BACKGROUND:Eplets, acting as specificity determinants on donor HLA antigen molecules, may induce the formation of de novo donor-specific antibodies (dnDSAs) by the recipient's B cells posttransplantation. HLA-DQ DSAs are the most common type of DSAs that target mismatched donor HLA-DQ antigens. The HLA-DQ protein exists as a heterodimer, consisting of HLA-DQA1 and HLA-DQB1 molecules. The specific risk of each mismatched HLA-DQ dimer contributing to dnDSA formation remains underexplored. METHODS:A total of 434 kidney transplant donor/recipient pairs from Zheng Zhou University First Affiliated Hospital, who underwent the transplant procedure between September 2016 and November 2020, were included in this analysis. All were DSA-negative pretransplant. For each HLA-DQ heterodimer, the total incidence of dnDSA, alpha chain-specific dnDSA, and beta chain-specific dnDSA after kidney transplant was analyzed. The dnDSA formation rates of different mismatched HLA-DQ dimers were compared and their haplotype association with specific HLA-DRB1 protein were determined. RESULTS:In this study, 38 HLA-DQ dnDSAs were detected, with 28 to HLA-DQB1 and 10 to HLA-DQA1. HLA-DQA1*03:02-DQB1*03:03 dimer had the highest dnDSA formation rate of 28.3%. The HLA-DQA1*05:xx-DQB1*03:01 and HLA-DQA1*05:xx-DQB1*02:xx, which are known for their high immunogenicity in previous studies, showed a relatively low DSA formation rate of 4.3%. The common DQB1*01:xx-DQB1*05:xx and DQA1*01:xx-DQB1*06:xx dimers had DSA formation rates of 1.8% and 3.9%, respectively. The high immunogenic HLA-DQA1*03:02-DQB1*03:03 dimer formed haplotype with DRB1*09:01 (98%), whereas the low immunogenic HLA-DQA1*02:01-DQB1*03:03 dimer formed exclusively haplotype with DRB1*07:01. CONCLUSIONS:The DQA1*03:02-DQB1*03:03 dimer and the HLA-DRB1*09:01-HLA-DQA1*03:02-DQB1*03:03 haplotype turned out to be the most immunogenic in inducing HLA-DQ dnDSA in this Northern Han Chinese cohort.

Lung transplantation is a life-saving therapeutic option for many chronic end-stage pulmonary diseases, but long-term survival may be limited by rejection of the transplanted organ. Since HLA disparity between donor and recipient plays a major role in rejection, we performed a single center, retrospective observational cohort analysis in our lung transplant cohort (n = 128) in which we calculated HLA compatibility scores for B-cell epitopes (HLAMatchmaker, HLA-EMMA), T-cell epitopes (PIRCHE-II) and missing self-induced NK cell activation (KIR Ligand Calculator). Adjusted Cox proportional hazards model was used to investigate the association between mismatched scores and time to development of donor-specific antibodies (DSA) post-transplant, time to first biopsy-proven acute rejection episode, freedom from CLAD, graft survival and overall survival. For time to first DSA, HLA-EMMA DQB1 scores and PIRCHE-II DQB1 scores were significantly associated with more rapidly developing anti-HLA-DQ antibodies. HLA-EMMA DQB1 score was significantly associated with worse survival. KIR ligand Host-versus-Graft (HvG) mismatches was significantly associated with worse graft survival (CLAD or death) and shorter time to first biopsy-proven rejection when 2 mismatches were present. We demonstrated that HLA-DQB1 compatibility scores and KIR ligand HvG 2 mismatches may allow for identification of recipients at risk of poor long-term outcomes after lung transplantation.

