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Background: Antibody-mediated rejection is a leading cause of kidney-transplant failure. The targeting of CD38 to inhibit graft injury caused by alloantibodies and natural killer (NK) cells may be a therapeutic option., Methods: In this phase 2, double-blind, randomized, placebo-controlled trial, we assigned patients with antibody-mediated rejection that had occurred at least 180 days after transplantation to receive nine infusions of the CD38 monoclonal antibody felzartamab (at a dose of 16 mg per kilogram of body weight) or placebo for 6 months, followed by a 6-month observation period. The primary outcome was the safety and side-effect profile of felzartamab. Key secondary outcomes were renal-biopsy results at 24 and 52 weeks, donor-specific antibody levels, peripheral NK-cell counts, and donor-derived cell-free DNA levels., Results: A total of 22 patients underwent randomization (11 to receive felzartamab and 11 to receive placebo). The median time from transplantation until trial inclusion was 9 years. Mild or moderate infusion reactions occurred in 8 patients in the felzartamab group. Serious adverse events occurred in 1 patient in the felzartamab group and in 4 patients in the placebo group; graft loss occurred in 1 patient in the placebo group. At week 24, resolution of morphologic antibody-mediated rejection was more frequent with felzartamab (in 9 of 11 patients [82%]) than with placebo (in 2 of 10 patients [20%]), for a difference of 62 percentage points (95% confidence interval [CI], 19 to 100) and a risk ratio of 0.23 (95% confidence interval [CI], 0.06 to 0.83). The median microvascular inflammation score was lower in the felzartamab group than in the placebo group (0 vs. 2.5), for a mean difference of -1.95 (95% CI, -2.97 to -0.92). Also lower was a molecular score reflecting the probability of antibody-mediated rejection (0.17 vs. 0.77) and the level of donor-derived cell-free DNA (0.31% vs. 0.82%). At week 52, the recurrence of antibody-mediated rejection was reported in 3 of 9 patients who had a response to felzartamab, with an increase in molecular activity and biomarker levels toward baseline levels., Conclusions: Felzartamab had acceptable safety and side-effect profiles in patients with antibody-mediated rejection. (Funded by MorphoSys and Human Immunology Biosciences; ClinicalTrials.gov number, NCT05021484; and EUDRACT number, 2021-000545-40.)., (Copyright © 2024 Massachusetts Medical Society.)

The kidney epithelium, with its intricate arrangement of highly specialized cell types, constitutes the functional core of the organ. Loss of kidney epithelium is linked to the loss of functional nephrons and a subsequent decline in kidney function. In kidney transplantation, epithelial injury signatures observed during post-transplantation surveillance are strong predictors of adverse kidney allograft outcomes. However, epithelial injury is currently neither monitored clinically nor addressed therapeutically after kidney transplantation. Several factors can contribute to allograft epithelial injury, including allograft rejection, drug toxicity, recurrent infections and postrenal obstruction. The injury mechanisms that underlie allograft injury overlap partially with those associated with acute kidney injury (AKI) and chronic kidney disease (CKD) in the native kidney. Studies using advanced transcriptomic analyses of single cells from kidney or urine have identified a role for kidney injury-induced epithelial cell states in exacerbating and sustaining damage in AKI and CKD. These epithelial cell states and their associated expression signatures are also observed in transplanted kidney allografts, suggesting that the identification and characterization of transcriptomic epithelial cell states in kidney allografts may have potential clinical implications for diagnosis and therapy., (© 2024. Springer Nature Limited.)

Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm 2 glomerular area (NK glom ) and PTC per mm 2 cortical area (NK PTC ) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NK glom Spearman's correlation coefficient [SCC] = 0.55, p < 0.001, NK PTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMR prob : NK glom 0.59, NK PTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NK PTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Diebold, Farkash, Barnes, Regele, Kozakowski, Schatzl, Mayer, Haindl, Vietzen, Hidalgo, Halloran, Eskandary and Böhmig.)

