Back to the future: the uncharted role of T cells in chronic and unexplained microvascular inflammation.

The 2022 Banff classification for kidney allograft pathology introduced the category "microvascular inflammation DSA-negative and C4d-negative" for cases without evidence of a humoral cause. Many questions remain about the etiology, prognosis, and treatment of this phenotype. Cristoferi et al. performed a molecular comparison of chronic active antibody-mediated rejection and its seronegative counterpart and suggest a central role for T cells in chronic donor-specific antibody-negative, C4d-negative microvascular inflammation. These results further question how we should classify rejection.

An observational cohort study examined the change point of kidney function stabilization in the initial period after transplantation.

Baseline kidney function following kidney transplantation is often used in research and clinical decision-making yet is not well defined. Here, a method to determine baseline function was proposed and validated on three single-center retrospective cohorts consisting of 922 patients from Belgium (main cohort) and two validation cohorts of 987 patients from the Netherlands and 519 patients from Germany. For each transplant, a segmented regression model was fitted on the estimated glomerular filtration rate (eGFR) evolution during the first-year post-transplantation. This yielded estimates for change point timing, rate of eGFR change before and after change point and eGFR value at change point, now considered the "baseline function". Associations of eGFR evolution with recipient/donor characteristics and the graft failure rate were assessed with linear regression and Cox regression respectively. The change point occurred on average at an eGFR value of 43.7±14.6 mL/min/1.73m2, at a median time of 6.5 days post-transplantation. Despite significant associations with several baseline donor-recipient characteristics (particularly, donor type; living vs deceased), the predictive value of these characteristics for eGFR value and timing of the change point was limited. This followed from a large heterogeneity within eGFR trajectories, which in turn indicated that favorable levels of kidney function could be reached despite a suboptimal initial evolution. Segmented regression consistently provided a good fit to early eGFR evolution, and its estimate of the change point can be a useful reference value in future analyses. Thus, our study shows that baseline kidney function after transplantation is heterogeneous and partly related to pretransplant donor characteristics.

Probable antibody-mediated rejection in kidney transplantation is a rare and challenging phenotype to define: Findings from a single-center study.

The Banff 2022 consensus introduced probable antibody-mediated rejection (AMR), characterized by mild AMR histologic features and human leukocyte antigen (HLA) donor-specific antibody (DSA) positivity. In a single-center observational cohort study of 1891 kidney transplant recipients transplanted between 2004 and 2021, 566 kidney biopsies were performed in 178 individual HLA-DSA-positive transplants. Evaluated at time of the first HLA-DSA-positive biopsy of each transplant (N = 178), 84 of the 178 (47.2%) of first biopsies were scored as no AMR, 22 of the 178 (12.4%) as probable AMR, and 72 of the 178 (40.4%) as AMR. The majority (77.3%) of probable AMR cases were first diagnosed in indication biopsies. Probable AMR was associated with lower estimated glomerular filtration rate (mL/min/1.73m2) than no AMR (20.2 [8.3-32.3] vs 40.1 [25.4-53.3]; P = .001). The one-year risk of (repeat) AMR was similar for probable AMR and AMR (subdistribution hazard ratio (sHR), 0.99; 0.42-2.31; P = .97) and higher than after no AMR (sHR, 3.05; 1.07-8.73; P = .04). Probable AMR had a higher five-year risk of transplant glomerulopathy vs no AMR (sHR, 4.29; 0.92-19.98; P = 06), similar to AMR (sHR, 1.74; 0.43-7.04; P = .44). No significant differences in five-year risk of graft failure emerged between probable AMR and AMR (sHR, 1.14; 0.36-3.58; P = .82) or no AMR (sHR, 2.46; 0.78-7.74; P = .12). Probable AMR is a rare phenotype, however, sharing significant similarities with AMR in this single-center study. Future studies are needed to validate reproducible diagnostic criteria and associated clinical outcomes to allow for defining best management of this potentially relevant phenotype.

The Banff 2022 Kidney Meeting Report: Reappraisal of microvascular inflammation and the role of biopsy-based transcript diagnostics.

