Partnerships with Member States: enhancing cooperation for EU food safety risk assessment.

This editorial summarises a longer note (95th Meeting of the EFSA Management Board, item 7. Available online: https://www.efsa.europa.eu/sites/default/files/event/mb95/item-07-doc1-partnership-with-mss-230622-d2.pdf) submitted to Management Board of the European Food Safety Authority (EFSA) for consideration prior to a discussion held in its 95th meeting, in June 2023. It presents the context and the framework of EFSA's current scientific cooperation activities and the vision of transitioning towards long-term partnerships as the necessary evolution for the sustainability of food safety risk assessment in the EU. This new approach needs strategic alignment and political will to ensure the involvement of risk assessment organisations in the Member States. For that purpose, Management Board members can act as key advocates towards national risk managers and policy makers.

IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.

The impact of donor-specific HLA antibody (DSA) following liver transplantation remains controversial. We hypothesized DSA IgG subclass characteristics, compared to total DSA IgG, might correlate with specific histopathological phenotype(s) of subclinical graft injury. We therefore studied 129 stable, arguably "clinically ideal," pediatric liver recipients at the time of a screening biopsy to enter an immunosuppression withdrawal trial. Sixty-five (50%) subjects tested positive for class II DSA. IgG subclass profile was characterized by mean fluorescence intensity (MFI) and normalized subclass composition (>5%). A prominent IgG4 DSA profile was strongly correlated with greater HLA mismatch, a histopathological phenotype characterized by the presence of interface activity (with variable degrees of fibrosis), and a transcriptional profile of attenuated T cell-mediated rejection. Specifically, compared to those without class II DSA, those with IgG4 class II DSA MFI sum >2000 exhibited an odds ratio (OR) of 20.79 (95% confidence interval [CI] 4.38-98.69) and IgG4 subclass composition >5% exhibited an OR of 8.99 (95% CI 2.70-29.9). Our data suggest that IgG4 DSA may serve as a useful biomarker to identify, among clinically and biochemically stable liver transplant recipients, a subset with histological and transcriptional features indicative of an active, suboptimally controlled alloimmune response.

Differences in immunogenicity of HLA antigens and the impact of cross-reactivity on the humoral response.

BACKGROUND: Information about differences in immunogenicity of various HLA antigens may help guide donor selection and identify mismatches to avoid for patients likely to need retransplantation. To date, antibody responses to a wide array of individual mismatched antigens have not been evaluated. METHODS: Frequencies of antibodies to mismatched HLA-A, HLA-B, HLA-DR, and HLA-DQ antigens were determined for 703 renal transplant patients who had no detectable donor-specific antibody before transplantation. The impact of cross-reactive group matching and production of antibodies cross-reactive with mismatched antigens were also assessed. Antibodies were identified using multiplexed bead assays. RESULTS: The overall mean frequencies were similar for HLA-A (53.2%), HLA-DR (52.6%), and HLA-DQ (59.0%) antibodies, but significantly lower for HLA-B antibodies (42.4%). However, the response to individual antigens ranged from 15.0% to 76.2%. Antibody frequencies were reduced significantly for 54 of 62 specificities when the patient possessed an antigen cross-reactive with the donor mismatch, but the magnitude of the effect was variable and ranged from 8% to 83%. Moreover, there was directionality in the protective effect of cross-reactive group matching. Overall mean donor-specific antibody frequencies were comparable for men and women except for a significantly higher frequency of antibodies to HLA-DR among men (56.6% vs. 47.8%, P=0.004). Overall mean frequencies in blacks were higher than, or comparable to those of, whites, but differences were not significant. CONCLUSION: There is considerable variability in the immunogenicity of different HLA antigens that is impacted by the presence or absence of cross-reactive antigens in the patient's phenotype. This information can be used to augment the immunologic evaluation of donor-recipient pairs.

Tetramer staining for the detection of HLA-specific B cells.

HLA-specific B cells can be identified, quantified, and isolated after staining with HLA tetramers. Quantification of these B cells can in turn identify individuals who are sensitized to HLA antigens and the isolation of these cells facilitates a variety of experimental investigations.

A flow cytometric crossmatch test using endothelial precursor cells isolated from peripheral blood.

Flow cytometric crossmatch tests provide a donor-specific, cell based method for the detection of alloreactive antibodies in the sera of transplant patients. Conventional crossmatch tests used in solid organ transplantation utilize lymphocytes as target cells to detect the presence of alloreactive HLA antibodies. Isolation of endothelial precursor cells (EPCs) from peripheral blood now allows testing for antibodies reactive with non-HLA endothelial cell antigens.

Rituximab prevents an anamnestic response in patients with cryptic sensitization to HLA.

