Antibodies to denatured human leukocyte antigen: relevant or just noise?

Introduction of solid phase assays and adoption of flowbased human leukocyte antigen (HLA) antigen-coated beads as the method of choice for detection of HLA antibodies heralded a new age in the pretransplant assessment of organ transplant recipients. For the first time, a technique was available which showed greater sensitivity and accuracy than the complement dependent cytotoxicity technique for both the detection and specificity determination of HLA antibodies. Although this development has been of benefit for the clinical management of the transplant recipient, it has raised questions regarding the clinical interpretation of the data generated (1). In this issue of Transplantation, Visentin et al. (2) have addressed a feature of HLA antigen-coated beads having both technical and clinical implications. Using bead antibody detection technology, they studied a cohort of sensitized renal transplant recipients for incidence and clinical relevance of HLA class 1 antibodies directed at cryptic epitopes on denatured HLA molecules (dHLA), not accessible to antibody when the HLA heavy chain is complexed with A-2-microglobulin in its native state (nHLA). The methods used for coating beads with HLA molecules for antibody detection (screening beads) and accurate antibody specificity assignment (single antigen beads [SAB]) rely on different technologies. In the former case, molecules are immunoprecipitated from cell lines, whereas SAB are bound with HLA molecules produced using recombinant technology. The dHLA molecules occur on SAB, most likely caused by the different methods of manufacture and the amount of dHLA binding varies from bead to bead (3). This issue has been addressed by the manufacturers who have produced iBeads, which have largely intact HLA molecules on their surface, although there is evidence that levels of nHLA differ between beads and may differ from normal SAB beads by having a lower density of HLA molecules on their surface (3), both factors impacting on the mean fluorescence intensity (MFI) obtained. Previous studies have demonstrated that antibodies to dHLA class 1 can be found in a proportion of normal nontransfused, nontransplanted males (4), suggesting they are not formed as a result of previous exposure to alloantigens. Cross-reactivity with epitopes found on microorganisms or allergens have been suggested as a likely source of immunization (4, 5). The question that concerns those involved in transplantation centers around the potential role of anti-dHLA in rejection and how they should be considered in the organ-matching process. Some reports (2, 5) suggest that they are not associated with renal transplant rejection, although Visentin et al. (2) were unable to show a statistically significant difference in rejection rates between those with donor-specific antibodies (DSA) to dHLA versus those with DSA to nHLA owing to small numbers of rejection cases in both groups. In contrast, Sicard et al. (6) showed that antibody-mediated rejection could occur in nonimmunized male patients with HLA class 1 or class 2 DSA detected by SAB, although they did not demonstrate that these antibodies were directed at dHLA. The findings demonstrating a lack of effect on rejection incidence of anti-dHLA is not unexpected given the lack of antibody accessibility to cryptic epitopes on an intact cell. Visentin et al. (2) however have suggested a multicenter study to further address the clinical significance of these antibodies. Such a study should also include thoracic transplant recipients and examine whether antibodies to dHLA are formed de novo after transplantation. The fact that dHLA antibodies occur commonly in nonimmunized male individuals does not preclude the fact that they could also be formed as a result of exposure to alloantigen by means of the indirect presentation route. The impact of antibodies to class 2 dHLA in graft outcome also requires further study. The second concern is the exclusion of patients with dHLA DSA in the matching process. The use of iBeads and, where applicable, the cell-based flow cross-match, where dHLA is not a confounding variable, can assist in interpretation. However, instances occur where the flow cross-match is negative in the presence of antibodies to nHLA, so caution must be exercised. In addition, the MFI of dHLA antibodies tend to be in the lower range (G3,000) (2), which can assist in the decision making process. These findings highlight again the importance of defining antibodies in epitope specificity rather than allele specificity. This is of importance in recipient-donor matching where an antibody to dHLA may co-occur in a patient with an antibody to a defined nHLA epitope on the same allele. ANALYSIS AND COMMENTARY