Challenging the golden standard in defining donor-specific antibodies: does the solid phase assay meet the expectations?

The purpose of the study was to compare three different methods defining donor-specific antibodies (DSA): complement-dependent cytotoxicity (CDC), the flow cytometry method (FCM), and a special for that purpose commercially available Luminex-based solid phase assay (SPA). A panel of human monoclonal antibodies (HuMabs) with well-defined human leukocyte antigen (HLA) specificities was used as antibody source and single HLA antigen expressing cell lines (SAL) were used as targets. Two methods yielded identical results (CDC and FCM). However, the SPA, the method by which solubilized HLA molecules from the SAL are captured by microspheres, showed two additional reactions which could not be explained, neither by the epitope recognized by the HuMab nor by the widely accepted sensitivity of the SPA methodology. These unexplained results suggest that by capturing solubilized HLA molecules on microspheres, conformational changes might occur. Positive results obtained by similar Luminex-based microsphere methods should be therefore taken with caution and the 'recognized' HLA antigens should not automatically be considered as unacceptable for transplantation.

New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.

Monitoring of indirect allorecognition: wishful thinking or solid data?

Monitoring of T cells involved in the alloimmune response after transplantation requires the availability of reliable in vitro assays for the detection of T cells with both direct and indirect allospecificity. While generally accepted assays exist to measure helper and cytotoxic T cells involved in direct allorecognition, consensus about an assay for monitoring indirect T-cell allorecognition in clinical transplantation is lacking. Many studies claim a relationship between the reactivity of T cells with indirect allospecificity and graft rejection, but different protocols are used and essential controls are often lacking. In this review, the disadvantages and pitfalls of the current approaches are discussed, in some cases supported by the results of our own in vitro experiments. We conclude that an international workshop is necessary to establish and validate a uniform, robust and reliable assay for the monitoring of transplant recipients and to study the actual role of indirect allorecognition in acute and chronic rejection.

Identification of acceptable HLA mismatches in immunized patients using single-antigen-expressing cell lines.

Sera of highly sensitized patients (HSP) contain complex human leukocyte antigen (HLA) antibodies, minimizing the chance to identify crossmatch-negative donors. Expression of 3-6 HLA class I antigens on lymphocytes hampers identification of acceptable mismatches (AMs) by conventional screening (C-SCR). The single-antigen-expressing cell line (SAL) concept circumvents this problem. As a proof of principle, 26 sera of sensitized patients were tested by flow cytometry for immunoglobulin G antibodies against 16 HLA-A and -B SALs. Results were compared with C-SCR. Mostly, SAL reactions confirmed presence/absence of HLA antibodies. While C-SCR sometimes failed to provide unambiguous antibody specificity, we defined 24 new HLA antibody specificities with SALs and proposed 33 new AM by non-reactivity with SALs. Thus, the SAL concept is useful for confirmation/identification of AM and will enhance transplantation of HSP.