Antibody-mediated rejection (ABMR) is a leading cause of graft failure. Emerging evidence suggests a significant contribution of natural killer (NK) cells to microvascular inflammation (MVI). We investigated the influence of genetically determined NK cell functionality on ABMR development and activity. The study included 86 kidney transplant recipients subjected to systematic biopsies triggered by donor-specific antibody detection. We performed killer immunoglobulin-like receptor typing to predict missing self and genotyped polymorphisms determining NK cell functionality (FCGR3A V/F158 [rs396991], KLRC2 wt/del , KLRK1 HNK/LNK [rs1049174], rs9916629-C/T). Fifty patients had ABMR with considerable MVI and elevated NK cell transcripts. Missing self was not related to MVI. Only KLRC2 wt/wt showed an association (MVI score: 2 [median; interquartile range: 0-3] vs 0 [0-1] in KLRC2 wt/del recipients; P = .001) and remained significant in a proportional odds multivariable model (odds ratio, 7.84; 95% confidence interval, 2.37-30.47; P = .001). A sum score incorporating all polymorphisms and missing self did not outperform a score including only KLRC2 and FCGR3A variants, which were predictive in univariable analysis. NK cell genetics did not affect graft functional decline and survival. In conclusion, a functional KLRC2 polymorphism emerged as an independent determinant of ABMR activity, without a considerable contribution of missing self and other NK cell gene polymorphisms., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Among all biopsies in the Trifecta-Kidney Study ( ClinicalTrials.gov NCT04239703), elevated plasma donor-derived cell-free DNA (dd-cfDNA) correlated most strongly with molecular antibody-mediated rejection (AMR) but was also elevated in other states: T cell-mediated rejection (TCMR), acute kidney injury (AKI), and some apparently normal biopsies. The present study aimed to define the molecular correlates of plasma dd-cfDNA within specific states., Methods: Dd-cfDNA was measured by the Prospera test. Molecular rejection and injury states were defined using the Molecular Microscope system. We studied the correlation between dd-cfDNA and the expression of genes, transcript sets, and classifier scores within specific disease states, and compared AMR, TCMR, and AKI to biopsies classified as normal and no injury (NRNI)., Results: In all 604 biopsies, dd-cfDNA was elevated in AMR, TCMR, and AKI. Within AMR biopsies, dd-cfDNA correlated with AMR activity and stage. Within AKI, the correlations reflected acute parenchymal injury, including cell cycling. Within biopsies classified as MMDx Normal and archetypal No injury (NRNI), dd-cfDNA still correlated significantly with rejection- and injury-related genes. TCMR activity (eg, the TCMR Prob classifier) correlated with dd-cfDNA, but within TCMR biopsies, top gene correlations were complex and not the top TCMR-selective genes., Conclusions: In kidney transplants, elevated plasma dd-cfDNA is associated with 3 distinct molecular states in the donor tissue: AMR, recent parenchymal injury (including cell cycling), and TCMR, potentially complicated by parenchymal disruption. Moreover, subtle rejection- and injury-related changes in the donor tissue can contribute to dd-cfDNA elevations in transplants considered to have no rejection or injury., Competing Interests: P.F.H. has shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company with an interest in molecular diagnostics, and is a consultant to Natera Inc. All Natera Inc authors are employees and own equity at Natera Inc. The other authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The Banff system for histologic diagnosis of rejection in kidney transplant biopsies uses guidelines to assess designated features-lesions, donor-specific antibody (DSA), and C4d staining. We explored whether using regression equations to interpret the features as well as current guidelines could establish the relative importance of each feature and improve histologic interpretation., Methods: We developed logistic regression equations using the designated features to predict antibody-mediated rejection (AMR/mixed) and T-cell-mediated rejection (TCMR/mixed) in 1679 indication biopsies from the INTERCOMEX study ( ClinicalTrials.gov NCT01299168). Equations were trained on molecular diagnoses independent of the designated features., Results: In regression and random forests, the important features predicting molecular rejection were as follows: for AMR, ptc and g, followed by cg; for TCMR, t > i. V-lesions were relatively unimportant. C4d and DSA were also relatively unimportant for predicting AMR: by AUC, the model excluding them (0.853) was nearly as good as the model including them (0.860). Including time posttransplant slightly but significantly improved all models. By AUC, regression predicted molecular AMR and TCMR better than Banff histologic diagnoses. More importantly, in biopsies called "no rejection" by Banff guidelines, regression equations based on histology features identified histologic and molecular rejection-related changes in some biopsies and improved survival predictions. Thus, regression can screen for missed rejection., Conclusions: Using lesion-based regression equations in addition to Banff histology guidelines defines the relative important of histology features for identifying rejection, allows screening for potential missed diagnoses, and permits early estimates of AMR when C4d and DSA are not available., Competing Interests: P.F.H. holds shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between TSI and Thermo Fisher Scientific, and by a research grant from Natera Inc. P.F.H. is a consultant to Natera Inc. The other authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

This review outlines the molecular disease states in kidney transplant biopsies as documented in the development of the Molecular Microscope Diagnostic System (MMDx). These states include T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), recent parenchymal injury, and irreversible atrophy-fibrosis. The MMDx project, initiated through a Genome Canada grant, is a collaboration involving many centers. MMDx uses genome-wide microarrays to measure transcript expression, interprets the results using ensembles of machine learning algorithms, and generates a report. Experimental studies in mouse models and cell lines were extensively used to annotate molecular features and interpret the biopsy results. Over time, MMDx revealed unexpected aspects of the disease states: for example, AMR is usually C4d-negative and often DSA-negative, and subtle "Minor" AMR-like states are frequent. Parenchymal injury correlates with both reduced glomerular filtration rate and increased risk of graft loss. In kidneys with rejection, injury features, not rejection activity, are the strongest predictors of graft survival. Both TCMR and AMR produce injury, but TCMR induces immediate nephron injury and accelerates atrophy-fibrosis, whereas AMR induces microcirculation and glomerular damage that slowly leads to nephron failure and atrophy-fibrosis. Plasma donor-derived cell-free DNA levels correlate strongly with AMR activity, acute kidney injury, and in a complex way with TCMR activity. Thus, the MMDx project has documented the molecular processes that underlie the clinical and histologic states in kidney transplants, and provides a diagnostic tool that can be used to calibrate biomarkers, optimize histology interpretation, and guide clinical trials., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Current knowledge about the factors correlating with functional decline and subsequent failure of kidney allografts in antibody-mediated rejection (ABMR) is limited. We conducted a cohort study involving 75 renal allograft recipients diagnosed with late ABMR occurring at least 6 months after transplantation. The study aimed to examine the correlation of molecular and histologic features with estimated glomerular filtration rate (eGFR) trajectories and death-censored graft survival. We focused on sum scores reflecting histologic ABMR activity versus chronicity and molecular scores of ABMR probability (ABMR Prob ), injury-repair response (IRRAT) and fibrosis (ciprob). In multivariable Cox analysis, a Banff lesion-based chronicity index (ci+ct+cg[x2]; hazard ratio per interquartile range [IQR]: 1.97 [95% confidence interval: 0.97 to 3.99]) and IRRAT (1.93 [0.96 to 3.89]) showed the strongest associations with graft failure. Among biopsy variables, IRRAT exhibited the highest relative variable importance and emerged as the sole independent predictor of eGFR slope (change per IQR: -4.2 [-7.8 to -0.6] mL/min/1.73 m 2 /year). In contrast, morphologic chronicity associated with baseline eGFR only. We conclude that the extent of molecular injury is a robust predictor of renal function decline. Transcriptome analysis has the potential to improve outcome prediction and possibly identify modifiable injury, guiding targeted therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Herz, Diebold, Kainz, Mayer, Doberer, Kozakowski, Halloran and Böhmig.)

