Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Randomized Trial to Assess the Clinical Utility of Renal Allograft Monitoring by Urine CXCL10 Chemokine.

SIGNIFICANCE STATEMENT: This study is the first randomized controlled trial to investigate the clinical utility of a noninvasive monitoring biomarker in renal transplantation. Although urine CXCL10 monitoring could not demonstrate a beneficial effect on 1-year outcomes, the study is a rich source for future design of trials aiming to explore the clinical utility of noninvasive biomarkers. In addition, the study supports the use of urine CXCL10 to assess the inflammatory status of the renal allograft. BACKGROUND: Urine CXCL10 is a promising noninvasive biomarker for detection of renal allograft rejection. The aim of this study was to investigate the clinical utility of renal allograft monitoring by urine CXCL10 in a randomized trial. METHODS: We stratified 241 patients, 120 into an intervention and 121 into a control arm. In both arms, urine CXCL10 levels were monitored at three specific time points (1, 3, and 6 months post-transplant). In the intervention arm, elevated values triggered performance of an allograft biopsy with therapeutic adaptations according to the result. In the control arm, urine CXCL10 was measured, but the results concealed. The primary outcome was a combined end point at 1-year post-transplant (death-censored graft loss, clinical rejection between month 1 and 1-year, acute rejection in 1-year surveillance biopsy, chronic active T-cell-mediated rejection in 1-year surveillance biopsy, development of de novo donor-specific HLA antibodies, or eGFR <25 ml/min). RESULTS: The incidence of the primary outcome was not different between the intervention and the control arm (51% versus 49%; relative risk (RR), 1.04 [95% confidence interval, 0.81 to 1.34]; P = 0.80). When including 175 of 241 (73%) patients in a per-protocol analysis, the incidence of the primary outcome was also not different (55% versus 49%; RR, 1.11 [95% confidence interval, 0.84 to 1.47]; P = 0.54). The incidence of the individual end points was not different as well. CONCLUSIONS: This study could not demonstrate a beneficial effect of urine CXCL10 monitoring on 1-year outcomes (ClinicalTrials.gov_ NCT03140514 ).

Screening strategy for de novo donor-specific HLA antibodies beyond the first year after kidney transplantation: Personalized or "one size fits all"?

Screening for de novo donor-specific HLA antibodies (DSAs) after kidney transplantation is widely recommended. The aim of this single-center, cross-sectional study was to investigate the frequency of therapeutic interventions triggered by de novo DSA screening. We included 464 patients screened for de novo DSA at annual visits after a median of 5 years post-transplant (range 1 to 19 years). Overall, de novo DSAs were detected in 55/464 patients (11.9%) with a stepwise increase of the prevalence from 4.9% at 1 year post-transplant to 18.9% at >10 years post-transplant. Subsequent allograft biopsies were performed in 24/55 patients (44%). The main reasons to omit biopsies were good/stable allograft function and anticipated lack of clinical consequences (eg, relevant comorbidities). Rejection processes were detected in 16/24 biopsies (67%). Therapeutic interventions were made in 18/464 screened patients (3.9%) with a significantly higher rate in the youngest quartile of patients (≤48 years; 7.9%) compared to the middle 50% (49-67 years; 3%) and the oldest quartile (≥68 years; 1.7%) (P = .03). Our study suggests that the frequency of therapeutic interventions triggered by de novo DSA screening after kidney transplantation is overall low, but significantly higher in younger patients, arguing for a personalized, age-adapted de novo DSA screening strategy.

Caveats of HLA antibody detection by solid-phase assays.

Solid-phase assays for human leukocyte antigens (HLA) antibody detection have clearly revolutionized the field of HLA diagnostics and transplantation. The key advantages are a high sensitivity and specificity for the detection of HLA antibodies compared with cell-based assays, as well as the potential for standardization. Solid-phase assays enabled the broad introduction of tools such as "virtual crossmatching" and "calculated panel reactive antibodies," which are essential components in many organ allocation systems, kidney-paired donation programs, and center-specific immunological risk stratification procedures. The most advanced solid-phase assays are the so-called single antigen beads (SAB). They are available now for more than 15 years, and the transplant community embraced their significant advantages. However, SAB analysis and interpretation is complex and many pitfalls have to be considered. In this review, we will discuss problems, limitations, and challenges using SAB. Furthermore, we express our wishes for the improvements of SAB as well as their future use for immunological assessment and research purposes.

