Antigenicity & Immunogenicity
To read more details about the Antigenicity & Immunogenicity theme within 19th IHIWS, click on each of the following subthemes. You will get to learn about the project leader, project description, milestones, data required and more.
.
Project name: HLA Loss Relapse after hematopoietic cell transplantation
Project leaders:
- Esteban Arrieta Bolaños, Senior Postdoctoral Researcher, Institute for Experimental Cellular Therapy, University Hospital Essen, Germany (Esteban.arrieta-bolanos@uk-essen.de)
- Katharina Fleischhauer, Director, Institute for Experimental Cellular Therapy, University Hospital Essen, Germany (katharina.fleischhauer@uk-essen.de)
- Luca Vago, Group Leader, Unit of Immunogenetics, Leukemia Genomics and Immunobiology and Department of Hematology and Bone Marrow Transplantation, San Raffaele Scientific Institute, Milan, Italy (vago.luca@hsr.it)
Detailed project description:
Relapse is the most frequent cause of death in patients treated for hematological malignancies by allogeneic hematopoietic cell transplantation (HCT) (Bolon et al, CIBMTR summary slides 2022). HLA loss relapse, first described by the project leaders of this component (Vago et al, N Engl J Med 2009), is a condition in which, after partially-mismatched HCT, tumor cells reemerge after deleting the HLA haplotype carrying the mismatched, patient-specific alleles, while the other haplotype is duplicated (uniparental disomy). HLA loss relapses therefore become invisible to alloreactive donor T cells. The most common way of treating relapse, donor lymphocyte infusions, is considered to be consequently largely ineffective, while maintaining toxicity against healthy tissues (ie, graft-versus-host disease), which retain a normal HLA asset. HLA loss occurs in up to 25% of relapses after HCT from haploidentical family donors, and in up to 10% of HCT from unrelated donors (Crucitti et al, Leukemia 2015). Diagnosis of this condition is challenging, especially at early stages, when the number of tumor cells is still low, resulting in a chimeric sample carrying very low amounts of patient-specific HLA alleles, along with the shared and/or donor-specific alleles. With the constant increase in the use of both haploidentical related and mismatched unrelated donors in current clinical practice, in particular under GvHD prophylaxis with post-transplantation cyclophosphamide, this phenomenon is likely to become more prevalent. Hence, raising awareness of its incidence, promoting its prompt detection in post-transplant follow-up, and developing better methods for its diagnosis as well as guidelines for their implementation, interpretation, and practical clinical implications are necessary. Current methods to detect HLA loss relapses by qPCR have been developed and rapidly became available as commercial kits, but these cover a limited number of HLA alleles (Ahci & Toffalori et al, Blood 2017). With the advent of NGS typing for HLA, accurate allele counting has become feasible. Nevertheless, the available HLA typing softwares are not tailored to detect chimeric conditions with more than two HLA alleles per locus. Some HLA typing laboratories with interest in HLA loss diagnostics have developed methods to circumvent these problems. This workshop component has the objective to
- determine the current prevalence of HLA loss testing among H&I laboratories
- collect the different methods used for HLA loss testing across laboratories
- cross-validate these methods on a reference panel of artificial DNA mixes
- establish validated protocols for HLA diagnostics to be made available to the community.
Milestones in years
2024: Survey about HLA loss diagnostics to Tissue Typing laboratories registered within EFI/ASHI/APHIA, enrollment of interested laboratories
2025: Exchange of protocols for HLA loss diagnostics. Exchange of reference panels of artificial DNA mixes and analysis by the participating laboratories.
2026: Data collection and establishment of validated protocols for HLA loss diagnostics.
Data Required (number, type of data, inclusion/exclusion criteria):
Protocols currently used for HLA loss diagnostics. Results of HLA diagnostics performed on the artificial DNA mixes that will be shared.
Samples Required (if applicable, number, type of samples, inclusion/exclusion criteria):
Artificial mixes of DNA (at least 10) extracted from PBMC of healthy blood donors will be prepared in the laboratories of the project leaders and sent to the participating laboratories.
Reagents/Additional Assays Required:
Local assays for HLA loss detection. For interested laboratories that do not currently have such assays in place, the relevant protocols will be shared by the project leaders.