In lung transplantation, antibody-mediated rejection (AMR) diagnosed using the International Society for Heart and Lung Transplantation criteria is uncommon compared with other organs, and previous studies failed to find molecular AMR (ABMR) in lung biopsies. However, understanding of ABMR has changed with the recognition that ABMR in kidney transplants is often donor-specific antibody (DSA)-negative and associated with natural killer (NK) cell transcripts. We therefore searched for a similar molecular ABMR-like state in transbronchial biopsies using gene expression microarray results from the INTERLUNG study (#NCT02812290). After optimizing rejection-selective transcript sets in a training set (N = 488), the resulting algorithms separated an NK cell-enriched molecular rejection-like state (NKRL) from T cell-mediated rejection (TCMR)/Mixed in a test set (N = 488). Applying this approach to all 896 transbronchial biopsies distinguished 3 groups: no rejection, TCMR/Mixed, and NKRL. Like TCMR/Mixed, NKRL had increased expression of all-rejection transcripts, but NKRL had increased expression of NK cell transcripts, whereas TCMR/Mixed had increased effector T cell and activated macrophage transcripts. NKRL was usually DSA-negative and not recognized as AMR clinically. TCMR/Mixed was associated with chronic lung allograft dysfunction, reduced one-second forced expiratory volume at the time of biopsy, and short-term graft failure, but NKRL was not. Thus, some lung transplants manifest a molecular state similar to DSA-negative ABMR in kidney and heart transplants, but its clinical significance must be established., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P F Halloran reports financial support was provided by Thermo Fisher Scientific. P F Halloran reports financial support was provided by Natera Inc. P F Halloran reports a relationship with Natera, Inc. that includes: consulting or advisory. Disclosure P F Halloran holds shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between TSI and Thermo Fisher Scientific, and by a research grant from Natera Inc. P.F. Halloran is a consultant to Natera Inc. The other authors have nothing to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: We studied the variation in molecular T cell-mediated rejection (TCMR) activity in kidney transplant indication biopsies and its relationship with histologic lesions (particularly tubulitis and atrophy-fibrosis) and time posttransplant., Methods: We examined 175 kidney transplant biopsies with molecular TCMR as defined by archetypal analysis in the INTERCOMEX study ( ClinicalTrials.gov #NCT01299168). TCMR activity was defined by a molecular classifier., Results: Archetypal analysis identified 2 TCMR classes, TCMR1 and TCMR2: TCMR1 had higher TCMR activity and more antibody-mediated rejection ("mixed") activity and arteritis but little hyalinosis, whereas TCMR2 had less TCMR activity but more atrophy-fibrosis. TCMR1 and TCMR2 had similar levels of molecular injury and tubulitis. Both TCMR1 and TCMR2 biopsies were uncommon after 2 y posttransplant and were rare after 10 y, particularly TCMR1. Within late TCMR biopsies, TCMR classifier activity and activity molecules such as IFNG fell progressively with time, but tubulitis and molecular injury were sustained. Atrophy-fibrosis was increased in TCMR biopsies, even in the first year posttransplant, and rose with time posttransplant. TCMR1 and TCMR2 both reduced graft survival, but in random forests, the strongest determinant of survival after biopsies with TCMR was molecular injury, not TCMR activity., Conclusions: TCMR varies in intensity but is always strongly related to molecular injury and atrophy-fibrosis, which ultimately explains its effect on survival. We hypothesize, based on the reciprocal relationship with hyalinosis, that the TCMR1-TCMR2 gradient reflects calcineurin inhibitor drug underexposure, whereas the time-dependent decline in TCMR activity and frequency after the first year reflects T-cell exhaustion., Competing Interests: P.F.H. holds shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, and is supported in part by a licensing agreement between TSI and Thermo Fisher and by a research grant from Natera. P.F.H. is a consultant to Natera. The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Blockade of interleukin-6 (IL-6) has emerged as a promising therapeutic option for antibody-mediated rejection. Subtherapeutic anti-IL-6 antibody level or treatment cessation following prolonged cytokine neutralization may result in proinflammatory rebound phenomena via accumulation of IL-6 and/or modulated gene expression of major components of the IL-6/IL-6 receptor (IL-6R) axis., Methods: We evaluated biologic material obtained from a randomized controlled, double-blind phase 2 trial designed to evaluate the safety and efficacy of the anti-IL-6 monoclonal antibody clazakizumab in late antibody-mediated rejection. Twenty kidney transplant recipients, allocated to clazakizumab or placebo, received 4-weekly doses over 12 wks, followed by a 40-wk extension where all recipients received clazakizumab. Serum proteins were detected using bead-based immunoassays and RNA transcripts using quantitative real-time polymerase chain reaction (peripheral blood) or microarray analysis (serial allograft biopsies)., Results: Clazakizumab treatment resulted in a substantial increase in median total (bound and unbound to drug) serum IL-6 level (1.4, 8015, and 13 600 pg/mL at 0, 12, and 52 wks), but median level of free (unbound to drug) IL-6 did not increase (3.0, 2.3, and 2.3 pg/mL, respectively). Neutralization of IL-6 did not boost soluble IL-6R or leukocyte or allograft expression of IL-6, IL-6R, and