Human platelet antigen antibody induction in uncomplicated pregnancy is associated with HLA sensitization.

BACKGROUND: Alloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS: In a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex-based multiplex assay. RESULTS: HPA mismatches were observed in 109 of 241 pregnancies (45%), but child-specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8-193; p = 0.006), whereas the number of pregnancies was not. CONCLUSION: Our observational data indicated a low immunogenicity of HPA and suggest that a broader immune response-inferred by HLA sensitization-is probably associated with HPA antibody induction.

Human pregnancy and generation of anti-angiotensin receptor and anti-perlecan antibodies.

Non-HLA antibodies against the angiotensin II type 1 receptor (AT1 R) and the C-terminal fragment of perlecan (i.e., LG3) are associated with the development of renal allograft rejection. It is currently unknown how humans develop anti-AT1 R or anti-LG3 antibodies. The aim of this study was to investigate whether pregnancy-as a model of sensitization to polymorphic proteins-induces anti-AT1 R and/or anti-LG3 antibodies. We included 104 samples from women obtained after physiologic full-term pregnancy and 80 samples from healthy nonsensitized controls (40 women and 40 men). Both anti-AT1 R and anti-LG3 antibody levels were lower in pregnancy samples than in controls (both P < 0.05). By multivariate analysis, male gender was an independent predictor for high anti-AT1 R antibody levels (OR 3.66, P = 0.04) and pregnancy was predictive for low anti-LG3 antibody levels (OR 6.53, P = 0.0001). There was no correlation of anti-AT1 R with anti-LG3 antibody levels, either in the pregnancy or in the control samples (r(2) ≤ 0.03, P ≥ 0.26). In conclusion, physiologic full-term pregnancy does not induce anti-AT1 R or anti-LG3 antibodies and may even lower their levels. Therefore, anti-AT1 R and anti-LG3 antibodies are likely not caused by allosensitization. The lack of correlation of anti-AT1 R with anti-LG3 antibodies suggests different mechanisms of generation, which remain to be elucidated.

Soluble CD30 correlates with clinical but not subclinical renal allograft rejection.

Soluble CD30 (sCD30) has been proposed as a promising noninvasive biomarker for clinical renal allograft rejection, but its diagnostic characteristics regarding detection of subclinical rejection have not been assessed. We investigated sCD30 in 146 consecutive kidney allograft recipients under tacrolimus-mycophenolate-based immunosuppression having 250 surveillance biopsies at 3 and 6 months as well as 52 indication biopsies within the first year post-transplant. Allograft histology results were classified as (i) acute Banff score zero or interstitial infiltrates only, (ii) tubulitis t1, (iii) tubulitis t2-3 and (iv) isolated vascular compartment inflammation. sCD30 correlated well with the extent of clinical (P < 0.0001), but not subclinical tubulointerstitial rejection (P = 0.06). To determine diagnostic characteristics of sCD30, histological groups were assigned to two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (tubulitis t1-3 and isolated vascular compartment inflammation). For clinical allograft inflammation, AUC was 0.87 (sensitivity 89%, specificity 79%; P = 0.0006); however, for subclinical inflammation, AUC was only 0.59 (sensitivity 50%, specificity 69%; P = 0.47). In conclusion, sCD30 correlated with clinical, but not subclinical renal allograft rejection limiting its clinical utility as a noninvasive rejection screening biomarker in patients with stable allograft function receiving tacrolimus-mycophenolate-based immunosuppression.

HLA antibody affinity determination: From HLA-specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum.

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.

Multicenter Validation of a Urine CXCL10 Assay for Noninvasive Monitoring of Renal Transplants.

