Characterisation of the HLA-DQ alpha eplet 52SK targeting the DQA1*01 alleles family.

Eplet 52SK is unique in the HLA eplet registry as targeting the whole family of DQA1*01 alleles. It is proposed as an antibody-verified eplet but has not been validated enough to deserve this label. Especially, confusion can occur with reactivity targeting the 52PQ eplet which is present on the DQB1*05 and DQB1*06 alleles families, as DQ molecule stability imposes DQA1*01 to selectively associate with these DQ-ß families only. Using two Luminex single antigen (LSA) assays from two vendors, beads bearing DR-α/DQ6 heterodimers, a special build LSA panel of additional DQ beads, and an adsorption/elution strategy relying on cells from deceased donors or recombinant cells solely expressing one DQ antigen, we definitely established the antibody-verified status of eplet 52SK using patients' sera reacting only against the DQ5 and DQ6 beads of the One Lambda LSA panel in routine patients' follow up. We also show that reactivity against this eplet is not a rare event among anti-DQ1 immunisation. This study further strengthens the importance of considering the DQA1 locus in immunological studies of HLA and in organ allocation strategies.

Bead-by-bead normalization of single antigen assays: A necessary step for accurate detection of weak anti-HLA antibodies.

Ascertaining the presence of weakly positive anti-HLA donor-specific antibodies (DSA) in organ transplantation with multiplex single antigen beads assays may be challenging despite their high sensitivity due to technical variability issues. Through extensive datasets of Next-Generation Sequencing HLA typings and single antigen analyses, we reassessed the mean fluorescence intensity (MFI) positivity threshold of the assay to enhance accuracy. By showing that some beads were more prone to false positivity than others, we propose a nuanced approach that accounts for nonspecific intrinsic reactivities at the HLA antigen level, that is, on a bead-by-bead basis, as it enhances assay precision and reliability. This is substantiated by a comprehensive statistical analysis of MFI values and the implementation of the determination of a "Quantile Adjusted Threshold 500" (QAT500) value for each bead. Applied to DSA detection during patients' follow-up, this approach discriminated better and earlier low-strength DSA that would later raise their MFI above the clinically relevant threshold of 3000. Moving from a subjective interpretation to a more objective and precise methodology allows for standardizing HLA antibody and DSA detection. The study emphasizes the need for further research with real clinical data to validate and refine this approach.

Refining cPRA calculation to improve HLA compatibility assessment in organ transplantation: A detailed picture of the Paris waiting list.

The determination of panel reactive antibodies (cPRA) scores plays a critical role in assessing the immunological compatibility between organ transplant recipients and potential donors. Traditional cPRA methods focus on a limited number of HLA loci using physical cytotoxicity tests. However, advancements such as the Luminex single antigen (LSA) assay, which uses mean fluorescence intensity (MFI) of individualised HLA antigens for antibody evaluation, provide a foundation for a more precise assessment. We developed cPRAdictor, a novel cPRA calculation tool using a large series of HLA-type individuals in France with NGS. cPRAdictor was applied to a cohort of 5962 kidney transplant candidates in Paris. We analysed how extending the range of HLA specificities could affect cPRA values. Implementing cPRAdictor revealed and allowed quantification of the significant discrepancies in cPRA values that appeared when HLA loci C and DP, and antigen-specific antibodies were taken into account. Notably, over 43% of the immunised transplant candidates showed an increase in calculated cPRA values when considering C/DP loci and antigen-specific antibodies, negatively impacting their eligibility and prioritisation in the transplantation programme. These findings highlight the necessity of revisiting cPRA calculation methodologies to include a broader spectrum of immunological data, as more exhaustive and precise information regarding anti-HLA antibodies in patients' sera and donor and recipient HLA typing are available prospectively. This will strongly improve both accuracy and equity at the organ allocation step, especially for highly sensitised candidates for whom organ offers are very limited in number.

