two different assays: an acid-treated SAFB assay (anti-dHLA DSA) and the iBeads assays (SAFB+/iBeads- DSA). Eighty-five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520-13 882]). Anti-dHLA and SAFB+/iBeads- DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500-1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti-dHLA DSA or only SAFB+/iBeads- DSA developed acute clinical antibody-mediated rejection in the first-year post-transplantation, and their five-yr death-censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T-cell flow cytometry cross-match. Therefore, both anti-dHLA DSA and SAFB+/iBeads- DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.
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http://dx.doi.org/10.1111/ctr.12529DOI Listing
May 2015

Denatured class I human leukocyte antigen antibodies in sensitized kidney recipients: prevalence, relevance, and impact on organ allocation.

Transplantation 2014 Oct;98(7):738-44

1 Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 2 UMR CNRS 5164, Université de Bordeaux, Bordeaux, France. 3 Agence de la Biomédecine, Saint-Denis La Plaine, France. 4 One Lambda, Inc., Canoga Park, CA. 5 Laboratoire d'histocompatibilité, Etablissement Français du Sang, Lyon, France. 6 Service de Néphrologie, Transplantation, Dialyse, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 7 Address correspondence to: Jean-Luc Taupin, Pharm.D., Ph.D., Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France.

Background: Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs).

Methods: Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated.

Results: Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P<0.0001). Removing the HLA-A and HLA-B antigens targeted by denatured HLA antibodies from unacceptable antigens lowered the calculated panel reactive antibody for 90 patients, sometimes dramatically. The iBeads assay demonstrated nearly the same ability to predict crossmatch results than the acid treatment assay.

Conclusion: Denatured class I HLA antibodies are common, but the antigens they target should not be considered as unacceptable in most cases, because they negatively impact access to a transplant while predominantly providing negative sensitive crossmatches. The iBeads assay seems to be a valuable alternative to better define unacceptable antigens.
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http://dx.doi.org/10.1097/TP.0000000000000229DOI Listing
October 2014

HLA-DR15-derived self-peptides are involved in increased autologous T cell proliferation in multiple sclerosis.

Brain 2013 Jun;136(Pt 6):1783-98

Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Centre for Molecular Neurobiology Hamburg, University Medical Centre Eppendorf, 20251 Hamburg, Germany.

The HLA-DR15 haplotype confers the largest part of the genetic risk to develop multiple sclerosis, a prototypic CD4+ T cell-mediated autoimmune disease. The mechanisms how certain HLA-class II molecules functionally contribute to autoimmune diseases are still poorly understood, but probably involve shaping an autoimmune-prone T cell repertoire during central tolerance in the thymus and subsequently maintaining or even expanding it in the peripheral immune system. Self-peptides that are presented by disease-associated HLA-class II molecules most likely play important roles during both processes. Here, we examined the functional involvement of the HLA-DR15 haplotype in autologous proliferation in multiple sclerosis and the contribution of HLA-DR15 haplotype-derived self-peptides in an in vitro system. We observe increased autologous T cell proliferation in patients with multiple sclerosis in relation to the multiple sclerosis risk-associated HLA-DR15 haplotype. Assuming that the spectrum of self-peptides that is presented by the two HLA-DR15 allelic products is important for sustaining autologous proliferation we performed peptide elution and identification experiments from the multiple sclerosis-associated DR15 molecules and a systematic analysis of a DR15 haplotype-derived self-peptide library. We identify HLA-derived self-peptides as potential mediators of altered autologous proliferation. Our data provide novel insights about perturbed T cell repertoire dynamics and the functional involvement of the major genetic risk factor, the HLA-DR15 haplotype, in multiple sclerosis.
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http://dx.doi.org/10.1093/brain/awt108DOI Listing
June 2013

Lot-to-lot variability in HLA antibody screening using a multiplexed bead-based assay.

Transfusion 2013 Sep 10;53(9):1940-7. Epub 2013 Jan 10.