Data Infrastructure Required:
Experience in high-resolution HLA typing, preferentially by NGS. Any other method for HLA loss detection (e.g., qPCR, STR) welcome but not required.
Additional Information:
NA.
Project Name: Immunogenicity of HLA-DQ antibodies (epitopes)
Project Leader: Anat R Tambur
Detailed Project Description:
The goal of this project is to stratify HLA-DQ mismatches that are more immunogenic versus those that are more permissible (under lower-level immunosuppression protocols). Only patients with de-novo DSA qualify for this project. We aim to validate preliminary observations from the 18th IHIWS and expand the knowledge derived from that dataset.
Milestones in Years:
- Centres’ enrolment – estimate of support required – by end of 2024 (continuous enrolment will be possible but we encourage centres to reach out early in the process so that we can secure the required reagents and other support)
- NGS typing (recipient and donor) as needed; repeat SAB as needed; clinical data collection; submission to IHIWS website – by end of 2025.
- Data analysis and preparation for presentation at 19th IHIWS – by May of 2026
Data Required (number, type of data, inclusion/exclusion criteria):
- Documentation that patient did not exhibit DSA prior to transplant – submitting pre-transplant SAB data within 1-month pre transplant; and having at least one additional one test with similar results prior to that (no need to submit prior samples’ data). To determine absence of DSA, MFI must be below 1000.
- Transplant date (not required if additional samples will have calculated time lapsed, if this helps in obtaining IRB approval)
- Raw SAB data documenting presence of dnDSA (including date to calculate time from transplant, or providing time lapsed). To determine presence of DSA we will consider MFI > 3000 unless there is an indication of presence of shared epitope, in which case lower MFIs may be considered.
- Two-field HLA typing information (at least for DRB1, DQA1, DQB1) for both recipient and donor.
- Demographic data: Gender, Race, Age
- Prior sensitization data: Transfusions, Pregnancy. Patients with prior Transplants should be excluded.
- Transplanted organ: Kidney, Heart, Lung, Pancreas, Liver
- Clinical data (if available): Biopsy proven ABMR, Clinically suspected ABMR, TCMR, Graft failure, Unknown, Well functioning graft (if GFR or Cr levels are known)
Samples Required (if applicable, number, type of samples, inclusion/exclusion criteria):
We will be happy to perform adsorption/elution experiments for a selected portion of the patients if exhibit unique antibody recognition patterns. To this aim we require 2 field typing resolution for both recipient and donor, and raw data of antibody recognition patter (raw SAB data). If chosen for additional studies – we require 500ul of sera to be shipped to our laboratory.
Reagents/Additional Assays Required:
NGS typing reagents (vendor per participating laboratory’s preference)
Single Antigen Bead assay (class II; vendor per participating laboratory’s preference)
Data Infrastructure Required:
Website to host the data requested above. Will be happy to discuss more.
Project Name: Definition of molecular mismatch immunogenicity
Project Leader(s): Sebastiaan Heidt, Eric Spierings & Cynthia Kramer
Detailed Project Description:
On the population level it is clear that a higher number of molecular mismatches (amino acid, eplet, PIRCHE II) increases the chance of donor specific antibody (DSA) formation. However, from the same data it is clear that not all molecular mismatches result in antibody formation, leading to the hypothesis that not all molecular mismatches are equally immunogenic. For HLA molecular matching to become a clinical reality it is vital to determine the most immunogenic molecular mismatches, which can then be avoided during future organ allocation. We also aim to define molecular mismatches that never induce an antibody response. To this aim, large numbers of first-transplant recipients, non-immunized at time of transplantation, who either became immunized due to the transplant or not, are required.
In this project, we will determine the amino acid, eplet, and PIRCHE II mismatches between donor and recipient based on second-field molecular HLA typing data for the loci HLA-A, -B, -C, -DRB1, -DRB345, -DQB1, -DQA1, -DPB1, and -DPA1. The status of being non-immunized at time of transplant must be confirmed by a negative Luminex single antigen bead status. The transplant-induced immunization has to be reported by Luminex single antigen bead data.