BACKGROUND: Urine CXCL10 (C-X-C motif chemokine ligand 10, interferon gamma-induced protein 10 [IP10]) outperforms standard-of-care monitoring for detecting subclinical and early clinical T-cell-mediated rejection (TCMR) and may advance TCMR therapy development through biomarker-enriched trials. The goal was to perform an international multicenter validation of a CXCL10 bead-based immunoassay (Luminex) for transplant surveillance and compare with an electrochemiluminescence-based (Meso Scale Discovery [MSD]) assay used in transplant trials. METHODS: Four laboratories participated in the Luminex assay development and evaluation. Urine CXCL10 was measured by Luminex and MSD in 2 independent adult kidney transplant trial cohorts (Basel and TMCT04). In an independent test and validation set, a linear mixed-effects model to predict (log 10 -transformed) MSD CXCL10 from Luminex CXCL10 was developed to determine the conversion between assays. Net reclassification was determined after mathematical conversion. RESULTS: The Luminex assay was precise, with an intra- and interassay coefficient of variation 8.1% and 9.3%; showed modest agreement between 4 laboratories (R 0.96 to 0.99, P < 0.001); and correlated with known CXCL10 in a single- (n = 100 urines, R 0.94 to 0.98, P < 0.001) and multicenter cohort (n = 468 urines, R 0.92, P < 0.001) but the 2 assays were not equivalent by Passing-Bablok regression. Linear mixed-effects modeling demonstrated an intercept of -0.490 and coefficient of 1.028, showing Luminex CXCL10 are slightly higher than MSD CXCL10, but the agreement is close to 1.0. After conversion of the biopsy thresholds, the decision to biopsy would be changed for only 6% (5/85) patients showing acceptable reclassification. CONCLUSIONS: These data demonstrate this urine CXCL10 Luminex immunoassay is robust, reproducible, and accurate, indicating it can be readily translated into clinical HLA laboratories for serial posttransplant surveillance.

Delayed graft function is not associated with an increased incidence of renal allograft rejection.

Delayed graft function (DGF) is considered as a risk factor for renal allograft rejection, but this association might be confounded by diagnostic biases (e.g., higher biopsy frequency in patients with DGF, inclusion of clinically diagnosed rejection episodes, and limited details on the rejection phenotype). This retrospective study including 329 deceased donor transplantations aimed to clarify a causal relationship between DGF and rejection. DGF occurred in 93/329 recipients (28%), whereas immediate graft function (IGF) in 236/329 recipients (72%). The percentage of patients with ≥1 allograft biopsy within the first year post-transplant was similar between the DGF and IGF group (96% vs. 94%; p=0.60). The cumulative one-yr incidence of biopsy-proven clinical (35% vs. 34%; p=0.62) and combined (sub)clinical rejection (58% vs. 60%; p=0.79) was not different between the two groups. Furthermore, there were no differences regarding rejection phenotypes/severities and time frame of occurrence. By multivariable Cox regression analysis, donor-specific HLA antibodies, younger recipient age, and immunosuppressive regimens were independent predictors for clinical rejection, while DGF was not. These results in an intermediate sized, but thoroughly investigated patient population challenge the concept that DGF is a risk factor for rejection and highlights the need for additional studies in this regard.

Snowflake epitope matching correlates with child-specific antibodies during pregnancy and donor-specific antibodies after kidney transplantation.

Development of donor-specific human leukocyte antigen (HLA) antibodies (DSA) remains a major risk factor for graft loss following organ transplantation, where DSA are directed towards patches on the three-dimensional structure of the respective organ donor's HLA proteins. Matching donors and recipients based on HLA epitopes appears beneficial for the avoidance of DSA. Defining surface epitopes however remains challenging and the concepts underlying their characterization are not fully understood. Based on our recently implemented computational deep learning pipeline to define HLA Class I protein-specific surface residues, we hypothesized a correlation between the number of HLA protein-specific solvent-accessible interlocus amino acid mismatches (arbitrarily called Snowflake) and the incidence of DSA. To validate our hypothesis, we considered two cohorts simultaneously. The kidney transplant cohort (KTC) considers 305 kidney-transplanted patients without DSA prior to transplantation. During the follow-up, HLA antibody screening was performed regularly to identify DSA. The pregnancy cohort (PC) considers 231 women without major sensitization events prior to pregnancy who gave live birth. Post-delivery serum was screened for HLA antibodies directed against the child's inherited paternal haplotype (CSA). Based on the involved individuals' HLA typings, the numbers of interlocus-mismatched antibody-verified eplets (AbvEPS), the T cell epitope PIRCHE-II model and Snowflake were calculated locus-specific (HLA-A, -B and -C), normalized and pooled. In both cohorts, Snowflake numbers were significantly elevated in recipients/mothers that developed DSA/CSA. Univariable regression revealed significant positive correlation between DSA/CSA and AbvEPS, PIRCHE-II and Snowflake. Snowflake numbers showed stronger correlation with numbers of AbvEPS compared to Snowflake numbers with PIRCHE-II. Our data shows correlation between Snowflake scores and the incidence of DSA after allo-immunization. Given both AbvEPS and Snowflake are B cell epitope models, their stronger correlation compared to PIRCHE-II and Snowflake appears plausible. Our data confirms that exploring solvent accessibility is a valuable approach for refining B cell epitope definitions.