The XVI-th Banff Meeting for Allograft Pathology was held at Banff, Alberta, Canada, from 19th to 23rd September 2022, as a joint meeting with the Canadian Society of Transplantation. To mark the 30th anniversary of the first Banff Classification, premeeting discussions were held on the past, present, and future of the Banff Classification. This report is a summary of the meeting highlights that were most important in terms of their effect on the Classification, including discussions around microvascular inflammation and biopsy-based transcript analysis for diagnosis. In a postmeeting survey, agreement was reached on the delineation of the following phenotypes: (1) "Probable antibody-mediated rejection (AMR)," which represents donor-specific antibodies (DSA)-positive cases with some histologic features of AMR but below current thresholds for a definitive AMR diagnosis; and (2) "Microvascular inflammation, DSA-negative and C4d-negative," a phenotype of unclear cause requiring further study, which represents cases with microvascular inflammation not explained by DSA. Although biopsy-based transcript diagnostics are considered promising and remain an integral part of the Banff Classification (limited to diagnosis of AMR), further work needs to be done to agree on the exact classifiers, thresholds, and clinical context of use.

Automated Urinary Chemokine Assays for Noninvasive Detection of Kidney Transplant Rejection: A Prospective Cohort Study.

RATIONALE & OBJECTIVE: Prior studies have demonstrated the diagnostic potential of urinary chemokines C-X-C motif ligand 9 (CXCL9) and CXCL10 for kidney transplant rejection. However, their benefit in addition to clinical information has not been demonstrated. We evaluated the diagnostic performance for detecting acute rejection of urinary CXCL9 and CXCL10 when integrated with clinical information. STUDY DESIGN: Single-center prospective cohort study. SETTING & PARTICIPANTS: We analyzed 1,559 biopsy-paired urinary samples from 622 kidney transplants performed between April 2013 and July 2019 at a single transplant center in Belgium. External validation was performed in 986 biopsy-paired urinary samples. TESTS COMPARED: We quantified urinary CXCL9 (uCXCL9) and CXCL10 (uCXCL10) using an automated immunoassay platform and normalized the values to urinary creatinine. Urinary chemokines were incorporated into a multivariable model with routine clinical markers (estimated glomerular filtration rate, donor-specific antibodies, and polyoma viremia) (integrated model). This model was then compared with the tissue diagnosis according to the Banff classification for acute rejection. OUTCOME: Acute rejection detected on kidney biopsy using the Banff classification. RESULTS: Chemokines integrated with routine clinical markers had high diagnostic value for detection of acute rejection (n=150) (receiver operating characteristic area under the curve 81.3% [95% CI, 77.6-85.0]). The integrated model would help avoid 59 protocol biopsies per 100 patients when the risk for rejection is predicted to be below 10%. The performance of the integrated model was similar in the external validation cohort. LIMITATIONS: The cross-sectional nature obviates investigating the evolution over time and prediction of future rejection. CONCLUSIONS: The use of an integrated model of urinary chemokines and clinical markers for noninvasive monitoring of rejection could enable a reduction in the number of biopsies. Urinary chemokines may be useful noninvasive biomarkers whose use should be further studied in prospective randomized trials to clarify their role in guiding clinical care and the use of biopsies to detect rejection after kidney transplantation. PLAIN-LANGUAGE SUMMARY: Urinary chemokines CXCL9 and CXCL10 have been suggested to be good noninvasive biomarkers of kidney transplant rejection. However, defining a context of use and integration with clinical information is necessary before clinical implementation can begin. In this study, we demonstrated that urinary chemokines CXCL9 and CXCL10, together with clinical information, have substantial diagnostic accuracy for the detection of acute kidney transplant rejection. Application of urinary chemokines together with clinical information may guide biopsy practices following kidney transplantation and potentially reduce the need for kidney transplant biopsies.

Data-Driven Chronic Allograft Phenotypes: A Novel and Validated Complement for Histologic Assessment of Kidney Transplant Biopsies.