Milestones in Years:
2023: Start development of traveling algorithm, contacting centers to participate
2024: Finalize development of traveling algorithm, start data collection
2025: Continuation of data collection and interim analyses
2026: Finalizing analyses
Recipient/Donor Description:
Inclusion criteria:
First transplant recipients: males, and females without a history of pregnancy
Non-sensitized at time of transplant as defined by:
- cPRA pre-transplant 0% based on Single Antigen Bead (SAB) analysis with all bead MFI less than 1000, or lower if a clear eplet specificity is present across many beads.
- All sera must have been tested in such a way that complement interference/prozone effect can be excluded.
- Molecular HLA typing of donor and recipient
- HLA class I – A, B, C
- HLA class II – DRB1, DRB3/4/5, DQB1, DQA1, DPB1, DPA1
- Two types of patients can be considered for entry into the study:
- Patients who developed de novo DSA post-transplant either:
- while on maintenance immunosuppression, or
- have documented graft loss from chronic antibody mediated rejection, and are now off immunosuppression
- Patients who did not develop a de novo DSA post-transplant despite at least 5 years of follow up.
- Patients who developed de novo DSA post-transplant either:
Exclusion criteria:
- Repeat transplant
- Prior pregnancy
- Combined transplants
- No HLA antigen/allele mismatches between donor and recipient
- Sensitized patients at the time of transplant defined by cPRA by SAB>0%
- Absence of molecular HLA typing for the donor and/or recipient
- Second-field molecular HLA typing with new alleles
- Patients who have not developed dnDSA but are less than 5 years post-transplant
Data Required (number, type of data, inclusion/exclusion criteria):
- Minimal requirements:
- Unambiguous second-field molecular HLA typing for HLA-A, -B, -C, -DRB1, -DRB3-5, -DQB1, -DQA1, -DPB1, and -DPA1 for both donor and recipient
- HML file, or
- GL string in absence of valid HML file but complete HLA typing
- Luminex SAB raw data Class I and Class II antibody testing files pre-transplant, including vendor and lot number
- Luminex SAB raw data Class I and Class II antibody testing files at time of first detection of de novo DSA, including vendor and lot number
- Specify antibody specificities
- Luminex SAB raw data Class I and Class II antibody testing files of patients who are listed as non-DSA formers at 5 years or last follow up, including vendor and lot number
- Specify antibody specificities
- Prevention of interference/prozone effect (specify method)
- Time antibody testing post-transplant by Luminex SAB by either
- transplant date + date antibody testing post-transplant or,
- timing antibody testing post-transplant (number of weeks after transplantation)
- Transplant organ type
- Recipient de novo DSA (yes or no)
- Whether patient was classified as adherent or non-adherent at time of post-transplant Luminex SAB (yes, no, unknown)
- Unambiguous second-field molecular HLA typing for HLA-A, -B, -C, -DRB1, -DRB3-5, -DQB1, -DQA1, -DPB1, and -DPA1 for both donor and recipient
Samples required (if applicable, number, type of samples, inclusion/exclusion criteria):
If no molecular typing at second field level is available for donor and/or recipient, DNA would be required for the typing to be performed. In case no Luminex SAB data is present, a serum sample from the appropriate time-point is required.
Reagents/Additional Assays Required:
For a proportion of laboratories, we expect to require additional second-field molecular HLA typing, as well as Luminex SAB analysis.
Data Infrastructure Required:
The data infrastructure of 18th IHIWS for uploading second-field HLA typing data through HML, Luminex single antigen bead data through .csv files, which will be converted to HAML (irrespective of vendor), and data matrix containing description of data are required for this project.
Quality check of files with error message.
During the 18th IHIW, acquisition of large data sets was significantly hampered by regulatory privacy barriers. Therefore, we aim to resolve this issue before data collection starts. In early 2024, we will organize a forum discussion with participating centers and those who are interested to participate to overcome this crucial issue. The aim of this discussion is to allow collective data usage at the highest level of detail within the various national and international regulations. Such solution could comprise a “travelling algorithm” in the analysis phase or from a processing of data before central submission. Approaches to consider include, but are not limited to Differential Privacy, Federated Learning and/or Secure Multi-Party Computation. Either way, the digital information structures need to be adapted to the outcome of this discussion.
