Projects

Structural Basis of HLA Compatibility

April, 2005 Update

A. Introduction

Although it is generally accepted that HLA plays a major role in determining graft outcome, the ongoing debate about the utility of HLA matching seems to reflect the inadequacy of merely counting the number of mismatched A, B, DR antigens, a system that has been used for more than thirty years.Because we know now so much more about the structure and function of HLA, the time has come to develop a better matching system that reflects our understanding of how the immune system perceives HLA incompatibility.Detailed information about the structure of HLA molecules and amino acid sequences of HLA alleles provide new opportunities to assess donor-recipient HLA compatibility in transplantation and transfusion.

The goal of this workshop project is to develop structurally based approaches to determine HLA compatibility.These studies focus on humoral immune responses to HLA because HLA-specific alloantibodies represent important risk factors for transplant failure and platelet transfusion refractoriness.Structural HLA matching considers amino acid sequence polymorphisms as potential epitopes for alloantibodies and several matching algorithms have been reported.The studies have been designed to increase our understanding of the antigenicity and immunogenicity of structurally defined polymorphisms and their clinical significance in HLA compatibility.There are three projects.

B. Workshop Projects

1. Determine The Structural Basis Of Epitopes Recognized By HLA-Specific Human Monoclonal Antibodies

This project deals with the analysis of reactivity patterns of HLA antibodies with DNA typed panels in complement-dependent lymphocytoxicity and solid phase assays. It will focus on human monoclonal antibodies generated from HLA-typed antibody producer-immunizer combinations and their specific reactivity with class I and class II structural polymorphisms.

Laboratories submit:

a. HLA typing information of antibody producer and immunizer (preferably typed by DNA-based methods at the allelic level)

b. Information about reactivity of monoclonal(s) with HLA-allele typed panels in lymphocytotoxicity methods and in binding assays such as ELISA and Flow beads.

c. Aliquots of monoclonals for further testing in participating laboratories.

Workshop participation:

a. Participating laboratories will test monoclonals with HLA-typed panels in lymphocytotoxicity and binding assays

b. Databases with reactivity patterns of monoclonals and other relevant information will be made available to participants who wish to analyze the data with locally developed structurally based algorithms for antibody analysis.

c. A data analysis group of participants will review and interpret the results of the various analysis programs to determine a consensus about the structural basis of HLA epitopes recognized by the monoclonals.The group will also prepare a workshop report.

2. Determine The Relative Immunogenicity Of Structurally Defined HLA Mismatches Following Kidney Transplantation.

Humoral immunization to HLA can occur under different conditions: during pregnancy, after blood transfusion and following a transplant.Although during these immunizing events, the antibody producer is often exposed to multiple HLA incompatibilities, the specificities of the antibodies are generally limited to a few epitopes. The goal of this project is to determine the relative immunogenicity of structurally defined HLA polymorphisms following kidney transplantation.

These studies deal with patients whose kidney transplant from an HLA mismatched donor has failed and was removed by nephrectomy after they had returned on the waiting list for a second transplant.Recent studies have shown that many patients exhibit an abrupt increase in their serum reactivity following transplant nephrectomy.We interpret this finding that the patient had made donor-specific HLA antibodies but they were undetectable because the kidney allograft had adsorbed them.

Serum samples before and after nephrectomy will be tested with DNA typed panels in complement-dependent lymphocytoxicity and antigen-binding assays.From the structural polymorphisms of the mismatched HLA antigens of the donor, we can determine which epitopes had elicited specific antibodies and which ones did not. The analysis of a large number of cases will provide estimates about epitope immunogenicity following kidney transplantation.