glycoprotein 130 mRNA. Cessation of treatment at the end of the trial did not result in a meaningful increase in C-reactive protein or accelerated progression of graft dysfunction during 12 mo of follow-up., Conclusion: Our results argue against clinically relevant rebound phenomena and modulation of major components of the IL-6/IL-6R axis following prolonged IL-6 neutralization with clazakizumab., Competing Interests: G.A.B. is a member of the steering committee for an ongoing pivotal phase 3 trial evaluating clazakizumab in chronic active antibody-mediated rejection ( ClinicalTrials.gov number, NCT03744910; sponsored by CSL Behring). E.C. was formerly an employee of Vitaeris Inc, a manufacturer of clazakizumab. The other authors have no conflicts of interest to declare., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The Molecular Microscope Diagnostic System (MMDx) may overcome histology shortcomings. Previous studies have simply examined discrepant findings but have not attempted to determine clinical endpoints. To measure performance, clinical outcomes are strongly required., Methods: This single-center cohort study described discrepancies between MMDx and histology from 51 kidney transplant recipients (KTRs) and analyzed 72 indication biopsies, including 21 follow-up biopsies. Clinical performance was assessed by a combined endpoint of graft failure, rejection on follow-up biopsy, de novo donor-specific antibody, and improvement of kidney allograft function upon antirejection treatment., Results: MMDx agreed in 33 (65%) and differed in 18 (35%) of 51 KTRs. Most discrepancies occurred in biopsies called no rejection by MMDx and rejection by histology (15/24, 63%). In contrast, in biopsies called rejection by MMDx, 3 were classified as no rejection by histology (3/27, 11%). Discrepant findings between MMDx and histology occurred following delayed graft function and MMDx from biopsies with a low percentage of cortex. Among 15 biopsies classified as no rejection by MMDx but rejection by histology, the clinical course suggested no rejection in 9 cases. Six KTRs reached the endpoint, showing predominant t ≥ 2 lesions., Conclusions: The most often occurring discrepancy is rejection by histology but no rejection by MMDx. As more KTRs do not meet the combined endpoint for rejection, MMDx might be clinically useful in these discrepant cases. Although strong histological findings have priority in indicating the treatment, clinical implementation of MMDx could strengthen treatment strategies., Competing Interests: P.F.H. has shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company with an interest in molecular diagnostics. The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Donor-derived cell-free DNA (dd-cfDNA) fraction and quantity have both been shown to be associated with allograft rejection. The present study compared the relative predictive power of each of these variables to the combination of the two, and developed an algorithm incorporating both variables to detect active rejection in renal allograft biopsies., Methods: The first 426 sequential indication biopsy samples collected from the Trifecta study ( ClinicalTrials.gov # NCT04239703) with microarray-derived gene expression and dd-cfDNA results were included. After exclusions to simulate intended clinical use, 367 samples were analyzed. Biopsies were assessed using the molecular microscope diagnostic system and histology (Banff 2019). Logistic regression analysis examined whether combining dd-cfDNA fraction and quantity adds predictive value to either alone. The first 149 sequential samples were used to develop a two-threshold algorithm and the next 218 to validate the algorithm., Results: In regression, the combination of dd-cfDNA fraction and quantity was found to be significantly more predictive than either variable alone ( P = 0.009 and P < 0.0001). In the test set, the area under the receiver operating characteristic curve of the two-variable system was 0.88, and performance of the two-threshold algorithm showed a sensitivity of 83.1% and specificity of 81.0% for molecular diagnoses and a sensitivity of 73.5% and specificity of 80.8% for histology diagnoses., Conclusions: This prospective, biopsy-matched, multisite dd-cfDNA study in kidney transplant patients found that the combination of dd-cfDNA fraction and quantity was more powerful than either dd-cfDNA fraction or quantity alone and validated a novel two-threshold algorithm incorporating both variables., Competing Interests: P.F.H. reports having shares in Transcriptome Sciences Inc, a University of Alberta research company with an interest in molecular diagnostics, and is a consultant to Natera. Inc. K.S.M.-T. is an employee of Transcriptome Sciences Inc. A.P. reports current employment with, and an ownership interest in, Natera, Inc, N.K., E.A., C.C., N.A.H.B., Z.D., N.L., R.K.S., B.G.Z., P.V.H., M.R., H.T., and P.G. are all employees of Natera, Inc, with stocks or options to buy stocks in the company. The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The INTERHEART study (ClinicalTrials.gov #NCT02670408) used genome-wide microarrays to detect rejection in endomyocardial biopsies; however, many heart transplants with no rejection have late dysfunction and impaired survival. We used the microarray measurements to develop a molecular classification of parenchymal injury., Methods: In 1320 endomyocardial biopsies from 645 patients previously studied for rejection-associated transcripts, we measured the expression of 10 injury-induced transcript sets: 5 induced by recent injury; 2 reflecting macrophage infiltration; 2 normal heart transcript sets; and immunoglobulin transcripts, which