Department of Laboratory Medicine and Pathology, Department of Biomedical Statistics & Informatics, Department of Anesthesiology, Division of Transfusion Medicine, Mayo Clinic, Rochester, Minnesota; Westat, Inc., Rockville, Maryland; One Lambda, Inc., Canoga Park, California; Deparment of Laboratory Medicine, University of California at San Francisco, Blood Systems Research Institute, San Francisco, California.

Background: Identifying antibodies to HLA (anti-HLA) by solid-phase assays is used to screen blood donors to mitigate transfusion-related acute lung injury risk. Various cutoffs for detection assays have been proposed in the literature; however, these do not take into consideration lot-to-lot variability of commercially available assays.

Study Design And Methods: Samples from 93 nontransfused males were tested using five different lots of a multiplex bead-based anti-HLA detection kit. A subset of 17 samples was tested on 5 days using a single lot. An additional 96 samples from donations with varied anti-HLA levels were tested using kits from two different lots. Results were reported as a normalized background (NBG) ratio.

Results: For the 93 nontransfused donors, NBG values generated using the reference lot were significantly higher than those obtained with three of the four comparator lots. However, for the 96 samples with low-, moderate-, and higher-level anti-HLA, Class I (CL-I) values were 1.4 times lower and Class II (CL-II) values were 1.2 times lower using the reference versus comparator lot. For CL-I antibodies the between-lot standard deviation (SD) was 1.36 (95% confidence interval [CI], 1.19-1.60), while the between-day SD was 1.27 (95% CI, 1.08-1.52). Similarly, for CL-II antibodies the between-lot SD was 0.81 (95% CI, 0.70-0.95), while the between-day SD was 0.50 (95% CI, 0.43-0.60).

Conclusions: There is interlot variability in the tested HLA detection assay as well as significant bias between lots. It may be reasonable to develop a new cutoff when a new lot is obtained.
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http://dx.doi.org/10.1111/trf.12064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626740PMC
September 2013

Clinical cytometry and progress in HLA antibody detection.

Methods Cell Biol 2011 ;103:285-310

Department of Pathology, Emory University, Atlanta, Georgia, USA.

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes.
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http://dx.doi.org/10.1016/B978-0-12-385493-3.00012-7DOI Listing
November 2011

Donor-specific antibody against denatured HLA-A1: clinically nonsignificant?

Hum Immunol 2011 Jun 9;72(6):492-8. Epub 2011 Mar 9.

Seattle Cancer Care Alliance, Clinical Research Division, Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01:01+ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pre-transplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre- and post-transplant sera versus DD cells was negative. Rescreening of multiple pre- and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patient's anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1+ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.
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http://dx.doi.org/10.1016/j.humimm.2011.02.012DOI Listing
June 2011

Agreement among HLA antibody detection assays is higher in ever-pregnant donors and improved using a consensus cutoff.

Transfusion 2011 May 18;51(5):1105-16. Epub 2010 Nov 18.

Blood Systems Research Institute and Blood Centers of the Pacific, San Francisco, California 94118, USA.

Background: HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). HLA antibody detection methods include ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay, and it is not obvious how to compare results across platforms.

Study Design And Methods: Five hundred twenty-five serum samples were selected from 7841 donors in the Leukocyte Antibody Prevalence Study (LAPS) repository based on risk for the development of HLA antibodies, using the number of pregnancies as the risk factor. Subjects included 81 males and females with 0 (n = 187), 1 (n = 67), or 2+ pregnancies (n = 190). Replicate frozen serum aliquots were sent blinded to four different HLA antibody assay manufacturers for detection using five different assays.

Results: The flow cytometry and multiplex bead based-assays typically resulted in a larger proportion of HLA antibody positive samples compared with ELISA based assays. Latent variable analysis was used to derive a new set of consensus cutoffs, which yielded similar sensitivities across test platforms and increased concordance amongst assays. Assay agreement was higher in ever pregnant females than in males and never-pregnant females.

Conclusions: Different assays resulted in varied positivity rates when the manufacturer's suggested cutoffs were used, demonstrating that care needs to be taken when comparing clinical outcomes data generated using different HLA antibody assays and testing platforms. The method used here, involving latent variable analysis, presents one possible approach to calculating comparable cutoffs that result in broad agreement across assays with respect to positivity designation.
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http://dx.doi.org/10.1111/j.1537-2995.2010.02938.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089710PMC
May 2011

Long-term in vitro reactivity for human leukocyte antigen antibodies and comparison of detection using serum versus plasma.