Serologic Antigen Assignments corresponding to HLA alleles.
PI: Kazutoyo Osoegawa, Marcelo Fernandez Vina
Steering committee: Robert Liwski, Massimo Mangiola, Semiramis Do Monte, Alberto Cardoso Martins Lima, Sebastiaan Heidt, Renato De Marco, Gottfried Fischer, Sandra Tafulo
Goals for this project:
- Validation and possible expansion of HLA Serotypes assigned in 2024
- Validation of amino acid residues considered for Serotype Assignment (residues Defining Epitopes; DEP)
- Correlation between Eplets, DEP and their combinations
Introduction:
The serologic HLA specificities recognized before 1996 were defined by serum cluster analyses and reactivity patterns. With the advent of HLA typing by nucleotide sequencing methods, serologic typing has been discontinued; as a result of these practices many HLA alleles defined and recognized in the last 30 years are lacking the counterpart serologic definition. In addition, it has been recognized that because of lack of serologic reagents and systematic analyses there were limitations in the definition of HLA serotypes. In order to address these limitations, it was proposed to define and extend the serotype definition of HLA alleles encoded at HLA loci considered for humoral compatibility. The criteria of the definition of serotypes included the analysis of amino acid replacements at well-recognized residues located at the distal membrane domains that define common epitopes Assignment (residues Defining Epitopes; DEP). All these residues are included in eplets located at these domains.
Logics for serotype assignments were recently published 1, this work included computational tools for systematically classifying HLA alleles into serological specificities. The criteria for definition of serotypes consider that alleles carrying the same or equivalent amino acid replacements at selected residue positions (DEP) belong to the same serotype; in other words a serotype is unit that represents all or the most immunogenic serologic epitope. Under this premises antigen preparations from alleles belonging to the same serotype should show high correlation (see Figure 1A) when tested with sera many patients that show sensitization against HLA molecules in solid phase assays including Single Antigen Beads (SABs). On the other hand, antigen preparations from alleles corresponding to different serotypes should show low correlation (see Figure 1B). In recent work, the definition of serotypes defined according to novel criteria was validated by evaluating correlations mean fluorescence intensity (MFI) values of Single Antigen Beads (SABs) tested by solid phase assays with >13,000 patients’ sera.
19th IHIWS Serology Study:
In order to extend our knowledge about serotypes and residues that define epitopes we propose organize collaborative exercises associated to the 19th IHIWS. We propose to participants to submit results from serologic tests to be collected followed by central analyses. The data will be made available to participants in order to perform sub-analyses to be presented and discussed on site at the 19th IHIWS.
- For the performing these studies we propose that participants perform anti-HLA antibody tests from sensitized patients with at least one of SAB panel 19th IHIWS reagents manufactured and provided by vendors. The SAB panels have been designed by the vendors including HLA antigen preparations corresponding to alleles with validated and not validated serotypes. In addition, the SAB panels may include antigen preparations from wild type alleles that have been mutated in vitro with the purpose of replacing amino acid residues that are thought to determine epitopes. When potential novel or unconfirmed serotypes are observed, we propose confirming them by surrogate flow cytometric crossmatches (FXM) and/or adsorption/elution antibody test in order to confirm the positive reactivity or to eliminate false positive reactivity. The results of these experiments will be evaluated in the context of the reactivity with monoclonal antibodies that distinguish closely related but distinct serotypes.
Essential data:
- The investigators should obtain authorization form their IRB. The samples will be de-identified and the data should be associated with a 19th IHIWS code identifier.
- The investigators that participate in these exercises will commit to submit antibody tests data obtained with these 19th IHIWS reagents.
- In addition, they should provide HLA typing results obtained by NGS testing from each of the patient’s tested for anti-HLA antibody screening.
Non-essential data:
- If the local IRB allows, it would be desirable to obtain information regarding type of patient (e.g. transplant type), source of immunization, and if pregnancy and HLA phenotype of the father if available.
- If post-transplant data is submitted, Donor’s HLA typing is valuable if allowed.