Laboratories submit:

a. Serum samples from patients with rejected their kidney transplant and who underwent transplant nephrectomy.These sera must be obtained at three time points: (1) pre-transplant, (2) after transplant failure and the patient was placed on the waiting list and (3) after transplant nephrectomy.

b. HLA typing information of patient and kidney donor (preferably typed by DNA-based methods at the allelic level)

c. Preliminary information about the panel reactivity of the submitted serum samples screened by lymphocytotoxicity and antigen-binding assays such as ELISA and Flow beads.Relevant information about the transplant such as survival time, date and reason for transplant nephrectomy and graft histopathology findings

Workshop participation:

a. Participating laboratories will screen patient sera with HLA-typed panels in lymphocytotoxicity and antigen-binding assays.

b. A large patient database with serum screening results, HLA types and other relevant information will be made available to participants for analysis with locally developed structurally based algorithms designed to analyze HLA antibody specificity patterns

c. A data analysis group of participants will review and interpret the results of the various analysis programs to determine what donor-specific epitopes are recognized the antibodies of each patient. A frequency determination of epitope-specific antibodies in a large group of patients will permit an assessment of the relative immunogenicity of structurally defined, mismatched HLA epitopes of the transplant donor.The group will also prepare a workshop report.

3.Determine The Role Of Structurally Defined HLA Polymorphisms In Platelet Transfusion Support Of Alloimmunized Thrombocytopenic Patients

Blood transfusions are a major cause of HLA alloimmunization. An important example is the refractory thrombocytopenic patient who has become HLA alloimmunized after transfusion with pooled platelet concentrates from multiple random donors and who has therefore, been exposed to many HLA mismatches. This project is designed to determine how structurally based HLA matching can be applied to optimize platelet transfusion support of refractory patients.Patient sera will be screened for antibody reactivity against structurally defined class I polymorphisms and the data will be compared with patient responses to single-donor HLA-typed platelet transfusions.

Laboratories submit:

a. Serum samples from alloimmunized thrombocytopenic patients who require matched platelet transfusions

b. Patient HLA types (preferably determined by DNA-based methods at the allelic level) and information about serum reactivity with local HLA-allele typed panels by lymphocytotoxicity and binding assays such as ELISA and Flow beads.

c. Clinical documentation of patient disease status, transfusion history and HLA alloimmunization-induced refractoriness in the absence of sepsis, hepatosplenomegaly and disseminated intravascular coagulopathy (DIC).

d. HLA types of transfused platelets and patient responses determined as 1-hour and 24-hour blood platelet increments.

Workshop participation:

a. Participating laboratories will test patient sera with HLA-typed panels in lymphocytotoxicity and antigen-binding assays.

b. Participants will continue to collect platelet transfusion reponses.

c. A large patient database with serum screening results, platelet transfusion responses and other relevant information will be made available to participants for analysis with locally developed structurally based algorithms. This analysis will include comparisons between in vitro and in vivo data.

d. A data analysis group of participants will review and interpret the results of the various analysis programs to arrive at a consensus how structurally based HLA matching can be implemented to optimize platelet transfusion support of refractory patients.This group will also prepare a workshop report

This project will be guided by the

Structural HLA Compatibility (SHC) Project Steering Committee:

ReneDuquesnoy (Pittsburgh),Chair

Frans Claas (Leiden, The Netherlands), Co-Chair

Rhonda Holdsworth (Melbourne, Australia)

Howard Gebel (Atlanta, GA)

Andrew Lobashevsky (Birmingham, AL)

Cristina Navarette (London, UK)

Steve Takemoto (Los Angeles, CA)

Ralf Wassmuth (Duesseldorf, Germany)

Contact Addresses:

Rene J. Duquesnoy, Ph.D.

Division of Transplantation Pathology
University of Pittsburgh Medical Center
Biomedical Science Tower, Room W1552
Pittsburgh, PA, 15261, USA
Phone:++1-412-647-6148 or ++1-412-624-1075
Fax:++1-412-647-1755
E-mail:Duquesnoyr@msx.upmc.edu

Frans H. J. Claas, Ph.D.

Department of Immunohematology and Blood Transfusion
Leiden University Medical Center
P.O. Box 9600
2300 RC Leiden, The Netherlands
Phone: ++31-71-526-3800
Fax: ++31-71-521-6751
E-mail: F.H.J.Claas@lumc.nl

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