BACKGROUND: Some patients sensitized to HLA antigens do not have antibody present in serum specimens that are available before transplantation. However, such patients are at risk for an anamnestic response resulting from a proinflammatory response to the trauma of transplant surgery. Quantifying HLA-specific B cells provides a way to identify these patients and provide treatment to prevent an anamnestic response. METHODS: B cells were isolated before transplantation from 59 patients, 20 of whom were treated with rituximab at the time of transplantation. Ninety-nine tests were performed to quantify HLA-specific B cells by staining with HLA tetramers. Patients were considered sensitized or nonsensitized based on the frequencies of HLA-specific B cells. Pretransplantation and posttransplantation sera were tested for the detection of antibody specific for the tetramer antigen. RESULTS: Of the 24 cases where patients were considered sensitized to HLA antigens but did not have antibody before transplantation, no posttransplantation antibody to the tetramer antigen was detected in 10 cases when patients were treated with rituximab, but antibody was detected in 13 of 16 cases when there was no rituximab treatment (P=0.00006). The mean frequencies of B cells specific for HLA-B7 were the same in rituximab-treated patients who did not make antibody and in nontreated patients who did make antibody (6.0% vs. 5.7%; P=0.8). CONCLUSIONS: Elimination of peripheral HLA-specific B cells in patients who are sensitized to HLA antigens but lacking detectable antibody abrogates an anamnestic response.

HLA antibody detection and characterization by solid phase immunoassays: methods and pitfalls.

Solid phase immunoassays for the detection and characterization of HLA-specific antibodies provide greatly increased sensitivity, specificity, and time and reagent efficiency, compared to the traditionally used cell-based methods. Testing is performed using commercially available test kits. The assays are of two general types: enzyme-linked immunosorbent assays and multianalyte bead. The types vary in both sensitivity and equipment requirements.While these assays afford great improvement over the cell-based assays, they can be confounded by interference from substances within the serum that result in high background reactivity. The high sensitivity of the assays also makes them more susceptible to environmental factors and operator variability. The user must be aware of the capabilities of the various formats, the factors that can affect test results, and lot to lot variability of any single product. Knowledge of the characteristics of each product and thorough and accurate analysis of the results are essential to the utility of these assays.

Clinical relevance and IgG subclass determination of non-HLA antibodies identified using endothelial cell precursors isolated from donor blood.

BACKGROUND: ABO and human leukocyte antigen (HLA) alloantibodies provide major immunologic barriers to successful transplantation; however, there is increasing recognition for the role of anti-endothelial cell antibodies (AECAs) in allograft rejection. We investigated the relationship between AECAs identified using donor-derived endothelial cell precursors (ECPs) and kidney allograft rejection and function. METHODS: Sixty live donor kidney recipients were tested pretransplant for AECAs and HLA-antibodies using flow cytometric crossmatch tests and solid-phase bead immunoassays. Renal allograft function was assessed by serum creatinine (SCr) values collected at early (mean, 50 days) and late (mean, 815 days) time points posttransplant and by incidence and type of rejection. Immunoglobulin G (IgG) subtype determination of both AECAs and HLA antibodies bound to ECPs was performed using flow cytometry. RESULTS: Fourteen patients (23%) tested positive for donor-reactive IgG AECAs and had statistically higher SCr values and incidences of cellular rejection early posttransplant compared with 46 patients who tested negative (P=0.014 and P<0.05). SCr values were not statistically different late posttransplant. IgG subclass determination showed AECAs to be enriched for IgG2 and IgG4, subclasses that do not activate complement effectively. Detection of donor-reactive immunoglobulin M (IgM) AECAs did not correlate with increased SCr or incidence of rejection. CONCLUSION: Crossmatch tests performed using donor-derived ECPs allow for the identification of alloantibodies that are associated with cellular rejection and are distinct from alloantibodies detected using lymphocytes.

Characterization of an antibody specific for HLA-A36 and not reactive with HLA-A1.

Humoral sensitization to HLA often results in antibodies to public determinants shared among two or more antigens. Although monoclonal antibodies to A36 have been produced, there are no reports of polyclonal antibodies that react with A36 but not A1. We report here sera from a heart transplant recipient that reacted with A36 but not A1 in tests with both phenotype and single antigen panels on the Luminex platform. Flow cytometric crossmatch tests yielded positive results with an A36 bearing phenotype but not with a phenotype containing A1. A36 reactivity in solid phase assays was abrogated by absorption with cells bearing A36, but not with A1-positive cells. The frequency of B cells in this patient specific for A1 was comparable to that for individuals not sensitized to A1. These data indicate that reactivity was to an epitope present on A36 but absent from A1.

Naturally occurring interference in Luminex assays for HLA-specific antibodies: characteristics and resolution.

Substances occurring naturally in the sera of patients can interfere with Luminex antibody assays, causing increased background and changes in antibody specificity. We present data on the effectiveness of hypotonic dialysis (HD) or dithiothreitol (DTT) treatment in eliminating this interference. HD significantly increased reaction strength of positive control beads and reduced reaction strength of negative control beads. HD also improved specificity identification, determination of donor-specific antibody (DSA) strength, and crossmatch predictability compared with values in untreated serum. DTT also increased the reaction strength of positive control beads, but in most cases, further increased reactivity of negative control beads. DTT improved crossmatch predictability but to a lesser extent than did HD and may differ with specificities defined in other assays. Because interference is frequently observed in sera from highly sensitized patients, it is important to recognize and eliminate interference in Luminex antibody assays for accurate and meaningful test interpretation.