Possible Findings:
To validate serotypes and identified novel serotypes we propose to systematically evaluate the concordance between antigen preparations. This work will allow for:
- Confirmation existing serotypes or define novel serotypes (Goal A listed above as Validation and possible expansion of HLA Serotypes assigned in 2024.
- Validation of the role of amino acid residues considered for Serotype Assignment and identify residues not listed in previous work (Goal B listed above as Validation of amino acid residues considered for Serotype Assignment (residues Defining Epitopes; DEP).
- Perform correlation analyses between Eplets and DEP and their combinations as defined above for Goal C. In order to perform these analyses, it will be necessary performing comprehensive annotations of eplets including each residue position and the corresponding amino acid replacement. These annotations will allow to perform systematic computer analyses that will allow to examine serotypes, concordances and discordances with individual and combinations of DEP. The inclusion of these analyses will likely prove to be useful for the assessment of DSA corresponding to alleles lacking full serotype coverage in the SAB panels.
The primary data obtained in these tests and experiments will be collected centrally and distributed to participants that interested to perform analyses to be presented and discussed at the 19th IHIWS. The data will also be analyzed centrally; the central data analyses will be provided to investigators expressing interested in participating in collaborative reports. Interim analyses will be performed in order to monitor project progress.
In addition, the primary data will be made available to other 19th IHIWS projects interested in analyzing eplets and examining their correlation with DEP and serotypes.
Milestones in years:
- 2024 first semester: Publish a manuscript from the initial validation data for the proof of concept
- 2024 second semester: Start distribution of reagents
- 2025: Collect data from the international participants and develop central data analysis pipeline
- 2026 first quarter: Complete the data submission and initiate data analysis
- 2026 May: Discuss and present the findings at IHIWS meeting
Figure 1A: Scatter plots and linear regression analyses for the SABs that belong to the same serotype/antigen: A*02:01 vs. A*02:06
Figure 1B: Scatter plots and linear regression analyses for the SABs that belong to the different serotypes/antigens: A*23:01 vs. A*24:02
Figure 1: Scatter plots are shown; comparisons made in order to identify a relationship between MFI values observed in two different SABs. Each dot represents two MFI values from two different SABs represented on X- and Y-axis per serum. Red dots show MFI values > 1000 in SAB represented on X-axis and MFI values < 1000 on Y-axis. Blue dots show MFI values < 1000 in SAB represented on X-axis and MFI values > 1000 on Y-axis. MFI values < 1000 on both SABs are omitted. A) High correlations are observed for the pairs of SABs belonging to the same serotype/antigen. B) Antigen preparations from alleles corresponding to different serotypes show low correlation.
REFERENCE
- Osoegawa K, Marsh SGE, Holdsworth R, Heidt S, Fischer G, Murphey C, Maiers M and Fernandez Vina MA. A new strategy for systematically classifying HLA alleles into serological specificities. HLA. 2022;100:193-231.
Project name:
Adsorption elution study for the antibody-verification of eplets
Project leader(s):
Sebastiaan Heidt, Cynthia Kramer, and Massimo Mangiola
Detailed project description:
For several solid organ transplant settings is has been shown on the population level that the number of eplet mismatches is associated with an increased chance of donor specific antibody (DSA) formation. However, from the same data it is clear that not all eplet mismatches result in antibody formation, leading to the hypothesis that not all eplet mismatches are equally immunogenic. In addition, eplets are theoretically defined and require experimental verification to establish if an antibody can actually bind to it. The verification status of eplets is listed in the Eplet Registry. Recently, Bezstarosti and colleagues (Front Immunol 2021 Vol. 12) showed that the verification status is based on varying levels of evidence and proposed that only eplets verified by human monoclonal antibodies and adsorption/elution studies from serum of immunized individuals unequivocally can verify eplets. The proposed level of evidence scheme, as well as the truly verified status will soon be adapted in the Eplet Registry.
Since the production of (recombinant) human monoclonal antibodies is time-consuming and requiring a specialized laboratory infrastructure, the number of eplets verified by monoclonal antibodies remains limited. On the other hand, adsorption/elution studies are relatively straightforward and can be performed in most routine HLA laboratories. Dedicated magnetic beads coated with single HLA specificities will be provided for this purpose.