Highly sensitized (HS) kidney transplant (KTx) candidates, that is, typically considered internationally as those with panel-reactive antibody levels of >85%, remain a substantial subpopulation of patients with low chance of receiving a compatible organ. Among its many objectives, Eurotransplant-an international transplant organ allocation network serving 8 European countries-aims to improve the management of HS KTx candidates through its prioritized "acceptable mismatch" (AM) program. However, despite this program, some HS patients within the Eurotransplant network who have panel-reactive antibodies >85% still cannot access donor kidneys. For patients who remain in the AM program for ≥3 y without undergoing transplantation, an additional prioritization strategy has been implemented. This involves defining further AMs to allow for desensitization with imlifidase within the AM program. While the AM desensitization program was being developed, the Belgian Imlifidase Scientific Expert Group within the Eurotransplant network independently recognized the need for guidelines on imlifidase desensitization for real-world use in HS KTx candidates (including both AM and Eurotransplant Kidney Allocation System patients). This article describes the consensus guidelines they subsequently developed, which represent a model that any center within the Eurotransplant region could adapt or apply in clinical practice when treating HS KTx candidates who require imlifidase desensitization. The consensus guidelines include patient eligibility criteria for imlifidase treatment that align with Eurotransplant allocation rules and incorporate posttransplant management strategies for HS patients. These guidelines are dynamic and will be reviewed and updated regularly as Eurotransplant rules change and imlifidase experience grows.

Highly sensitized (HS) patients in need of kidney transplantation (KTx) typically spend a longer time waiting for compatible kidneys, are unlikely to receive an organ offer, and are at increased risk of antibody-mediated rejection (AMR). Desensitization using imlifidase, which is more rapid and removes total body immunoglobulin G (IgG) to a greater extent than other methods, enables transplantation to occur between HLA-incompatible (HLAi) donor-recipient pairs and allows patients to have greater access to KTx. However, when the project was launched there was limited data and clinical experience with desensitization in general and with imlifidase specifically. Hence, this Delphi methodology was used to reach a consensus from a multi-disciplinary team (MDT) of experts from 15 countries on the management of HS patients undergoing imlifidase HLAi from a deceased donor (DD) KTx. This Delphi consensus provides clinical practice guidance on the use of imlifidase in the end-to-end management of HS patients undergoing an HLAi DD KTx and supports centers in the development of guidelines for the utilization and integration of imlifidase into clinical practice.

In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex‐defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope‐specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan–Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre‐transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations ( p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA‐DR, and 1 of HLA‐DQ, and most antibodies were directed to HLA‐B (47%). Fifty‐three patients (69.7%) had DESA against one donor epitope (range 1–5). Long‐term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84–3.25) in deceased donation, and 2.22 (1.25–3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA‐based risk analysis on antigen level.

On 24 January 2023, Eurotransplant has introduced the virtual crossmatch for kidney and pancreas allocation as a better alternative for the physical Complement Dependent Cytotoxicity (CDC) crossmatches at the donor centre, which were associated with a longer cold ischaemia time and false positive reactions. For the time being, the physical CDC crossmatch at the recipient centre will remain in place as the final histocompatibility check. While Eurotransplant is certainly not the first organ allocation organisation to introduce virtual crossmatching, several novel aspects have been introduced, such as calculation of the virtual panel reactive antibody (vPRA) on 11 loci at the second-field level in addition to the serological broad and split level, electronic HLA typing data transmission using Histoimmunogenetics Markup Language (HML) file format, and the actual virtual crossmatch based on ambiguous, second-field HLA typing of the donor on all 11 loci. This short communication will focus on these novel aspects of the virtual crossmatch in Eurotransplant.

In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.