BACKGROUND: No validated system currently exists to realistically characterize the chronic pathology of kidney transplants that represents the dynamic disease process and spectrum of disease severity. We sought to develop and validate a tool to describe chronicity and severity of renal allograft disease and integrate it with the evaluation of disease activity. METHODS: The training cohort included 3549 kidney transplant biopsies from an observational cohort of 937 recipients. We reweighted the chronic histologic lesions according to their time-dependent association with graft failure, and performed consensus k-means clustering analysis. Total chronicity was calculated as the sum of the weighted chronic lesion scores, scaled to the unit interval. RESULTS: We identified four chronic clusters associated with graft outcome, based on the proportion of ambiguous clustering. The two clusters with the worst survival outcome were determined by interstitial fibrosis and tubular atrophy (IFTA) and by transplant glomerulopathy. The chronic clusters partially overlapped with the existing Banff IFTA classification (adjusted Rand index, 0.35) and were distributed independently of the acute lesions. Total chronicity strongly associated with graft failure (hazard ratio [HR], 8.33; 95% confidence interval [CI], 5.94 to 10.88; P<0.001), independent of the total activity scores (HR, 5.01; 95% CI, 2.83 to 7.00; P<0.001). These results were validated on an external cohort of 4031 biopsies from 2054 kidney transplant recipients. CONCLUSIONS: The evaluation of total chronicity provides information on kidney transplant pathology that complements the estimation of disease activity from acute lesion scores. Use of the data-driven algorithm used in this study, called RejectClass, may provide a holistic and quantitative assessment of kidney transplant injury phenotypes and severity.

Allorecognition and the spectrum of kidney transplant rejection.

Detection of mismatched human leukocyte antigens by adaptive immune cells is considered as the main cause of transplant rejection, leading to either T-cell mediated rejection or antibody-mediated rejection. This canonical view guided the successful development of immunosuppressive therapies and shaped the diagnostic Banff classification for kidney transplant rejection that is used in clinics worldwide. However, several observations have recently emerged that question this dichotomization between T-cell mediated rejection and antibody-mediated rejection, related to heterogeneity in the serology, histology, and prognosis of the rejection phenotypes. In parallel, novel insights were obtained concerning the dynamics of donor-specific anti-human leukocyte antigen antibodies, the immunogenicity of donor-recipient non-human leukocyte antigen mismatches, and the autoreactivity against self-antigens. Moreover, the potential of innate allorecognition was uncovered, as exemplified by natural killer cell-mediated microvascular inflammation through missing self, and by the emerging evidence on monocyte-driven allorecognition. In this review, we highlight the gaps in the current classification of rejection, provide an overview of the expanding insights into the mechanisms of allorecognition, and critically appraise how these could improve our understanding and clinical approach to kidney transplant rejection. We argue that consideration of the complex interplay of various allorecognition mechanisms can foster a more integrated view of kidney transplant rejection and can lead to improved risk stratification, targeted therapies, and better outcome after kidney transplantation.

Biological pathways and comparison with biopsy signals and cellular origin of peripheral blood transcriptomic profiles during kidney allograft pathology.

Kidney transplant injury processes are associated with molecular changes in kidney tissue, primarily related to immune cell activation and infiltration. How these processes are reflected in the circulating immune cells, whose activation is targeted by strong immunosuppressants, is poorly understood. To study this, we analyzed the molecular alterations in 384 peripheral blood samples from four European transplant centers, taken at the time of a kidney allograft biopsy, selected for their phenotype, using RNA-sequencing. In peripheral blood, differentially expressed genes in 136 rejection and 248 no rejection samples demonstrated upregulation of glucocorticoid receptor and nucleotide oligomerization domain-like receptor signaling pathways. Pathways enriched in antibody-mediated rejection (ABMR) were strongly immune-specific, whereas pathways enriched in T cell-mediated rejection were less immune related. In polyomavirus infection, upregulation of mitochondrial dysfunction and interferon signaling pathways was seen. Next, we integrated the blood results with transcriptomics of 224 kidney allograft biopsies which showed consistently upregulated genes per phenotype in both blood and biopsy. In single-cell RNASeq (scRNASeq) analysis of seven kidney allograft biopsies, the consistently overexpressed genes in ABMR were mostly expressed by infiltrating leukocytes in the allograft. Similarly, in peripheral blood scRNASeq analysis, these genes were overexpressed in ABMR in immune cell subtypes. Furthermore, overexpression of these genes in ABMR was confirmed in independent cohorts in blood and biopsy. Thus, our results highlight the immune activation pathways in peripheral blood leukocytes at the time of kidney allograft pathology, despite the use of current strong immunosuppressants, and provide a framework for future therapeutic interventions.