Anti-HLA serologic response to CD38-targeting desensitization therapy is challenged by peripheral memory B cells in highly sensitized kidney transplant candidates.

High human leukocyte antigen (HLA) sensitization limits access to compatible transplantation. New CD38-targeting agents have been shown to reduce anti-HLA antibodies, although with important interpatient variability. Thus, pretreatment identification of responder and nonresponder (NR) patients is needed for treatment decision-making. We analyzed 26 highly sensitized (HS) patients from 2 desensitization trials using anti-CD38 monoclonal antibodies. Hierarchical clustering identified 3 serologic responder groups: high responders, low responders, and NR. Spectral flow cytometry and functional HLA-specific memory B cell (mBC) assessment were first conducted on peripheral blood mononuclear cells and bone marrow samples from 16 patients treated with isatuximab (NCT04294459). Isatuximab effectively depleted bone marrow plasma cells, peripheral CD38-expressing plasmablasts, plasma cells, transitional B cells, and class-switch mBCs, ultimately reducing frequencies of HLA-specific immunoglobulin G (IgG)-producing mBCs. Multidimensional spectral flow cytometry with partial least squares discriminant analysis revealed that pretreatment abundance of specific circulating mBC phenotypes, especially CD38neg class-switch mBCs, accurately distinguished between high serologic responders and low responders or NR (AUC 0.958, 0.860-1.000, P = .009), who also displayed significantly lower frequencies of HLA-specific IgG-producing mBCs (P < .0001). This phenotypical mBC signature predicting response to therapy was validated in an external HS patient cohort (n = 10) receiving daratumumab (NCT04204980). This study identifies critical circulating mBC subset phenotypes that distinguish HS patients with successful serologic responses to CD38-targeting desensitization therapies, potentially guiding treatment decision-making.

Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQß-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQß eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Improving HLA typing imputation accuracy and eplet identification with local next-generation sequencing training data.

Assessing donor/recipient HLA compatibility at the eplet level requires second field DNA typings but these are not always available. These can be estimated from lower-resolution data either manually or with computational tools currently relying, at best, on data containing typing ambiguities. We gathered NGS typing data from 61,393 individuals in 17 French laboratories, for loci A, B, and C (100% of typings), DRB1 and DQB1 (95.5%), DQA1 (39.6%), DRB3/4/5, DPB1, and DPA1 (10.5%). We developed HaploSFHI, a modified iterative maximum likelihood algorithm, to impute second field HLA typings from low- or intermediate-resolution ones. Compared with the reference tools HaploStats, HLA-EMMA, and HLA-Upgrade, HaploSFHI provided more accurate predictions across all loci on two French test sets and four European-independent test sets. Only HaploSFHI could impute DQA1, and solely HaploSFHI and HaploStats provided DRB3/4/5 imputations. The improved performance of HaploSFHI was due to our local and nonambiguous data. We provided explanations for the most common imputation errors and pinpointed the variability of a low number of low-resolution haplotypes. We thus provided guidance to select individuals for whom sequencing would optimize incompatibility assessment and cost-effectiveness of HLA typing, considering not only well-imputed second field typing(s) but also well-imputed eplets.

Two-field resolution on-call HLA typing for deceased donors using nanopore sequencing.

The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies.

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

Characterization of the novel HLA-C*08:258 and -C*12:374 alleles by two different sequencing-based typing techniques.

The novel HLA-C*08:258 and -C*12:374 alleles differ from HLA-C*08:02:01:01 and -C*12:03:01:01 by one nucleotide substitution.

Identification of a novel HLA-C allele, HLA-C*03:618N.

The novel HLA-C*03:618N allele has one change in exon 1 leading to a premature stop codon.

Identification of the new HLA-DRB1*15:213 allele in a French cardiac transplant recipient.

The new HLA-DRB1*15:213 allele results from one nucleotide substitution in exon 3 of HLA-DRB1*15:02:01.

