Bead-by-bead normalization of single antigen assays: A necessary step for accurate detection of weak anti-HLA antibodies.

Ascertaining the presence of weakly positive anti-HLA donor-specific antibodies (DSA) in organ transplantation with multiplex single antigen beads assays may be challenging despite their high sensitivity due to technical variability issues. Through extensive datasets of Next-Generation Sequencing HLA typings and single antigen analyses, we reassessed the mean fluorescence intensity (MFI) positivity threshold of the assay to enhance accuracy. By showing that some beads were more prone to false positivity than others, we propose a nuanced approach that accounts for nonspecific intrinsic reactivities at the HLA antigen level, that is, on a bead-by-bead basis, as it enhances assay precision and reliability. This is substantiated by a comprehensive statistical analysis of MFI values and the implementation of the determination of a "Quantile Adjusted Threshold 500" (QAT500) value for each bead. Applied to DSA detection during patients' follow-up, this approach discriminated better and earlier low-strength DSA that would later raise their MFI above the clinically relevant threshold of 3000. Moving from a subjective interpretation to a more objective and precise methodology allows for standardizing HLA antibody and DSA detection. The study emphasizes the need for further research with real clinical data to validate and refine this approach.

Refining cPRA calculation to improve HLA compatibility assessment in organ transplantation: A detailed picture of the Paris waiting list.

The determination of panel reactive antibodies (cPRA) scores plays a critical role in assessing the immunological compatibility between organ transplant recipients and potential donors. Traditional cPRA methods focus on a limited number of HLA loci using physical cytotoxicity tests. However, advancements such as the Luminex single antigen (LSA) assay, which uses mean fluorescence intensity (MFI) of individualised HLA antigens for antibody evaluation, provide a foundation for a more precise assessment. We developed cPRAdictor, a novel cPRA calculation tool using a large series of HLA-type individuals in France with NGS. cPRAdictor was applied to a cohort of 5962 kidney transplant candidates in Paris. We analysed how extending the range of HLA specificities could affect cPRA values. Implementing cPRAdictor revealed and allowed quantification of the significant discrepancies in cPRA values that appeared when HLA loci C and DP, and antigen-specific antibodies were taken into account. Notably, over 43% of the immunised transplant candidates showed an increase in calculated cPRA values when considering C/DP loci and antigen-specific antibodies, negatively impacting their eligibility and prioritisation in the transplantation programme. These findings highlight the necessity of revisiting cPRA calculation methodologies to include a broader spectrum of immunological data, as more exhaustive and precise information regarding anti-HLA antibodies in patients' sera and donor and recipient HLA typing are available prospectively. This will strongly improve both accuracy and equity at the organ allocation step, especially for highly sensitised candidates for whom organ offers are very limited in number.

Improving HLA typing imputation accuracy and eplet identification with local next-generation sequencing training data.

Assessing donor/recipient HLA compatibility at the eplet level requires second field DNA typings but these are not always available. These can be estimated from lower-resolution data either manually or with computational tools currently relying, at best, on data containing typing ambiguities. We gathered NGS typing data from 61,393 individuals in 17 French laboratories, for loci A, B, and C (100% of typings), DRB1 and DQB1 (95.5%), DQA1 (39.6%), DRB3/4/5, DPB1, and DPA1 (10.5%). We developed HaploSFHI, a modified iterative maximum likelihood algorithm, to impute second field HLA typings from low- or intermediate-resolution ones. Compared with the reference tools HaploStats, HLA-EMMA, and HLA-Upgrade, HaploSFHI provided more accurate predictions across all loci on two French test sets and four European-independent test sets. Only HaploSFHI could impute DQA1, and solely HaploSFHI and HaploStats provided DRB3/4/5 imputations. The improved performance of HaploSFHI was due to our local and nonambiguous data. We provided explanations for the most common imputation errors and pinpointed the variability of a low number of low-resolution haplotypes. We thus provided guidance to select individuals for whom sequencing would optimize incompatibility assessment and cost-effectiveness of HLA typing, considering not only well-imputed second field typing(s) but also well-imputed eplets.

Allogeneic CD4 T Cells Sustain Effective BK Polyomavirus-Specific CD8 T Cell Response in Kidney Transplant Recipients.

Introduction: BK polyomavirus-associated nephropathy (BKPyVAN) is a significant complication in kidney transplant recipients (KTRs), associated with a higher level of plasmatic BK polyomavirus (BKPyV) replication and leading to poor graft survival. Methods: We prospectively followed-up with 100 KTRs with various degrees of BKPyV reactivation (no BKPyV reactivation, BKPyV-DNAuria, BKPyV-DNAemia, and biopsy-proven BKPyVAN [bp-BKPyVAN], 25 patients per group) and evaluated BKPyV-specific T cell functionality and phenotype. Results: We demonstrate that bp-BKPyVAN is associated with a loss of BKPyV-specific T cell proliferation, cytokine secretion, and cytotoxic capacities. This severe functional impairment is associated with an overexpression of lymphocyte inhibitory receptors (programmed cell death 1 [PD1], cytotoxic T lymphocyte-associated protein 4, T cell immunoreceptor with Ig and ITIM domains, and T cell immunoglobulin and mucin domain-containing-3), highlighting an exhausted-like phenotype of BKPyV-specific CD4 and CD8 T cells in bp-BKPyVAN. This T cell dysfunction is associated with low class II donor-recipient human leukocyte antigen (HLA) divergence. In contrast, in the context of higher class II donor-recipient HLA (D/R-HLA) divergence, allogeneic CD4 T cells can provide help that sustains BKPyV-specific CD8 T cell responses. In vitro, allogeneic HLA-mismatched CD4 T cells rescue BKPyV-specific CD8 T cell responses. Conclusion: Our findings suggest that in KTRs, allogeneic CD4 T cells can help to maintain an effective BKPyV-specific CD8 T cell response that better controls BKPyV replication in the kidney allograft and may protect against BKPyVAN.