Milestones in years:
- 2023: identification of (core) laboratories to perform adsorption/elution studies
- 2024: adsorption/elution studies, gathering of data
- 2025: adsorption/elution studies, gathering of data, central data analysis for confirmatory purposes, data aggregation. Writing of paper and updating the Eplet Registry
- 2026: presentation of data at IHIWS meeting
Data required (number, type of data, inclusion/exclusion criteria):
SAB data (HAML format) of sera pre-adsorption, as well as the SAB data (HAML format) of post-adsorption. If available, channel shift data of flow cytometry crossmatching using informative cells, including positive and negative controls. Second field typing data of cells used should be reported.
Preferentially, second field HLA typing data of self/antibody-producer (HML format) should be reported and if present second field HLA typing data of immuniser.
Samples required (if applicable, number, type of samples, inclusion/exclusion criteria):
Sera from immunized individuals displaying reactivity patterns suspicious reactivity against yet unverified eplets (as defined by Bezstarosti et al., Front Immunol 2021). Ideally, availability of second cells expressing the reactive alleles for confirmation of binding to natively expressed HLA by flow cytometry.
Reagents/additional assays required:
Magnetic beads for adsorption will be provided by Thermo Fischer One Lambda. Luminex SAB kits for reactivity pattern analysis.
Data infrastructure required:
Database of 19th IHIWS.
PI:
- Massimo Mangiola
- Semiramis Do Monte
- Mario Coelho Marroquim
- For support, contact support@epregistry.com.br
Goals for this Project:
- Verification of the provisional HLA eplets
- Identification of surfaced-exposed amino-acids constituting functional epitopes (eplets)
Introduction:
The HLA Eplet Registry is the official repository database for known HLA eplets. HLA eplets are considered a powerful biomarker for T cell rejection, antibody formation, antibody-mediated rejection and allograft loss. Therefore, the determination of the eplet mismatch load between a patient and a donor is a powerful biomarker of rejection and is currently used by many Transplant Centers to inform their treatment and post-transplant monitoring decision making. Additionally, several Centers are actively engineering transplants to be zero or low eplet mismatch at HLA-DR and HLA-DQ. Several prospective clinical trials to study immunosuppression reduction are undergoing or are being prepared.
Of the 521 eplets listed in the Registry, 146 (28%) have one of the verification statuses as reported by Bezstarosti et.al (PMID 35154076) and 375 (72%) are currently listed as “provisional”. Out of 252 Class I (HLA-A, -B, -C) eplets, 72 (28.6%) are antibody-verified. On Class II, 28.8 % (36/124) of HLA-DR, 32.5% (27/83) HLA-DQ, and 17.7% (11/62) of HLA-DP eplets have been antibody-verified. To advance the field of B cell molecular compatibility and achieve the goal of applying this powerful biomarker to organ allocation, confirmation of the antibody-verified eplets and verification of the provisional eplets is of paramount importance.
A collaborative effort to continuously update the HLA Eplet Registry is of extreme importance to the Transplant field, and a synergistic effort with the IHIWS is essential to achieve this goal.
19th IHIWS Eplet Registry Update:
To extend our knowledge about HLA eplets and their role in allograft survival, a continuous update of the HLA Eplet Registry is essential. We propose to make the update of the Eplet Registry not only a project of the 19th IHIWS, but also a constitutive project of this workshop.
For the 19th IHIWS, and in synergy with Projects 1 and 2, we propose to refine the HLA eplet databases and to clarify the existence of provisional HLA eplets. The data made available from Projects 1 and 2 will allow us to update and upgrade the HLA Eplet registry as follow:
Essential changes:
- Define an official HLA eplet nomenclature. The new nomenclature will be based on data obtained from monoclonal/mutagenesis studies and the results from the Projects 1 and 2.
- Upgrade and confirm antibody-verified HLA eplets.
- Confirm, refine and identify essential amino-acid residues defining eplets.
- Identify secondary contact points that may define HLA epitopes.
- Identify incorrect eplets currently listed.