Association of Predicted HLA T-Cell Epitope Targets and T-Cell-Mediated Rejection After Kidney Transplantation.

RATIONALE & OBJECTIVE: The relationship between human leukocyte antigen (HLA) molecular mismatches and T-cell-mediated rejection (TCMR) is unknown. We investigated the associations between the different donor HLA-derived T-cell targets and the occurrence of TCMR and borderline histologic changes suggestive of TCMR after kidney transplantation. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: All kidney transplant recipients at a single center between 2004 and 2013 with available biopsy data and a DNA sample for high-resolution HLA donor/recipient typing (N = 893). EXPOSURE: Scores calculated by the HLA matching algorithm PIRCHE-II and HLA eplet mismatches. OUTCOME: TCMR, borderline changes suggestive of TCMR, and allograft failure. ANALYTICAL APPROACH: Multivariable cause-specific hazards models were fit to characterize the association between HLA epitopes targets and study outcomes. RESULTS: We found 277 patients developed TCMR, and 134 developed only borderline changes suggestive of TCMR on at least 1 biopsy. In multivariable analyses, only the PIRCHE-II scores for HLA-DRB1 and HLA-DQB1 were independently associated with the occurrence of TCMR and with allograft failure; this was not the case for HLA class I molecules. If restricted to rejection episodes within the first 3 months after transplantation, only the T-cell epitope targets originating from the donor's HLA-DRB1 and HLA-DQB1, but not class I molecules, were associated with the early acute TCMR. Also, the median PIRCHE-II score for HLA class II was statistically different between the patients with TCMR compared to the patients without TCMR (129 [IQR, 60-240] vs 201 [IQR, 96-298], respectively; P < 0.0001). These differences were not observed for class I PIRCHE-II scores. LIMITATIONS: Observational clinical data and residual confounding. CONCLUSIONS: In the absence of HLA-DSA, HLA class II but not class I mismatches are associated with early episodes of acute TCMR and allograft failure. This suggests that current immunosuppressive therapies are largely able to abort the most deleterious HLA class I-directed alloimmune processes; however, alloresponses against HLA-DRB1 and HLA-DQB1 molecular mismatches remain insufficiently suppressed. PLAIN-LANGUAGE SUMMARY: Genetic differences in the human leukocyte antigen (HLA) complex between kidney transplant donors and recipients play a central role in T-cell-mediated rejection (TCMR), which can lead to failure of the transplanted kidney. Evaluating this genetic disparity (mismatch) in the HLA complex at the molecular (epitope) level could contribute to better prediction of the immune response to the donor organ posttransplantation. We investigated the associations of the different donor HLA-derived T-cell epitope targets and scores obtained from virtual crossmatch algorithms with the occurrence of TCMR, borderline TCMR, and graft failure after kidney transplantation after taking into account the influence of donor-specific anti-HLA antibodies. This study illustrates the greater importance of the molecular mismatches in class II molecules compared to class I HLA molecules.

Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases.

Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy specimen at a single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. This review provides an overview of the technical challenges that should be addressed when designing single-cell transcriptomics experiments that focus on glomerulopathies. The isolation of glomerular cells from core needle biopsy specimens for single-cell transcriptomics remains difficult and depends upon five major factors. First, core needle biopsies generate little tissue material, and several samples are required to identify glomerular cells. Second, both fresh and frozen tissue samples may yield glomerular cells, although every experimental pipeline has different (dis)advantages. Third, enrichment for glomerular cells in human tissue before single-cell analysis is challenging because no effective standardized pipelines are available. Fourth, the current warm cell-dissociation protocols may damage glomerular cells and induce transcriptional artifacts, which can be minimized by using cold dissociation techniques at the cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells; however, the efficacy of snRNA-seq on core needle biopsy specimens remains to be proven. The field of single-cell omics is rapidly evolving, and the integration of these techniques in multiomics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases.