Risk stratification by the virtual crossmatch: a prospective study in 233 renal transplantations.

The virtual crossmatch (virtual-XM) has been proposed for accurate identification of donor-specific HLA-antibodies, but large prospective studies assessing its value for pretransplant risk stratification are lacking. A total of 233 consecutive renal allograft recipients were prospectively stratified according to the virtual-XM. In patients with a negative virtual-XM (n=190, 82%), prospective cytotoxicity crossmatches (CDC-XM) were omitted, and they received standard immunosuppression. Virtual-XM positive patients were only transplanted if CDC-XM were negative. They received additional induction with anti-T-lymphocyte-globulin and intravenous immunoglobulins (n=43, 18%). The cumulative incidence of clinical/subclinical antibody-mediated rejection (AMR) at 1 year was lower in the negative virtual-XM than in the positive virtual-XM group [15/190 (8%) vs. 18/43 (42%); P<0.0001]. After a median follow-up of 2.6 years, allograft loss because of AMR occurred less often in the negative virtual-XM group (1% vs. 7%; P=0.04) and death-censored allograft survival at 2 years was higher (98% vs. 91%; P=0.02). Serum creatinine was not different at the last follow-up (129 µm vs. 130 µm; P=0.58). We conclude that a negative virtual-XM defines patients at low risk for AMR and early allograft loss, while a positive virtual-XM represents a significant risk for AMR despite enhanced induction therapy. This supports the utility of the virtual-XM for risk stratification and treatment allocation.

Peptides Derived From Mismatched Paternal Human Leukocyte Antigen Predicted to Be Presented by HLA-DRB1, -DRB3/4/5, -DQ, and -DP Induce Child-Specific Antibodies in Pregnant Women.

Predicted Indirectly ReCognizable Human Leukocyte Antigen (HLA) Epitopes (PIRCHE) are known to be a significant risk factor for the development of donor HLA-specific antibodies after organ transplantation. Most previous studies on PIRCHE limited their analyses on the presentation of the HLA-DRB1 locus, although HLA-DRB3/4/5, -DQ, and -DP are also known for presenting allopeptides to CD4+ T cells. In this study, we analyzed the impact of predicted allopeptides presented by these additional loci on the incidence of HLA-specific antibodies after an immunization event. We considered pregnancy as a model system of an HLA immunization and observed child-specific HLA antibody (CSA) development of 231 mothers during pregnancy by samples being taken at delivery. Our data confirm that PIRCHE presented by HLA-DRB1 along with HLA-DRB3/4/5, -DQ, and -DP are significant predictors for the development of CSA. Although there was limited peptidome overlap observed within the mothers' presenting HLA proteins, combining multiple presenting loci in a single predictor improved the model only marginally. Prediction performance of PIRCHE further improved when normalizing scores by the respective presenters' binding promiscuity. Immunogenicity analysis of specific allopeptides could not identify significant drivers of an immune response in this small cohort, suggesting confirmatory studies.

Outcome of Husband-to-Wife Kidney Transplantation With Mutual Children: Single Center Experience Using T Cell-Depleting Induction and Review of the Literature.

Few data on husband-to-wife transplantations with mutual children (H2W) exist in the current era. We investigated the outcome of H2W transplantations (n = 25) treated with T cell-depleting induction compared to women with prior pregnancies also receiving their first HLA-mismatched kidney transplant, but from a different donor source: (i) other living donor (n = 52) and (ii) deceased donor (n = 120). Seventy-four percent of the women had ≥2 pregnancies; median follow-up time was 5 years. Death-censored allograft survival was significantly lower in the H2W group compared to the other two groups (p = 0.03). Three of four graft losses in the H2W group were due to rejection. 5-year patient survival in the H2W group was high and similar compared to the other living donor group (100 vs. 98%; p = 0.28). The incidence of (sub)clinical antibody-mediated rejection was higher in the H2W group (36 vs. 20 vs. 18%) (p = 0.10). The frequency of infections was similar among the three groups. No immunological parameter was predictive for rejection or graft loss in H2W transplantations. In conclusion, H2W transplantation is a valuable option, but associated with a higher risk for allograft loss due to rejection despite T cell-depleting induction. Further research is required for better risk prediction on an individual patient level.