correlate with time. We used archetypal clustering to assign injury groups., Results: Injury transcript sets correlated with impaired function. Archetypal clustering based on the expression of injury transcript sets assigned each biopsy to 1 of 5 injury groups: 87 Severe-injury, 221 Late-injury, and 3 with lesser degrees of injury, 376 No-injury, 526 Mild-injury, and 110 Moderate-injury. Severe-injury had extensive loss of normal transcripts (dedifferentiation) and increase in macrophage and injury-induced transcripts. Late-injury was characterized by high immunoglobulin transcript expression. In Severe- and Late-injury, function was depressed, and short-term graft failure was increased, even in hearts with no rejection. T cell-mediated rejection almost always had parenchymal injury, and 85% had Severe- or Late-injury. In contrast, early antibody-mediated rejection (AMR) had little injury, but late AMR often had the Late-injury state., Conclusions: Characterizing heart transplants for their injury state provides new understanding of dysfunction and outcomes and demonstrates the differential impact of T cell-mediated rejection versus AMR on the parenchyma. Slow deterioration from AMR emerges as a major contributor to late dysfunction., Competing Interests: P.F.H. holds shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, and is supported in part by a licensing agreement between TSI and Thermo Fisher and by a research grant from Natera. P.F.H. is a consultant for Natera. The other authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases., (© 2022 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Circulating donor-derived cell-free DNA (cfDNA), a minimally invasive diagnostic tool for kidney transplant rejection, was validated using traditional histology. The molecular microscope diagnostic system (MMDx) tissue gene expression platform may provide increased precision to traditional histology., Methods: In this single-center prospective study of 208 biopsies (median = 5.8 mo) posttransplant, we report on the calibration of cfDNA with simultaneous biopsy assessments using MMDx and histology by area under the curve (AUC) analyses for optimal criterion, as well as for, previously published cfDNA cutoffs ≤ 0.21% to "rule-out" rejection and ≥1% to "rule-in" rejection., Results: There were significant discrepancies between histology and MMDx, with MMDx identifying more antibody-mediated rejection (65; 31%) than histology (43; 21%); the opposite was true for T cell-mediated rejection [TCMR; histology: 27 (13%) versus MMDx: 13 (6%)]. Most of the TCMR discrepancies were seen for histologic borderline/1A TCMR. AUC for cfDNA and prediction of rejection were slightly better with MMDx (AUC = 0.80; 95% CI: 0.74-0.86) versus histology (AUC = 0.75; 95% CI: 0.69-0.81). A cfDNA ≤ 0.21% had similar sensitivity (~91%) to "rule-out" rejection by histology and MMDx. Specificity was slightly higher with MMDx (92%) compared with histology (85%) to "rule-in" rejection using cfDNA criterion ≥1%. Strong positive quantitative correlations were observed between cfDNA scores and molecular acute kidney injury for both "rejection" and "nonrejection" biopsies., Conclusions: Molecular diagnostics using tissue gene expression and blood-based donor-derived cell-free DNA may add precision to some cases of traditional histology. The positive correlation of cfDNA with molecular acute kidney injury suggests a dose-dependent association with tissue injury irrespective of rejection characteristics., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Antibody-mediated rejection (ABMR) is a cardinal cause of renal allograft loss. This rejection type, which may occur at any time after transplantation, commonly presents as a continuum of microvascular inflammation (MVI) culminating in chronic tissue injury. While the clinical relevance of ABMR is well recognized, its treatment, particularly a long time after transplantation, has remained a big challenge. A promising strategy to counteract ABMR may be the use of CD38-directed treatment to deplete alloantibody-producing plasma cells (PC) and natural killer (NK) cells., Methods: This investigator-initiated trial is planned as a randomized, placebo-controlled, double-blind, parallel-group, multi-center phase 2 trial designed to assess the safety and tolerability (primary endpoint), pharmacokinetics, immunogenicity, and efficacy of the fully human CD38 monoclonal antibody felzartamab (MOR202) in late ABMR. The trial will include 20 anti-HLA donor-specific antibody (DSA)-positive renal allograft recipients diagnosed with active or chronic active ABMR ≥ 180 days post-transplantation. Subjects will be randomized 1:1 to receive felzartamab (16 mg/kg per infusion) or placebo for a period of 6 months (intravenous administration on day 0, and after 1, 2, 3, 4, 8, 12, 16, and 20 weeks). Two follow-up allograft biopsies will be performed at weeks 24 and 52. Secondary endpoints (preliminary assessment) will include morphologic and molecular rejection activity in renal biopsies, immunologic biomarkers in the blood and urine, and surrogate parameters predicting the progression to allograft failure (slope of renal function; iBOX prediction score)., Discussion: Based on the hypothesis that felzartamab is able to halt the progression of ABMR via targeting antibody-producing PC and NK cells, we believe that our trial could potentially provide the first proof of concept of a new treatment in ABMR based on a prospective randomized clinical trial., Trial Registration: EU Clinical Trials Register (EudraCT) 2021-000545-40 . Registered on 23 June 2021., Clinicaltrials: gov NCT05021484 . Registered on 25 August 2021., (© 2022. The Author(s).)