Missing Self-Induced Microvascular Rejection of Kidney Allografts: A Population-Based Study.

BACKGROUND: Circulating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I-mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA-negative MVI. However, missing self's clinical importance as a nonhumoral trigger of allograft rejection remains unclear. METHODS: In a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene. RESULTS: We identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA-associated MVI. CONCLUSION: Missing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA-negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.

Data-driven Derivation and Validation of Novel Phenotypes for Acute Kidney Transplant Rejection using Semi-supervised Clustering.

BACKGROUND: Over the past decades, an international group of experts iteratively developed a consensus classification of kidney transplant rejection phenotypes, known as the Banff classification. Data-driven clustering of kidney transplant histologic data could simplify the complex and discretionary rules of the Banff classification, while improving the association with graft failure. METHODS: The data consisted of a training set of 3510 kidney-transplant biopsies from an observational cohort of 936 recipients. Independent validation of the results was performed on an external set of 3835 biopsies from 1989 patients. On the basis of acute histologic lesion scores and the presence of donor-specific HLA antibodies, stable clustering was achieved on the basis of a consensus of 400 different clustering partitions. Additional information on kidney-transplant failure was introduced with a weighted Euclidean distance. RESULTS: Based on the proportion of ambiguous clustering, six clinically meaningful cluster phenotypes were identified. There was significant overlap with the existing Banff classification (adjusted rand index, 0.48). However, the data-driven approach eliminated intermediate and mixed phenotypes and created acute rejection clusters that are each significantly associated with graft failure. Finally, a novel visualization tool presents disease phenotypes and severity in a continuous manner, as a complement to the discrete clusters. CONCLUSIONS: A semisupervised clustering approach for the identification of clinically meaningful novel phenotypes of kidney transplant rejection has been developed and validated. The approach has the potential to offer a more quantitative evaluation of rejection subtypes and severity, especially in situations in which the current histologic categorization is ambiguous.

Missing Self-Induced Activation of NK Cells Combines with Non-Complement-Fixing Donor-Specific Antibodies to Accelerate Kidney Transplant Loss in Chronic Antibody-Mediated Rejection.

BACKGROUND: Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR. METHODS AND RESULTS: Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (n=73, 54%) had a higher risk of transplant failure (P=0.002). Among the remaining patients with complement-independent chronic AMR (n=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (P=0.02). In multivariable analysis, only proteinuria (HR: 7.24; P=0.01) and the presence of missing self (HR: 3.57; P=0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage. CONCLUSIONS: The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.

Risk factors, histopathological features, and graft outcome of transplant glomerulopathy in the absence of donor-specific HLA antibodies.

Transplant glomerulopathy is established as a hallmark of chronic antibody-mediated rejection in kidney transplant patients with donor-specific HLA antibodies (HLA-DSA). The clinical importance of transplant glomerulopathy in the absence of HLA-DSA is not well established. To help define this, 954 patients (encompassing 3744 biopsies) who underwent kidney transplantation 2004-2013 were studied with retrospective high-resolution HLA genotyping of both donors and recipients. The risk factors, histopathological appearance and prognosis of cases with transplant glomerulopathy in the absence of HLA-DSA were compared to those cases with HLA-DSA, and the impact of the PIRCHE-II score and eplet mismatches on development of transplant glomerulopathy evaluated. In this cohort, 10.3% developed transplant glomerulopathy, on average 3.2 years post-transplant. At the time of glomerulopathy, 23.5% had persistent pre-transplant or de novo HLA-DSA, while 76.5% were HLA-DSA negative. Only HLA-DSA was identified as a risk factor for glomerulopathy development as eplet mismatches and the PIRCHE-II score did not associate. HLA-DSA negative biopsies with glomerulopathy had less interstitial inflammation, less glomerulitis, and less C4d deposition in the peritubular capillaries compared to the HLA-DSA positive biopsies with glomerulopathy. While graft function was comparable between the two groups, HLA-DSA positive glomerulopathy was associated with a significantly higher risk of graft failure compared to HLA-DSA negative glomerulopathy (Hazard Ratio 3.84; 95% confidence interval 1.94-7.59). Landmark analysis three-years post-transplant showed that HLA-DSA negative patients with glomerulopathy still had a significant increased risk of graft failure compared to patients negative for glomerulopathy (2.62; 1.46-4.72). Thus, transplant glomerulopathy often occurs in the absence of HLA-DSA, independent of HLA molecular mismatches, and represents a different phenotype with less concomitant inflammation and better graft survival compared to that developed in the presence of HLA-DSA.

