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.

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)

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 18^th 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.

Project name: Clinical Histocompatibility Laboratory Informatics 

Project leaders:  Loren Gragert and Nicholas Brown 

Detailed project description:  

The project will build key infrastructure to standardize collection and reporting of clinical histocompatibility data and improve analysis tools and resources to aid in virtual crossmatch assessments.

We will query the databases underlying the histocompatibility laboratory information systems (LIS) to extract detailed information on molecular HLA typing (high resolution NGS typing and intermediate resolution deceased donor typing), solid phase antibody screens, flow crossmatch, and post-transplant donor-specific antibody (DSA) assessments. 

This more detailed information usually does not leave the HLA laboratory in an electronic form and is not being adequately captured in organ allocation systems and outcomes registries. To make it easier to transfer HLA lab information into electronic medical records (EMRs), biobanks for clinical research, and transplant registries, we are developing data standards for reporting results from HLA antibody screens and histocompatibility assessments. The project will continue development of first XML-based format for HLA antibody data, HLA antibody markup language (HAML), initially created by Eric Spierings, Gottfried Fischer, and Loren Gragert. 

For organ allocation systems, we plan to build informatics tools that analyze and integrate histocompatibility data between donors and recipients to aid in virtual crossmatch assessments. We also plan to build tools that would help perform data cleaning/curation for large-scale reanalysis of historical histocompatibility data for research. 

In addition to organ allocation systems, with multiple mismatched unrelated donors (MMUD) becoming more common in hematopoietic stem cell transplantation (HSCT), registries such as National Marrow Donor Program (NMDP) and World Marrow Donor Association (WMDA) are recognizing increased needs to have their donor selection systems capture HLA antibody screen data and utilize it to automatically screen off incompatible donors from the search.  

Our team plans to make all our informatics tools available to the transplant and immunogenetics community to benefit other research consortia, including Clinical Trials in Organ Transplantation (CTOT). 

Milestones in years: 

• 2023:  Scripts developed to extract detailed information from histocompatibility lab information systems from leading vendors on antibody screens, intermediate resolution molecular typing, and crossmatch results. 
• 2024: Publication of HLA antibody markup language (HAML) XML standard 
• 2025: Publish standards for transmitting histocompatibility data in electronic medical records (HL7 FHIR Orders and Observations implementation guide for communicating HLA antibody data and histocompatibility assessments)   
• 2026: Test advanced virtual crossmatch tools on histocompatibility for simulated donor and recipient pairings. 

Data required (number, type of data, inclusion/exclusion criteria): 

• Historical data on HLA typing (molecular and antigen level), antibody assays, and crossmatch results 
• Scripts will be provided for extracting and de-identifying detailed data from laboratory information systems of leading vendors 

Samples required (if applicable, number, type of samples, inclusion/exclusion criteria): 

• Physical samples are not required for this project. The project will involve secondary data analysis. 

Reagents/additional assays required: 

• Participants will not be required to run additional assays or utilize reagents. 

Data infrastructure required:  

• Participants will need access to their clinical histocompatibility information systems. 
• The project will host a web server for providing web-based analysis tools for aiding in virtual cross-matching and user-authenticated access to de-identified datasets.  

Project name:

Adsorption elution study for the antibody-verification of eplets

Project leader(s):

Sebastiaan Heidt

Cynthia Kramer

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 19^th IHIWS.