Development of an immunogenicity score for HLA-DQ eplets: A conceptual study.

Eplets are defined as distinct amino acid configurations on the surface of HLA molecules. The aim of this study was to estimate the immunogenicity of HLA-DQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches. We defined the immunogenicity of an eplet by the frequency of antibody responses against it. Around 90% of all listed DQB1 or DQA1 eplets were at least five times mismatched and thus included for the calculation of their immunogenicity. The DQB1 eplets with the five highest immunogenicity scores were 55PP, 52PR, 52PQ, 85VG and 45EV; 25% of all DQB1 eplets were not reacting. The DQA1 eplets with the five highest immunogenicity scores were 25YS, 47QL, 55RR, 187T and 18S; 17% of all DQA1 eplets were not reacting. The immunogenicity score had a slightly higher area under the curve to predict development of child-specific antibodies than various molecular mismatch scores (eg, eplet mismatch load, amino acid mismatch load). Overlapping eplets were identified as a barrier to unambiguously assign the immunogenicity score based on HLA antibody reaction patterns. In this conceptual study, we explored the immunogenicity of HLA-DQ eplets and created a map of potentially immunogenic regions on HLA-DQ molecules, which requires validation in clinical transplant cohorts.

Technical Considerations and Confounders for Urine CXCL10 Chemokine Measurement.

BACKGROUND: The urine C-X-C motif chemokine 10 (CXCL10) is a promising screening biomarker for renal allograft rejection. The aim of the study was to investigate important technical and biological aspects as well as potential confounders when measuring urine CXCL10. METHODS: We analyzed 595 urine samples from 117 patients, who participated in a randomized controlled trial investigating the clinical utility of urine CXCL10 monitoring for posttransplant management. Urine CXCL10 was measured by an immunoassay using electrochemiluminescence. RESULTS: Intraassay coefficient of variation was 2.5%, and interassay coefficient of variation was 10%. Urine CXCL10 remained stable (ie, <10% degradation) for 8 hours at 25°C or 37°C and for 3 days at 4°C. CXCL10 concentrations [pg/mL] strongly correlated with urine CXCL10/creatinine ratios [ng/mmol] (r2 = 0.98; P < 0.0001). Leucocyturia and active BK-polyomavirus infection are associated with higher CXCL10 concentrations, while allograft function, serum CRP, patient age, proteinuria, urine pH, hematuria, squamous epithelia cell count, and bacteriuria did not correlate with urine CXCL10 concentrations. In 145 paired samples obtained within 1-2 weeks, 80% showed a CXCL10/creatinine ratio change of < ±2 ng/mmol or ±50%, respectively. CONCLUSIONS: Urine CXCL10 measurement on the used platform is accurate and robust. Leucocyturia and active BK-polyomavirus infection are major confounders, which can be easily detected but represent important diagnostic "blind spots" when using urine CXCL10 to screen for allograft rejection. The intraindividual biological variability of urine CXCL10 within 1-2 weeks is mostly below ±50%, which is still much higher than the technical variability due to sample handling/processing (<20%).

Toward defining the immunogenicity of HLA epitopes: Impact of HLA class I eplets on antibody formation during pregnancy.