Revisiting the changes in the Banff classification for antibody-mediated rejection after kidney transplantation.

The Banff classification for antibody-mediated rejection (ABMR) has undergone important changes, mainly by inclusion of C4d-negative ABMR in Banff'13 and elimination of suspicious ABMR (sABMR) with the use of C4d as surrogate for HLA-DSA in Banff'17. We aimed to evaluate the numerical and prognostic repercussions of these changes in a single-center cohort study of 949 single kidney transplantations, comprising 3662 biopsies that were classified according to the different versions of the Banff classification. Overall, the number of ABMR and sABMR cases increased from Banff'01 to Banff'13. In Banff'17, 248 of 292 sABMR biopsies were reclassified to No ABMR, and 44 of 292 to ABMR. However, reclassified sABMR biopsies had worse and better outcome than No ABMR and ABMR, which was mainly driven by the presence of microvascular inflammation and absence of HLA-DSA, respectively. Consequently, the discriminative performance for allograft failure was lowest in Banff'17, and highest in Banff'13. Our data suggest that the clinical and histological heterogeneity of ABMR is inadequately represented in a binary classification system. This study provides a framework to evaluate the updates of the Banff classification and assess the impact of proposed changes on the number of cases and risk stratification. Two alternative classifications introducing an intermediate category are explored.

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

Eplet Mismatch Load and De Novo Occurrence of Donor-Specific Anti-HLA Antibodies, Rejection, and Graft Failure after Kidney Transplantation: An Observational Cohort Study.

BACKGROUND: In kidney transplantation, evaluating mismatches of HLA eplets-small patches of surface-exposed amino acids of the HLA molecule-instead of antigen mismatches might offer a better approach to assessing donor-recipient HLA incompatibility and improve risk assessment and prediction of transplant outcomes. METHODS: To evaluate the effect of number of eplet mismatches (mismatch load) on de novo formation of donor-specific HLA antibodies (DSAs) and transplant outcomes, we conducted a cohort study that included consecutive adult kidney recipients transplanted at a single center from March 2004 to February 2013. We performed retrospective high-resolution genotyping of HLA loci of 926 transplant pairs and used the HLAMatchmaker computer algorithm to count HLA eplet mismatches. RESULTS: De novo DSAs occurred in 43 (4.6%) patients. Multivariable analysis showed a significant independent association between antibody-verified eplet mismatch load and de novo DSA occurrence and graft failure, mainly explained by DQ antibody-verified eplet effects. The association with DQ antibody-verified eplet mismatches was linear, without a safe threshold at which de novo DSA did not occur. Odds for T cell- or antibody-mediated rejection increased by 5% and 12%, respectively, per antibody-verified DQ eplet mismatch. CONCLUSIONS: Eplet mismatches in HLA-DQ confer substantial risk for de novo DSA formation, graft rejection, and graft failure after kidney transplantation. Mismatches in other loci seem to have less effect. The results suggest that antibody-verified HLA-DQ eplet mismatch load could be used to guide personalized post-transplant immunosuppression. Adoption of molecular matching for DQA1 and DQB1 alleles could also help to minimize de novo DSA formation and potentially improve transplant outcomes.

Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.