- Identify redundant HLA eplets currently listed.
- Introduce new HLA eplets that are identifies by Projects 1 and 2.
- Clarify the Luminex antibody pattern observed for each verified HLA eplet.
Secondary changes:
- Upgrade the database tables to include the frequency by which HLA antibody occur in the patient population (from Project 2).
- Create an “Eplet Card” to display the essential information (monoclonal antibody results, mutagenesis results, AXE results, Luminex patterns from both vendors).
By synergizing with monoclonal studies, mutagenesis studies, and Project 1 and 2, we will be able to update and upgrade the HLA Eplet Registry. By improving the accuracy of the data in the Registry, we will improve the predictability of the eplet mismatch and advance the field of B cell molecular compatibility. Differently from Component 1 and 2, this Component will require more time to complete since it will rely of analysis of data from other IHIWS Components before implementation.
Project name:
Matched kidney donor project: Immunogenicity study of the same kidney donor in two different recipients
Project leader:
Lloyd D’Orsogna
Detailed project description:
HLA matching has played a critical role in the success of clinical kidney transplantation, and it has clearly been shown at the population level that an increasing number of HLA mismatches is associated with de novo donor-specific antibody (DSA) formation, antibody mediated rejection (AMR) and inferior graft survival. The resultant HLA sensitisation can also be a major barrier to re-transplantation in the event of graft failure. While best outcomes are achieved with fully HLA matched donor and recipient pairs, the highly polymorphic nature of HLA means that most recipients will never receive a fully matched organ.
Donor-specific antibodies (DSA) recognise specific polymorphic amino acids on the donor HLA, termed eplets, when not present at any of the recipient HLA loci (1-2). Multiple studies have reported that the mismatched eplet load is a better predictor of DSA formation and graft outcome than total HLA antigen mismatches only, especially at the class II loci (3-5). However, HLA antibodies often develop against only a single or limited number of the total mismatched eplets present in a specific donor-recipient pair (6).
Multiple confounding factors can also affect the immunogenicity of a mismatched HLA eplet within an individual recipient. Certain specific eplets have been reported to be more immunogenic than others (6). The physiochemical properties of amino acid mismatches, HLA allele expression and the individual HLA locus can all affect the probability of developing de novo DSA. For example, recent data suggest HLA-DQ mismatch can be extremely immunogenic and HLA-DQ DSA may be powerful predictors of poor graft outcome and graft loss. Certain HLA-C alleles are more highly expressed than other HLA-C alleles, and therefore more likely to elicit DSA formation when present on the donor. Low expression loci such as HLA-DRB3/4/5 and HLA-DP may be less likely to trigger rejection episodes. The same eplet can also be present across different HLA loci. Furthermore, the probability of DSA formation is also affected by non-immunological factors in the donor such as cold ischaemia time, donor age, blood flow and by deceased donor type, amongst others.
Therefore, it is still currently impossible to predict which HLA eplet mismatches will elicit donor-specific antibodies within an individual transplant pair.
This project proposes to study eplet immunogenicity between “matched” recipients who have each received a kidney from the same donor. Both kidneys are harvested from the majority of deceased organ donors and transplanted into two separate recipients. The matched donor and recipient cohort will be recruited as part of the 19th International Histocompatibility Workshop (IHIWS) and all recipients and donors will have high resolution (NGS) HLA typing results at all loci including HLA-DRB345, -DQA1 and -DPA1 and all recipients will have SAB assay results available with correction for prozone effect, as well as important clinical data. Transplant pairs will undergo detailed analysis including for HLA antigen matching, mismatched eplets, PIRCHE score, analysis of physicochemical properties and DSA, amongst others. We also aim to correlate our immunological findings with graft outcome, including kidney transplant biopsy histopathological results in a subset of patients.