HSCT with mismatched unrelated donors (MMUD) is associated with inferior outcome compared to matched unrelated donors. We aimed to identify permissible mismatches using HLA-EMMA, which determines permissibility by analyzing amino-acid (AA) sequences, in a single center cohort of 70 pediatric 9/10 MMUD HSCTs and 157 10/10 MUDs for comparison. AA matching was evaluated for the whole HLA protein, the α-helices, and the β-sheets, in both Host versus Graft (HvG) and Graft vs Host (GvH) direction. Superior EFS was found in 13 patients permissibly mismatched in the HvG direction (totalHvG, 92% vs 58% at 1 year, p=0.009), and in 21 patients matched for AA on the α-helices (αHvG, 90% vs 53%, p=0.002), similar to EFS with 10/10 MUDs (90% vs 80%, p=0.60). EFS was not related to β-sheet AA matching, nor to matching in the GvH direction. OS trended similarly as EFS for AA mismatches (totalHvG, 92% vs 74%, p=0.075 and αHvG90% vs 71%, p=0.072). These findings were reproduced in an EBMT inborn errors cohort of 271 pediatric 9/10 MMUD HSCTs and 929 10/10 MUD HSCTs, showing a significant effect of αHvG matching on both OS and EFS and similar OS and EFS between αHvG matched MMUDs and 10/10 MUDs. In summary, HvG-AA matching on the α-helices identifies 9/10 MMUD with permissible mismatches correlated with a favorable transplant outcome similar to matched donors.

Eurotransplant is responsible for the international allocation of organs between eight countries in Europe. All HLA laboratories affiliated to Eurotransplant must be EFI or ASHI-accredited and must participate in the Eurotransplant external proficiency testing (EPT) program, organized by the Eurotransplant Reference Laboratory (ETRL). EPT within Eurotransplant has a long tradition, starting in 1978. The current EPT program consists of the following schemes: HLA typing including serology, CDC crossmatching, HLA-specific antibody detection, and identification. Participants enter the results of laboratory tests using a web-based application. Assessed results are visible on the website. An additional component called “patient-based cases” runs since 2016. Results are summarized and published on the EPT website. Furthermore, these results are discussed during the annual extramural tissue typers meeting, which is organized by the ETRL. Thanks to this EPT program, the performance of all HLA laboratories affiliated to Eurotransplant can be monitored and corrected, if necessary. Because all affiliated laboratories are assessed in the same EPT program, where these laboratories show to be consistent in most of their results, Eurotransplant EPT has proven to be an efficient tool to create a more uniform level of quality of histocompatibility testing within Eurotransplant.

BACKGROUND:Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis. METHODS:We compared HLA expression between primary metastasized cSCCs, their metastases, and non-metastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all non-metastasized cSCCs. RESULTS:The mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to non-metastasized cSCCs (p = 0.065, 95 % CI -0.18-4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs. CONCLUSION:Contrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients.

The presence of donor‐specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody‐mediated rejection (ABMR) remains an important barrier to optimal long‐term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA‐producing B cells. We have genetically engineered an HLA‐A2‐specific CHAR (A2‐CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti‐HLA‐A2 antibody‐expressing target cells. In addition, we have performed A2‐CHAR‐Tc cytotoxic assays in an immunodeficient mouse model. A2‐CHAR expressing T cells could selectively eliminate HLA‐A2 antibody‐producing B cells in vitro. The cytotoxic capacity of A2‐CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2‐CHAR‐T cells could identify and eradicate HLA‐A2 antibody‐producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody‐producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation.