Natural killer (NK) cells may contribute to antibody-mediated rejection (ABMR) of renal allografts. The role of distinct NK cell subsets in this specific context, such as NK cells expressing the activating receptor NKG2C, is unknown. Our aim was to investigate whether KLRC2 gene deletion variants which determine NKG2C expression affect the pathogenicity of donor-specific antibodies (DSA) and, if so, influence long-term graft survival. We genotyped the KLRC2 wt/del variants for two distinct kidney transplant cohorts, (i) a cross-sectional cohort of 86 recipients who, on the basis of a positive post-transplant DSA result, all underwent allograft biopsies, and (ii) 1,860 recipients of a deceased donor renal allograft randomly selected from the Collaborative Transplant Study (CTS) database. In the DSA+ patient cohort, KLRC2 wt/wt (80%) was associated with antibody-mediated rejection (ABMR; 65% versus 29% among KLRC2 wt/del subjects; P =0.012), microvascular inflammation [MVI; median g+ptc score: 2 (interquartile range: 0-4) versus 0 (0-1), P =0.002], a molecular classifier of ABMR [0.41 (0.14-0.72) versus 0.10 (0.07-0.27), P =0.001], and elevated NK cell-related transcripts ( P =0.017). In combined analyses of KLRC2 variants and a functional polymorphism in the Fc gamma receptor IIIA gene ( FCGR3A -V/F158), ABMR rates and activity gradually increased with the number of risk genotypes. In DSA+ and CTS cohorts, however, the KLRC2 wt/wt variant did not impact long-term death-censored graft survival, also when combined with the FCGR3A- V158 risk variant. KLRC2 wt/wt may be associated with DSA-triggered MVI and ABMR-associated gene expression patterns, but the findings observed in a highly selected cohort of DSA+ patients did not translate into meaningful graft survival differences in a large multicenter kidney transplant cohort not selected for HLA sensitization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vietzen, Döhler, Tran, Süsal, Halloran, Eskandary, Herz, Mayer, Kozakowski, Wahrmann, Ely, Haindl, Puchhammer-Stöckl and Böhmig.)

Background: The relationship between the donor-derived cell-free DNA fraction (dd-cfDNA[%]) in plasma in kidney transplant recipients at time of indication biopsy and gene expression in the biopsied allograft has not been defined., Methods: In the prospective, multicenter Trifecta study, we collected tissue from 300 biopsies from 289 kidney transplant recipients to compare genome-wide gene expression in biopsies with dd-cfDNA(%) in corresponding plasma samples drawn just before biopsy. Rejection was assessed with the microarray-based Molecular Microscope Diagnostic System using automatically assigned rejection archetypes and molecular report sign-outs, and histology assessments that followed Banff guidelines., Results: The median time of biopsy post-transplantation was 455 days (5 days to 32 years), with a case mix similar to that of previous studies: 180 (60%) no rejection, 89 (30%) antibody-mediated rejection (ABMR), and 31 (10%) T cell-mediated rejection (TCMR) and mixed. In genome-wide mRNA measurements, all 20 top probe sets correlating with dd-cfDNA(%) were previously annotated for association with ABMR and all types of rejection, either natural killer (NK) cell-expressed ( e.g ., GNLY , CCL4 , TRDC , and S1PR5 ) or IFN-γ-inducible ( e.g ., PLA1A , IDO1 , CXCL11 , and WARS ). Among gene set and classifier scores, dd-cfDNA(%) correlated very strongly with ABMR and all types of rejection, reasonably strongly with active TCMR, and weakly with inactive TCMR, kidney injury, and atrophy fibrosis. Active ABMR, mixed, and active TCMR had the highest dd-cfDNA(%), whereas dd-cfDNA(%) was lower in late-stage ABMR and less-active TCMR. By multivariate random forests and logistic regression, molecular rejection variables predicted dd-cfDNA(%) better than histologic variables., Conclusions: The dd-cfDNA(%) at time of indication biopsy strongly correlates with active molecular rejection and has the potential to reduce unnecessary biopsies., Clinical Trial Registration Number: NCT04239703., (Copyright © 2022 by the American Society of Nephrology.)

Allograft rejection is defined as tissue injury in a transplanted allogeneic organ produced by the effector mechanisms of the adaptive alloimmune response. Effector T lymphocytes and IgG alloantibodies cause two different types of rejection that can occur either individually or simultaneously: T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). In TCMR, cognate effector T cells infiltrate the graft and orchestrate an interstitial inflammatory response in the kidney interstitium in which effector T cells engage antigen-presenting myeloid cells, activating the T cells, antigen-presenting cells, and macrophages. The result is intense expression of IFNG and IFNG-induced molecules, expression of effector T cell molecules and macrophage molecules and checkpoints, and deterioration of parenchymal function. The diagnostic lesions of TCMR follow, i.e. interstitial inflammation, parenchymal deterioration, and intimal arteritis. In ABMR, HLA IgG alloantibodies produced by plasma cells bind to the donor antigens on graft microcirculation, leading to complement activation, margination, and activation of NK cells and neutrophils and monocytes, and endothelial injury, sometimes with intimal arteritis. TCMR becomes infrequent after 5-10 years post-transplant, probably reflecting adaptive mechanisms such as checkpoints, but ABMR can present even decades post-transplant. Some rejection is triggered by inadequate immunosuppression and non-adherence, challenging the clinician to target effective immunosuppression even decades post-transplant., (© 2021. Springer Nature Switzerland AG.)

