Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation

Abstract

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ࣙ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum.Keywords: Anti-denatured HLA antibodiesHLA antibodiesHLA epitopesLuminex single antigen bead assay Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionTypically produced following exposure to foreign HLAs during pregnancy, after transfusion or upon organ transplantation, alloCorrespondence: Prof. Jean-Luc Taupin e-mail: jean-luc.taupin@chu-bordeaux.fr geneic anti-HLA antibodies are deleterious for the function and survival of transplanted organs when they are donor-specific (the so-called anti-HLA donor-specific antibodies or DSA) [1][2][3][4][5][6]. Understanding of the alloantibody response at antigen and epitope levels has been hampered since the origins of histocompatibility testing owing to the lack of sensitivity and resolution of the available tools. A major step forward was the release of the Luminex single antigen beads (LSAB) assays, which made possiblewww.eji-journal.eu 2112 J. Visentin et al. Eur. J. Immunol. 2015. 45: 2111-2121 the simultaneous analysis of nearly a hundred different class I or class II HLA alleles with a highly sensitive flow cytometric readout [7,8]. Using this tool with monoclonal anti-HLA antibodies or alloantibodies eluted after binding on cell lines expressing a single recombinant HLA allele, Terasaki's group indexed about two hundred distinct HLA epitopes accessible for antibody recognition [9]. The same task was achieved by Duquesnoy's group using a theoretical algorithm that predicts HLA epitopes on the molecular surface and relies on stereochemical modeling of the epitope-paratope interfaces of antigen-antibody complexes [10]. O...

Caveats of HLA antibody detection by solid‐phase assays

“…The clinical significance of these “natural” antibodies is a matter of debate and studies revealed conflicting results, even though the clinical impact might be rather limited . Interestingly, one study reported that antibodies against denatured HLA antigens may also be able to bind to intact HLA molecules .…”

The Road to HLA Antibody Evaluation: Do Not Rely on MFI

“…Still another concern regarding MFI values stems from interferences due to antibody-dependent complement activation. However, Visentin et al reported that sera from some patients contain antibodies to both native and denatured HLA antigens of the same specificity or specificities (13). These events can artifactually diminish MFI values to well below a cutoff threshold.…”

“…The limited data published on this topic suggest that reactivity to denatured proteins does not contribute to poor graft outcome (11). However, Visentin et al reported that sera from some patients contain antibodies to both native and denatured HLA antigens of the same specificity or specificities (13). Thus, unless and until reactivity to native and denatured HLA antigens can be easily discerned, it is premature to consider reactivity to denatured antigens as clinically innocuous.…”

Lung intragraft donor-specific antibodies as a risk factor for graft loss

“…[11][12][13][14][15][16] However, detection of serum DSA with SAFB has technical limitations, falsenegative and false-positive results being respectively caused by complement interference [17][18][19] and by anti-HLA antibodies of ill-defined pathogenic role recognizing denatured class I HLA molecules. [20][21][22][23][24][25] The presence of DSA is not synonymous with lung allograft injury. 26 The lack of direct association may be due to the inability of the DSA to bind to the graft because of the absence of expression of cognate HLA molecules, or because of insufficient binding strength between a low affinity DSA and its target.…”