Transfusion 2009 Feb 29;49(2):243-51. Epub 2008 Oct 29.

Blood Systems Research Institute, University of California, San Francisco, USA.

Background: Human leukocyte antigen (HLA) antibodies are a possible cause of transfusion-related acute lung injury (TRALI), and fluorescent bead assays are often used for antibody detection. Serum is the manufacturer's recommended sample, but plasma may be easier to obtain for studies of HLA antibody prevalence and TRALI case investigations.

Study Design And Methods: Specimens were obtained from 44 multiparous females positive for the presence of HLA antibodies by lymphocytotoxicity testing at least 13 years prior and from 1000 contemporary blood donors. Screening tests were performed using a multiplex bead-based assay. In addition to comparing results obtained with paired plasma and serum samples, the effects of storage at 4 degrees C for 1 week and of multiple freeze-thaw cycles were evaluated.

Results: Of 42 evaluable subjects with HLA antibodies documented more than 13 years earlier, only 1 showed loss of detectable antibodies, with 39 (93%) positive in the screening assay for HLA Class I and 24 (57%) positive in the screening assay for HLA Class II antibodies. In 968 evaluable contemporary donors, 291 screened positive for the presence of HLA Class I and 206 for HLA Class II antibodies using a low assay cutoff. Screening test concordance using paired plasma and serum samples was high, particularly for subjects with higher-level antibodies. Refrigeration of samples for 1 week did not significantly affect assay results, while repeated freeze-thaw cycles caused a decrement in signal level.

Conclusion: Serum and plasma samples gave concordant results in the majority of cases, particularly for specimens with higher-level antibodies. High-level HLA antibodies were present in most individuals for more than 13 years.
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http://dx.doi.org/10.1111/j.1537-2995.2008.01955.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058293PMC
February 2009

"Natural" human leukocyte antigen antibodies found in nonalloimmunized healthy males.

Transplantation 2008 Oct;86(8):1111-5

Department of Nephrology and Mineral Metabolism, National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico City, Mexico.

Background: Human leukocyte antigen (HLA) antibodies were normally not found in subjects who have not been immunized by pregnancies, transfusions, or transplants. But with new methodology, we now see that HLA antibodies are often found in nonalloimmunized males.

Methods: The sera of 424 healthy male donors were tested with single antigen Luminex beads.

Results: Human leukocyte antigen antibodies were detected in 63% of 424 male blood donors when a fluorescent value of more than 1000 was used as the cutoff. Antibodies to class I was found in 42%, class II in 11% and both in 12%. Five males who were tested eight times over a 6-month period consistently had the same specificity at similar strength levels at each testing. The antibodies reacted with specificities that are rare in the general population: 18.9% had antibodies to A*3002; more than 10% had antibodies to A*3101, B76, B*8201, and Cw*1701. About half of the donors with antibodies had one or two specificities; the other half had three or more specificities. Among those with class II specificities, 20.5% had antibodies to DPA1*0201/DPB1*0101, and 10.8% to DQA1*0503/DQB1*0301. Because the above data were obtained by testing sera of 424 Mexican donors, as a check, 29 males in Los Angeles were tested and shown to have similar specificities at roughly similar frequencies.

Conclusions: Normal males were found to have HLA antibodies to infrequent HLA specificities. It is likely that these HLA antibodies are produced to cross-reactive epitopes found in microorganisms, ingested proteins and allergens-making them natural antibodies.
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http://dx.doi.org/10.1097/TP.0b013e318186d87bDOI Listing
October 2008

HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens.

Hum Immunol 2007 Mar 22;68(3):170-80. Epub 2006 Dec 22.

One Lambda Inc., Canoga Park, CA 90064, USA.

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.
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http://dx.doi.org/10.1016/j.humimm.2006.11.006DOI Listing
March 2007

Detection of antibodies to HLA-DP in renal transplant recipients using single antigen beads.