Identification of a novel HLA-B allele, HLA-B*08:304.

The novel allele B*08:304 differs from B*08:01:01:01 by one nucleotide substitution in exon 2.

Identification of a novel HLA-A null allele, HLA-A*01:420N allele.

The novel HLA-A*01:420N allele has two changes in exon 4 leading to premature stop codon.

The novel HLA-C*17:64Q allele characterized by two different sequencing-based typing techniques.

The novel allele HLA-C*17:64Q differs from HLA-C*17:01:01:02 by insertion of a Lysine in exon 2.

HLA graph, a free and ready-to-use bioinformatics tool to explore anti-HLA eplets reactivity pattern.

INTRODUCTION: HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti-HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti-HLA antibodies are not specific for HLA antigens, but recognize B-cell epitopes present on HLA molecules. METHODS: To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti-HLA antibodies identified by a single-antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single-antigen class I and class II assays. RESULTS AND DISCUSSION: The resulting MFI correlations reflect HLA antibody cross-reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single-antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA-DQ and HLA-DP loci were also developed. CONCLUSION: HLA Graph is a free and ready-to-use bioinformatics tool that can be used by all laboratories performing anti-HLA antibody identification by immunobead assay.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

Utility of assessing CD3+ cell chimerism within the first months after allogeneic hematopoietic stem-cell transplantation for acute myeloid leukemia.

After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3+ cell chimerism within the first 6 months post alloHSCT. One hundred and thirty five patients diagnosed with acute myeloid leukemia were enrolled in this study. We observed significantly lower overall survival and relapse free survival for patients without full donor chimerism (<95%, <98%, <99%) in whole blood at Day 30, as well as at Day 90 after alloHSCT, than for patients with full donor chimerism. This outcome was not observed when assessing selected CD3+ cells. However, at Day 90, patients with discordant whole blood versus selected CD3+ cell chimerism showed both significantly lower overall survival and relapse free survival, giving an interest to assess selected cells chimerism.

Characterization of the novel HLA-DRB1*04:326 allele by next generation sequencing.

HLA-DRB1*04:326 differs from HLA-DRB1*04:01:01 by one nucleotide substitution in codon 23 in exon 2.

Characterization of the novel HLA-C*04:450 allele by next-generation sequencing.

HLA-C*04:450 differs from HLA-C*04:01:01 by one nucleotide substitution in codon 328 in exon 7.

Antibodies against HLA cross-reactivity groups: From single antigen bead assay to immunoinformatics interpretation of epitopes.

Identification of anti-human leukocyte antigen (HLA) antibodies (Abs) is based on Luminex™ technology. We used bioinformatics to (i) study the correlations of mean fluorescence intensities (MFIs) for all the possible allele pairs, and (ii) determine the degree of epitope homology between HLA antigens. Using MFI data on anti-HLA Abs from 6000 Luminex™ assays, we provide an updated overview of class I and II HLA antigen cross-reactivity in which each node corresponded to an allele and each link corresponded to a strong correlation between two alleles (Spearman's ρ > 0.8). We compared these correlations with the serological groups and the results of an epitope analysis. The strongest correlations concerned allele-specific Abs directed against the same antigen. For the HLA-A locus, the highest values of Spearman's ρ reflected broad specificity. For the HLA-B locus, graphs defined the HLA-Bw4 public epitope, and correlations between HLA-A and -B alleles were only present for beads with the same Bw4 public epitope. For the HLA-C locus, we identified two groups that differed with regard to their KIR ligand subclassification. Lastly, the HLA-DRB1 subgroups were part of a network. In the epitope analysis, Spearman's ρ was related to the number of matched epitopes within pairs of alleles. The combination of Spearman's ρ with simple, undirected graphing constitutes an effective tool for understanding routinely encountered cross-reactivity profiles. Based on this model, we have implemented an online data visualization tool available at http://cusureau.pythonanywhere.com/.