Background: Donor -specific HLA antibody (DSA) is present in many kidney transplant patients whose biopsies are classified as no rejection (NR). We explored whether in some NR kidneys DSA has subtle effects not currently being recognized., Methods: We used microarrays to examine the relationship between standard-of-care DSA and rejection-related transcript increases in 1679 kidney transplant indication biopsies in the INTERCOMEX study (ClinicalTrials.gov NCT01299168), focusing on biopsies classified as NR by automatically assigned archetypal clustering. DSA testing results were available for 835 NR biopsies and were positive in 271 (32%)., Results: DSA positivity in NR biopsies was associated with mildly increased expression of antibody-mediated rejection (ABMR)-related transcripts, particularly IFNG-inducible and NK cell transcripts. We developed a machine learning DSA probability (DSA Prob ) classifier based on transcript expression in biopsies from DSA-positive versus DSA-negative patients, assigning scores using 10-fold cross-validation. This DSA Prob classifier was very similar to a previously described "ABMR probability" classifier trained on histologic ABMR in transcript associations and prediction of molecular or histologic ABMR. Plotting the biopsies using Uniform Manifold Approximation and Projection revealed a gradient of increasing molecular ABMR-like transcript expression in NR biopsies, associated with increased DSA ( P <2 × 10 -16 ). In biopsies with no molecular or histologic rejection, increased DSA Prob or ABMR probability scores were associated with increased risk of kidney failure over 3 years., Conclusions: Many biopsies currently considered to have no molecular or histologic rejection have mild increases in expression of ABMR-related transcripts, associated with increasing frequency of DSA. Thus, mild molecular ABMR-related pathology is more common than previously realized., (Copyright © 2021 by the American Society of Nephrology.)

Background: BK nephropathy (BKN) in kidney transplants diagnosed by histology is challenging because it involves damage from both virus activity and cognate T cell-mediated inflammation, directed against alloantigens (rejection) or viral antigens. The present study of indication biopsies from the Integrated Diagnostic System in the International Collaborative Microarray Study Extension study measured major capsid viral protein 2 (VP2) mRNA to assess virus activity and a T cell-mediated rejection (TCMR) classifier to assess cognate T cell-mediated inflammation., Methods: Biopsies were assessed by local standard-of-care histology and by genome-wide microarrays and Molecular Microscope Diagnostic System (MMDx) algorithms to detect rejection and injury. In a subset of 102 biopsies (50 BKN and 52 BKN-negative biopsies with various abnormalities), we measured VP2 transcripts by real-time polymerase chain reaction., Results: BKN was diagnosed in 55 of 1679 biopsies; 30 had cognate T cell-mediated activity assessed by by MMDx and TCMR lesions, but only 3 of 30 were histologically diagnosed as TCMR. We developed a BKN probability classifier that predicted histologic BKN (area under the curve = 0.82). Virus activity (VP2 expression) was highly selective for BKN (area under the curve = 0.94) and correlated with acute injury, atrophy-fibrosis, macrophage activation, and the BKN classifier, but not with the TCMR classifier. BKN with molecular TCMR had more tubulitis and inflammation than BKN without molecular TCMR. In 5 BKN cases with second biopsies, VP2 mRNA decreased in second biopsies, whereas in 4 of 5 TCMR classifiers, scores increased. Genes and pathways associated with BKN and VP2 mRNA were similar, reflecting injury, inflammation, and macrophage activation but none was selective for BKN., Conclusions: Risk-benefit decisions in BKN may be assisted by quantitative assessment of the 2 major pathologic processes, virus activity and cognate T cell-mediated inflammation., Competing Interests: P.F.H. held a Canada Research Chair in Transplant Immunology until 2008 and currently holds the Muttart Chair in Clinical Immunology. P.F.H. holds shares in Transcriptome Sciences Inc., a University of Alberta research company with an interest in molecular diagnostics, has given lectures for Thermo Fisher Scientific, and is a consultant for CSL Behring. The other authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

We previously characterized the molecular changes in acute kidney injury (AKI) and chronic kidney disease (CKD) in kidney transplant biopsies, but parenchymal changes selective for specific types of injury could be missed by such analyses. The present study searched for injury changes beyond AKI and CKD related to specific scenarios, including correlations with donor age. We defined injury using previously defined gene sets and classifiers and used principal component analysis to discover new injury dimensions. As expected, Dimension 1 distinguished normal vs. injury, and Dimension 2 separated early AKI from late CKD, correlating with time posttransplant. However, Dimension 3 was novel, distinguishing a set of genes related to epithelial polarity (e.g., PARD3) that were increased in early AKI and decreased in T cell-mediated rejection (TCMR) but not in antibody-mediated rejection. Dimension 3 was increased in kidneys from older donors and was particularly important in survival of early kidneys. Thus high Dimension 3 scores emerge as a previously unknown element in the kidney response-to-injury that affects epithelial polarity genes and is increased in AKI but depressed in TCMR, indicating that in addition to general injury elements, certain injury elements are selective for specific pathologic mechanisms. (ClinicalTrials.gov