Eplets are functional units of structural epitopes on donor HLA, potentially recognized by complementarity-determining regions of the paratope of the recipients' B-cell receptors or antibodies (Ab). Their individual immunogenicity is poorly described, yet this feature would be of clinical importance for pretransplant risk assessment. The aim of this study was to determine the relative immunogenicity of HLA class I eplets in the pregnancy setting, where mismatched eplets are present on paternal HLA antigens of the unborn child. One hundred fifty-nine predominantly Caucasian mothers giving birth at the University Hospital Basel and their first newborns were HLA-typed at high-resolution by next-generation sequencing (NGS) (NGSgo Workflow and NGSengine from GenDx; sequencing with a Miseq from Illumina) and eplets were determined using HLAMatchmaker. HLA class I specific IgG Ab was assessed in maternal sera drawn immediately after full-term delivery, by OneLambda LABScreen single antigen ibeads. The Ab profile was subsequently evaluated for eplet-associated patterns. All 72 currently Ab-verified HLA class I eplets were examined for their immunogenicity according to the frequency of child-specific HLA Ab (CSA) directed against their structures. Four hundred twelve of 477 (86.4%) paternal HLA-A, -B or -C alleles were mismatched. CSA were present in 46 mothers (28.9%), directed against 80 (19.4%) of these mismatches. The 10 most immunogenic eplets were 62GK, 145KHA, 144TKH, 62GE, 107W, 80I, 82LR, 41T, 127K, 45KE with immunogenicity rates between 45.8% and 27.3%. This pregnancy study also identified five non-reactive eplets: 62RR, 76ESN, 80TLR, 156DA, 163RW. Based on our results, immunogenic hot and cold spots on the surface of HLA class I molecules were localized and visualized on 3D models. This study strengthens the presumption that different eplets represent different immunogenic potentials. Validation of these results in the clinical transplant setting is an essential next step in identifying those eplets representing a particularly high-risk potential.

Utility of the virtual crossmatch in solid organ transplantation.

PURPOSE OF REVIEW: Solid-phase assays covered with single HLA molecules - such as single-antigen flow-beads (SAFBs) - allow determining the presence of donor-specific HLA antibodies (HLA-DSAs) 'virtually' by comparison of the HLA-antibody specificities of the recipient with the HLA typing of the donor. In this review, prospects and current limitation of the virtual crossmatch are discussed. RECENT FINDINGS: Several prospective and retrospective studies indicate that a negative virtual crossmatch is associated with a very low risk of early rejection and good long-term allograft survival. By contrast, a positive virtual crossmatch is associated with a significant risk for early rejection and decreased allograft survival. However, these studies revealed that not all HLA-DSAs detected by SAFB have a detrimental clinical impact. SUMMARY: The virtual crossmatch has emerged as a very useful tool for pretransplant risk assessment and organ allocation. Further advances of the virtual crossmatch approach will require improvements on the technical part of SAFB analysis and a better understanding and definition of pathogenic factors of HLA-DSA. Together with an extended HLA typing of the donor, this scenario will provide us the full benefits of applications based on virtual crossmatching.

Differential Impact of Delayed Graft Function in Deceased Donor Renal Transplant Recipients With and Without Donor-specific HLA-antibodies.

BACKGROUND: Delayed graft function (DGF) and pretransplant donor-specific HLA-antibodies (DSA) are both regarded as risk factors for rejection and lower graft survival. However, the combined impact of DGF and DSA has not been studied in detail. METHODS: We investigated 375 deceased donor kidney transplantations, which had DSA assignment by single-antigen bead technology and which had surveillance biopsies at 3 of 6 months. Median follow-up time was 6.1 years. RESULTS: DGF occurred in 137 of 375 patients (37%), and DSA were present in 85 of 375 patients (23%). The incidence of DGF was similar in DSA-positive (DSApos)-patients and DSA-negative (DSAneg)-patients (40% versus 36%; P = 0.45). In DSAneg-patients, 5-year graft survival was not different with/without DGF (81% versus 83%; P = 0.48). By contrast, in DSApos-patients, 5-year graft survival was significantly lower with DGF (64% versus 79%; P = 0.01). Moreover, DSApos-patients with DGF had a higher 1-year incidence of subclinical rejection, which were mostly antibody-mediated or mixed rejection phenotypes. Graft loss due to rejection was significantly more frequent in DSApos-patients with DGF (5/34; 15%) compared to DSApos-patients without DGF (2/51; 4%), and DSAneg-patients with/without DGF (3/103; 3% and 4/187; 2%, respectively) (P = 0.005). In a multivariate Cox model, DSA with DGF was an independent predictor for graft (hazard ratio = 2.84 [95% confidence interval, 1.54-5.06]; P = 0.001) and death-censored graft loss (hazard ratio = 4.65 [95% confidence interval, 1.83-11.51]; P = 0.002). CONCLUSIONS: DGF has a much more detrimental impact in DSApos-patients than in DSAneg-patients, which is likely related to a higher incidence of antibody-mediated rejection. If possible, the combined risks of DGF and DSA should be avoided.