Transplantation 2005 Nov;80(10):1511-3

Terasaki Foundation Laboratory, Los Angeles, CA 90064, USA.

With the development of single DP-antigen beads, antibodies to DP could clearly be segregated from other HLA antibodies. We studied 323 sera of patients from four different centers with functioning or rejected kidney grafts. DP antibodies were found in 5.1% of 138 patients with functioning grafts, and 19.5 % of 185 patients with rejected grafts (P<0.001)). 42.9% and 63.9% of the DP antibody positive patients had DR/DQ antibodies among the functioning and rejected group. Among patients who did not have Class I and DR/DQ antibodies, 13% of those who rejected a graft had DP antibodies, compared to 3.5% of patients with functioning grafts (P<0.05). DP 0301 had the highest specificity frequency in the rejected group. In conclusion, HLA-DP antibodies were detected at a higher frequency in patients who have rejected their grafts than those with functioning grafts. For regrafts, DP tissue typing is recommended to avoid presensitized DP alleles.
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http://dx.doi.org/10.1097/01.tp.0000181384.49832.3aDOI Listing
November 2005

Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities.

Transplantation 2003 Jan;75(1):43-9

Research Department, One Lambda. Inc., Canoga Park, CA, USA.

Background: It is difficult to assign antibody specificity for highly sensitized patients using a cell panel with multiple antigens per reaction. We describe here a single antigen bead panel for accurate identification of human leukocyte antigen (HLA) antibody specificities by flow cytometry.

Methods: A total of 110 single recombinant HLAs, including 34 A locus alleles, 57 B locus alleles, and 19 C locus alleles, were produced by a mammalian expression system. These single antigens were coated onto eight different colored microbeads, which were mixed together in one tube for simultaneous detection of HLA antibodies against eight different antigens per flow cytometry test.

Results: Single HLA reacted specifically with the serologically defined monoclonal antibodies. The single antigen panel provided higher resolution than the regular cell panel for antibody detection by uncovering the masked specificities. Single antigens also provided higher sensitivity than the multiple antigens coated onto beads for HLA antibody detection as demonstrated by serum dilution studies. In 10 sera from patients who had rejected a kidney transplant, single antigen beads identified antibodies to 31 of 35 antigens that were mismatched in the donor. Most important, none of the reactions were against antigens present in the recipient.

Conclusion: An accurate and sensitive HLA antibody detection method is described using flow cytometry beads coated with single HLAs produced by recombinant technology. The single antigen beads should be useful in predicting negative crossmatch in highly sensitized organ recipients and highly sensitized patients requiring platelets.
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http://dx.doi.org/10.1097/00007890-200301150-00008DOI Listing
January 2003

Molecular characterization of the HLA-Cw*0409N allele.

Hum Immunol 2002 Apr;63(4):295-300

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

Various molecular methods in conjunction with serologic assays are used for clinical human leukocyte antigen (HLA) typing. Although serologic reagents detect most HLA-A and -B allospecificities, serologic HLA-C typing is hampered by the lack of informative antisera for many of the known HLA-C gene products. HLA antigens not detected by serology, but detected by molecular methods, are often referred to as "blank" antigens. Their lack of reactivity with antibodies in serological assays often reflects the presence of null alleles. The present study has characterized an HLA-Cw*04 allele (Cw*0409N) detected by DNA typing but not by serology. In cultured B-lymphoid 13W09501 cells carrying this Cw*04 null allele, isoelectric focusing analysis could not detect any component with a pattern compatible with that of the product of the HLA-Cw*0401 allele, but detected components reacting with an anti-HLA-Cw4 and Cw6 monoclonal antibody. Sequencing analysis of the full length HLA-Cw4 cDNA amplified from the cell line 13W095-01 revealed a base deletion at codon 365 in exon 7, resulting in a reading frame shift that added 32 amino acids at the C-terminal of the HLA-Cw4 heavy chain. These results indicate that the HLA-Cw*0409N allele may produce a putative long HLA-Cw4 heavy chain that is not expressed on the cell surface.
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http://dx.doi.org/10.1016/s0198-8859(02)00376-2DOI Listing
April 2002