Proper invasion of the semi-allogeneic fetal trophoblast cells in the maternal-fetal interface (decidua) is essential for healthy placentation and pregnancy. Determining the characteristics and interplay of the maternal immune cells with fetal trophoblast cells at the maternal-fetal interface is key to understand their role in healthy and complicated pregnancy. To comprehend the composition and spatial orientation of maternal immune cells and fetal trophoblasts in situ, we applied imaging mass cytometry (IMC) on the decidua of the three trimesters of healthy pregnancy. We visualized the composition and interaction profiles of maternal immune cell populations, stromal cells, and fetal trophoblasts using a 41-marker IMC panel. We analyzed 148,177 segmented cells of first (n=3), second (n=5), and third trimester (n=5) placentas using phenotyping tools and interaction analysis programs including ImaCytE. We found a higher frequency of myeloid cells within the CD45+ population in the decidua using our in-situ approach compared to approaches using cell suspensions following enzymatic cell isolation from decidua (1st trim.: median 35.8% vs 17.6%, p=0.004; 2nd trim.: 52.5% vs 26.8%, p=0.004; Term: 60% vs 9.4%, p=0.001). Moreover, myeloid cells were the most pronounced cell type in the microenvironment of other decidual cells including fetal trophoblasts. In first trimester but not at term, HLA-DR-negative myeloid cells interact with CD4+ T cells, decidual NK cells, and trophoblasts. At term, T cell interactions with other immune cells became more prominent. By using IMC we confirmed previously described data but most notably further extended the comprehension of the decidual landscape. Our results highlight the dynamic role of myeloid cells at the human maternal-fetal interface throughout gestation. Furthermore, the results can be used to set up substantiated in vitro culture experiments to identify the consequences of cell interactions as found in the current study.

INTRODUCTION:Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta characterized by an infiltrate of CD68+ cells in the intervillous space. CHI is associated with adverse pregnancy outcomes such as miscarriage, fetal growth restriction, and (late) intrauterine fetal death. The adverse pregnancy outcomes and a variable recurrence rate of 25-100% underline its clinical relevance. The pathophysiologic mechanism of CHI is unclear, but it appears to be immunologically driven. The aim of this study was to obtain a better understanding of the phenotype of the cellular infiltrate in CHI.METHOD:We used imaging mass cytometry to achieve in-depth visualization of the intervillous maternal immune cells and investigated their spatial orientation in situ in relation to the fetal syncytiotrophoblast.RESULTS:We found three phenotypically distinct CD68+HLA-DR+CD38+ cell clusters that were unique for CHI. Additionally, syncytiotrophoblast cells in the vicinity of these CD68+HLA-DR+CD38+ cells showed decreased expression of the immunosuppressive enzyme CD39.DISCUSSION:The current results provide novel insight into the phenotype of CD68+ cells in CHI. The identification of unique CD68+ cell clusters will allow more detailed analysis of their function and could result in novel therapeutic targets for CHI.

The Eurotransplant Senior Program (ESP) has expedited the chance for elderly patients with kidney failure to receive a timely transplant. This current study evaluated survival parameters of kidneys donated after brain death with or without matching for HLA-DR antigens. This cohort study evaluated the period within ESP with paired allocation of 675 kidneys from donors 65 years and older to transplant candidates 65 years and older, the first kidney to 341 patients within the Eurotransplant Senior DR-compatible Program and 334 contralateral kidneys without (ESP) HLADR antigen matching. We used Kaplan-Meier estimates and competing risk analysis to assess all cause mortality and kidney graft failure, respectively. The log-rank test and Cox proportional hazards regression were used for comparisons. Within ESP, matching for HLA-DR antigens was associated with a significantly lower five-year risk of mortality (hazard ratio 0.71; 95% confidence interval 0.530.95) and significantly lower cause-specific hazards for kidney graft failure and return to dialysis at one year (0.55; 0.35-0.87) and five years (0.73; 0.53-0.99) post-transplant. Allocation based on HLA-DR matching resulted in longer cold ischemia (mean difference 1.00 hours; 95% confidence interval: 0.32-1.68) and kidney offers with a significantly shorter median dialysis vintage of 2.4 versus 4.1 yrs. in ESP without matching. Thus, our allocation based on HLA-DR matching improved five-year patient and kidney allograft survival. Hence, our paired allocation study suggests a superior outcome of HLA-DR matching in the context of old-for-old kidney transplantation.

