Mapping of Serologic Epitopes
Studies in this component of the workshop will be focused on defining HLA serologic epitopes contributing to antibody mediated rejection in renal and cardiac transplant recipients (because the pathology is best worked out in these groups).
The widespread use of highly sensitive and specific solid phase assays for HLA antibody definition has revealed that antibody reactivity is much more complicated than previously appreciated when only cytotoxic panels or ELISA techniques were used. It is clear that there are allele specific antibodies, functionally diverse antibodies with respect to complement, ‘natural’ antibodies for which no known sensitizing event occurred, etc. Historically defined CREG reactivity, while still present, represents only a portion of the antibody profiles commonly seen suggesting the wider sharing of specific epitopes than previously appreciated. In other instances, the antibody recognition appears to be very narrow. There is also a lack of predictable correlation between MFI values and crossmatch results by any method. The diversity in the ways the assays are performed, lab to lab, likely contribute to this lack of correlation.
The availability of new tools and methods for antibody assessment, new methods for donor specific HLA crossmatching, and the capability of having full length HLA genomic and complete exonic sequences will provide an exceptional opportunity to both define and do fine mapping of serologically reactive epitopes. Using cohorts of cardiac and renal transplant pairs in which DSA was present pre-transplant or produced de novo post-transplant and patients were assessed for antibody mediated rejection on biopsy, we have an opportunity to answer the many questions outlined below. This component of the workshop will use standardized and uniform custom and commercial reagents under strict protocols so that results can be compared reliably. SOPs will be provided as will custom reagents from the Stanford Core laboratory. Participants will be required to pass initial and intermittent blinded control exercises in order for their data to be included in the final analysis.
Participating labs will be expected to submit complete data on at least 10 donor/recipient pairs (more is better) who had/have DSA pre- or post-transplant, crossmatch data, and have been assessed for AMR on biopsy with availability of cells. Labs with at least 10 donor/recipient pairs who had/have DSA pre- or post-transplant and crossmatch data, but without access to biopsy information, and for whom sera and DNA are available may also participate. Labs that cannot perform all of the required tests or who do not have enough/any samples to contribute should partner with other labs having these capabilities and together will be considered as being one group. Labs who do not have access to partner labs may inquire whether it is possible to submit samples for testing to the Stanford Core laboratory.
Results will be submitted on a specified time schedule to the central database for analysis. Data will be sent to the core data repository (Stanford). The data will be on an access controlled, secure server, housed at Stanford and no storage will be in the Cloud. Data sharing among participating labs will only be with written consent of all labs who agree to share, and with whom they agree to share.
A fee of $500 (USD) will be required to participate, to cover the cost of the custom reagents and shipping.
Characterization of serologic HLA epitopes
Mapping serologic epitopes using NGS, and single antigen bead technologies is one of the components of the 17th IHIWS. Its main goal is to define HLA serologic epitopes contributing to antibody mediated rejection in renal and cardiac transplant recipients. To analyze the data generated by the projects under this component, it is important that we eliminate variables related to technique and reagents by using uniform methods, strict protocols and standardized reagents. To evaluate the performance of same technique and sample/reagents tested by different laboratories, we have launched an Epitopes Pilot project in which 13 international labs perform IgG SAB I, IgG SAB II, Bio-C1q SAB I, Bio-C1q SAB II on the same set of samples. Samples, custom and commercial reagents and SOPs will be provided by the Workshop organizers.
Projects in this component:
- Analysis of DSA epitopes from donor/recipient transplant pairs: Analysis will be performed based on the results obtained using standardized solid phase procedures/reagents, NGS HLA typing HLA typing combined with analysis on multiple available programs to translate into serological epitopes. The analysis will focus primarily on mapping serologically reactive epitopes, identify “acceptable” epitopes (NGS HLA defined epitopes to which highly sensitized patients failed to make antibodies) and study the different HLA-DQ epitopes to define the crucial amino acids for antibody reactivity to DQ molecule.
- Analysis of DSA epitopes and transplant outcomes from donor/recipient transplant pairs: Analysis will evaluate the benefit of adopting strategies for matching transplant recipients with potential donors, from HLA allele to HLA epitope matching by analyzing DSA epitopes and transplant outcomes. Analysis will evaluate the contribution of individual vs cumulative DSA epitope mismatches, immunodominance and immunogenicity of epitopes, as well as the clinical relevance of IgG-binding versus complement fixing DSA antibodies.
- Analysis of DSA epitopes and FXM: DSA-FXM is a flow cytometry based DSA detection assay combing cellular flow cytometric crossmatch (FCXM) with microbeads array technology to selectively detect DSA in the recipient serum. One common obstacle to predicting graft outcome with the results of the crossmatch is the lack of standardization of FXM techniques as well as the lack of predictable correlation between MFI values and crossmatch results by any method. This project will evaluate the correlation between DSA-FXM, other XM techniques and SAB results with transplant outcome data.
Basic questions to answer:
- What serologic HLA epitopes (as opposed to molecules) does a recipient recognize in an allotransplant setting which lead to AMR?
- Does epitope recognition of class I, or class II, or both preferentially lead to AMR?
- Are epitopes different for class I than for class II?
- Is there a frequency difference in epitope recognition of class I vs class II?
- Are HLA epitopes recognized by autoantibodies? How do these differ from alloantibodies?
- What epitopes are recognized by ‘natural’ antibodies? Do these have a physiologic or environmental (e.g., molecular mimicry) basis?
- What is the contribution of individual vs cumulative DSA epitope mismatches on immunodominance?
- What regions of the molecules are being recognized?
- Is there preferential reactivity to a region on the 3D molecule?
- Is there a region that contributes to immunodominant epitopes?
- Can we relate affinity vs avidity to results with SAB, or different techniques (e.g., IgG vs C1q)?
- How does a donor HLA specific FXM (DSA-FXM) correlate with the results from the SAB?
- What are the non-permissible mismatches
- Which type of epitope MM will preferentially (if not always) lead to adverse graft outcome such as, e.g., AMR, graft survival, graft loss?
- Is there a difference in outcomes for DSA epitopes recognized pre-transplant than for those found in de novo DSA post-transplant?
- Is there a correlation between the timing, type or number of epitope MM with respect to the transplant and the kinetics of adverse graft outcome?
- Is there epitope spreading or narrowing in AMR?
- Are the epitope targets different in accelerated acute, early, and late AMR?
- Nomenclature is inadequate
- Refers to the molecule or a particular chain of the molecule but not the reactive determinant in many cases.
- No way to discuss epitopes created by heterodimers
- Linear array does not equal a 3D epitope
- AMR is difficult to diagnose, consisting of histology, C4d, DSA and clinical features. (Would need to subgroup patients depending on constellation of these parameters observed.)
- No standardization of techniques for LMX-SAB or FXM and lack of correlation between them
- Lack of crystallized antibody/antigen complexes of HLA
How to indicate interest in participation in this component:
A questionnaire will be sent to any lab wishing to participate in this component. To register your interest, please click here.
Component Leaders: Dolly Tyan PhD (Chair), Marcelo Fernandez-Vina PhD, Robert Bray PhD, Peter Nickerson MD, Frans Claas PhD, Ms. Rhonda Holdsworth
Liaison: Donna Phelan CHS