Therefore this project represents a unique opportunity to study the immunogenicity of specific HLA and eplet mismatches, while controlling for other factors that can effect the probability of DSA formation. By using the matched kidney as control then both recipients will have received an organ with identical donor status, similar cold ischaemia time and there will be shared eplet mismatches with identical physiochemical properties and similar HLA expression levels. This will allow more detailed, accurate and unbiased analyses of how recipient eplet profile may affect the immunogenicity of mismatched eplets. Understanding which immunogenetic factors in the recipient lead to immunogenicity (or tolerance) of specific HLA eplet mismatches will have major implications for clinical transplantation including better matching, prevention of sensitization and the definition of acceptable mismatches for highly sensitized patients. In particular, our analysis will focus on cases where one recipient has made DSA against a mismatched donor eplet, while the other kidney recipient of the same donor has not made DSA to the identical mismatched eplet.
We propose that detailed study of the two different recipients that have received a kidney from the same donor has the possibility to greatly increase the current understanding of eplet immunogenicity and could lead to important improvements in allocation and clinical outcomes.
Milestones in years:
2024: Centre enrolment and data submission. Project leader will also contact centres to participate.
2025: Data submission and interim analysis. Ongoing enrolment is allowed.
2026: Final analysis, data preparation and presentation at IHIWS May 2026. Publication(s) after IHIWS workshop.
Data required (number, type of data, inclusion/exclusion criteria):
Essential data required:
- Matched donor pairs i.e. data from single donor that has given kidneys to 2 separate recipients. Individual donor will be assigned a single (same) donor identifier number, with individual (different) recipient identifier number assigned for each of the two recipients from the same donor.
- Minimum 2 field resolution HLA typing results all loci for donor and recipient (NMDP codes not acceptable). We are also collecting 3 or 4 field data if available.
- Luminex SAB raw data file (csv), with prozone measures reported (EDTA, dilution etc). All available SAB results including sample date.
- Date of transplantation
- Donor data: Donor sex and age (DOB) and donor type (DBD or DCD)
- Recipient data: Recipient sex and age (DOB), pregnancies, blood transfusions, return to dialysis date (if applicable), date of death (if applicable)
Additional data (NOT mandatory):
- Donor data: Blood group
- Recipient data: Blood group, immunosuppression regimen at induction, pathology findings from kidney transplant biopsy (Banff score)
Samples required (if applicable, number, type of samples, inclusion/exclusion criteria):
Most laboratories will submit data only.
If no molecular typing at second field level is available for donor and/or recipient, DNA sample will be required for high resolution HLA typing to be performed. In case no Luminex SAB data is available, a serum sample from the appropriate time-point is required.
We will be happy to perform adsorption/elution experiments for a selected portion of the patients if exhibit unique antibody recognition patterns.
Reagents/additional assays required:
NGS typing reagents (vendor per participating laboratory’s preference) – High resolution typing data to minimum 2 field at all HLA loci, including HLA-C, DRB3/4/5, DQA1 and DPA1.
Single Antigen Bead assay (vendor per participating laboratory’s preference)
For a proportion of laboratories, we expect to require additional second-field molecular HLA typing, and Luminex SAB analysis. We will endeavour for all willing laboratories to be able to participate irrespective of current assay platforms and resources.
We will be happy to perform adsorption/elution experiments for a selected portion of the patients if exhibit unique antibody recognition patterns.
Data infrastructure required:
The data infrastructure of 18th IHIWS for uploading second-field HLA typing data through HML, Luminex single antigen bead data through .csv files, which will be converted to HAML (irrespective of vendor), and data matrix containing description of data are required for this project. Quality check of files with error message.
REFERENCES
- Claas et al. Epitope-based HLA matching: A useful strategy with many shortcomings to overcome. Transplantation 2017; 101: 1744-1745
- Tambur et al. Toward HLA epitope matching in clinical transplantation. AJT 2013; 13: 3059-3060
- Wiebe et al. Class II epitope matching – strategy to minimize De Novo donor-specific antibody development and improve outcomes. AJT 2013; 13: 3114-3122
- Lim et al. HLA-DQ mismatches and rejection in kidney transplant recipients. CJASN 2016; 11: 875-883
- Sapir-Pichhadze et al. HLA-DR and HLA-DQ eplet mismatches and transplant glomerulopathy: A nested case-control study. AJT 2015; 15: 137-148
- Claas et al. Differential immunogenicity of HLA mismatches in clinical transplantation. Transplant Immunol 2005; 14: 187-191