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody‐verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody‐induction and binding are limited. In this proof‐of‐concept study, we performed site‐directed mutagenesis to narrow down the antibody‐verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

Background. The presence of donor-specific HLA antibodies before transplantation is associated with poor transplantation outcomes. Unacceptable antigens can be assigned for Eurotransplant kidney transplant candidates to prevent kidney offers against which the candidate has developed clinically relevant HLA antibodies. This retrospective cohort study aimed to assess to what degree unacceptable antigens affect access to transplantation in the Eurotransplant Kidney Allocation System (ETKAS). Methods. Candidates who underwent kidney-only transplantation between 2016 and 2020 were included (n=19240). Cox regression was used to quantify the relationship between the relative transplantation rate and virtual panel-reactive antibodies (vPRAs), which is the percentage of the donor pool with unacceptable antigens. Models used accrued dialysis time as the timescale; were stratified by country and blood group of patient and were adjusted for non-transplantable status, patient age, sex, history of kidney transplantations, and prevalence of 0 HLA-DR-mismatched donors. Results. Transplantation rates were 23% lower for vPRA 0.1% to 50%, 51% lower for vPRA 75% to 85%, and decreased rapidly for vPRA of >85%. Prior studies showed significantly lower ETKAS transplantation rates only for highly sensitized patients (vPRA of >85%). The inverse relationship between transplantation rate and vPRA is independent of Eurotransplant country, listing time, and 0 HLA-DR-mismatched donor availability. Results were similar when quantifying the relationship between vPRA and attainment of a sufficiently high rank for an ETKAS offer, suggesting lower transplantation rates for immunized patients are due to current ETKAS allocation. Conclusions. Immunized patients face lower transplantation rates across Eurotransplant. The current ETKAS allocation mechanism inadequately compensates immunized patients for reduced access to transplantation.

Organs transplanted across donor‐specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA‐affinity as well as DSA‐concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA‐specific monoclonal antibodies and assessed the technology‐specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio‐layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow‐induced dispersion analysis (FIDA). While the first three (solid‐phase) technologies revealed comparable high binding‐strengths, suggesting measurement of avidity, the latter (in‐solution) approach revealed slightly lower binding‐strengths, presumably indicating measurement of affinity. We believe that our newly developed in‐solution FIDA‐assay is particularly suitable to provide useful clinical information by not just measuring DSA‐affinities in patient serum samples but simultaneously delivering a particular DSA‐concentration. Here, we investigated DSA from 20 pre‐transplant patients, all of whom showed negative CDC‐crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA‐concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449‐fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre‐transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA‐concentration and DSA‐affinity.

Chronic histiocytic intervillositis (CHI) is a histopathological lesion in the placenta, characterized by an abundant intervillous cell infiltrate of CD68-positive macrophages of maternal origin, accompanied by adverse pregnancy outcomes. The pathophysiologic mechanism is unclear. Therefore, studied the placental intervillous cell infiltrate in three dizygotic twin couples with one-sided CHI: one had CHI, the other represented the non-affected control. Imaging mass cytometry (IMC), using a 42-marker panel, enabled in-depth assessment of phenotype and spatial orientation of the intervillous maternal immune cells. Approximately 80.4% of the intervillous immune cells were macrophages, 19.0% T-cells, 0.5% B-cells and 0.1% NK cells. The absolute number of macrophages and T cells was higher in CHI compared to controls. IMC data showed a difference in the activation status of macrophages between the affected and unaffected placentas: 44.5% of the CD68+ macrophages in the CHI cases demonstrated expression of the activation markers HLA-DR and CD38, whereas this was not expressed by the macrophages in the control twins. The fetal syncytiotrophoblasts cells that the HLA-DR+/CD38+/CD68+ macrophages are in proximity with show reduced CD39 expression. We confirmed our findings on a cohort of 11 placenta with CHI, compared to a control group. Since maternal intervillous blood is part of the maternal circulation and paired twin couples were studied here, it is likely that macrophages are activated locally in CHI. Further study should point out if HLA-DR+ macrophages are adhering to the fetal trophoblast cells and if they are locally affecting other immune cells of fetal cells.