NCT01299168)., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The tubulitis with/without interstitial inflammation not meeting criteria for T-cell-mediated rejection (minimal allograft injury) is the most frequent histological findings in early transplant biopsies. The course of transcriptional changes in sequential kidney graft biopsies has not been studied yet. Molecular phenotypes were analyzed using the Molecular Microscope ® Diagnostic System (MMDx) in 46 indication biopsies (median 13 postoperative days) diagnosed as minimal allograft injury and in corresponding follow-up biopsies at 3 months. All 46 patients with minimal injury in early biopsy received steroid pulses. MMDx interpreted indication biopsies as no-rejection in 34/46 (74%), T-cell-mediated rejection (TCMR) in 4/46 (9%), antibody-mediated rejection in 6/46 (13%), and mixed rejection in 2/46 (4%) cases. Follow-up biopsies were interpreted by MMDx in 37/46 (80%) cases as no-rejection, in 4/46 (9%) as TCMR, and in 5/46 (11%) as mixed rejection. Follow-up biopsies showed a decrease in MMDx-assessed acute kidney injury (P = 0.001) and an increase of atrophy-fibrosis (P = 0.002). The most significant predictor of MMDx rejection scores in follow-up biopsies was the tubulitis classifier score in initial biopsies (AUC = 0.84, P = 0.002), confirmed in multivariate binary regression (OR = 16, P = 0.016). Molecular tubulitis score at initial biopsy has the potential to discriminate patients at risk for molecular rejection score at follow-up biopsy., (© 2021 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Background: Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. Blocking IL-6 has been proposed as a promising therapeutic strategy., Methods: We performed a phase 2 randomized pilot trial to evaluate the safety (primary endpoint) and efficacy (secondary endpoint analysis) of the anti-IL-6 antibody clazakizumab in late ABMR. The trial included 20 kidney transplant recipients with donor-specific, antibody-positive ABMR ≥365 days post-transplantation. Patients were randomized 1:1 to receive 25 mg clazakizumab or placebo (4-weekly subcutaneous injections) for 12 weeks (part A), followed by a 40-week open-label extension (part B), during which time all participants received clazakizumab., Results: Five (25%) patients under active treatment developed serious infectious events, and two (10%) developed diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab displayed significantly decreased donor-specific antibodies and, on prolonged treatment, modulated rejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a negative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), and resolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the mean eGFR decline during part A was slower with clazakizumab compared with placebo (-0.96; 95% confidence interval [95% CI], -1.96 to 0.03 versus -2.43; 95% CI, -3.40 to -1.46 ml/min per 1.73 m 2 per month, respectively, P =0.04). During part B, the slope of eGFR decline for patients who were switched from placebo to clazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab., Conclusions: Although safety data indicate the need for careful patient selection and monitoring, our preliminary efficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression., (Copyright © 2021 by the American Society of Nephrology.)

Purpose of Review: Chronic antibody-mediated rejection (AMR) is a cardinal cause of transplant failure, with currently no proven effective prevention or treatment. The present review will focus on new therapeutic concepts currently under clinical evaluation., Recent Findings: One interesting treatment approach may be interference with interleukin-6 (IL-6) signaling to modulate B-cell immunity and donor-specific antibody (DSA) production. Currently, a large phase III randomized controlled trial is underway to clarify the safety and efficacy of clazakizumab, a high-affinity anti-IL-6 antibody, in chronic AMR. A prevention/treatment strategy may be costimulation blockade using belatacept to interfere with germinal center responses and DSA formation. In a recent uncontrolled study, belatacept conversion was shown to stabilize renal function and dampen AMR activity. Moreover, preliminary clinical results suggest efficacy of CD38 antibodies to deplete plasma and natural killer cells to treat AMR, with anecdotal reports demonstrating at least transient resolution of active rejection., Summary: There are promising concepts on the horizon for the prevention and treatment of chronic AMR. The design of adequately powered placebo-controlled trials to clarify the safety and efficacy of such new therapies, however, remains a big challenge, and will rely on the definition of precise surrogate endpoints predicting long-term allograft survival. Mapping the natural history of AMR would greatly help the understanding of who would derive benefits from treatment., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

The Banff 2019 kidney allograft pathology update excluded isolated tubulitis without interstitial inflammation (ISO-T) from the category of borderline (suspicious) for acute T cell-mediated rejection due to its proposed benign clinical outcome. In this study, we explored the molecular assessment of ISO-T. ISO-T or interstitial inflammation with tubulitis (I + T) was diagnosed in indication biopsies within the first 14 postoperative days. The molecular phenotype of ISO-T was compared to I + T either by using RNA sequencing (n = 16) or by Molecular Microscope Diagnostic System (MMDx, n = 51). RNA sequencing showed lower expression of genes related to interferon-y (p = 1.5 *10 -16 ), cytokine signaling (p = 2.1 *10 -20 ) and inflammatory response (p = 1.0*10 -13 ) in the ISO-T group than in I + T group. Transcripts with increased expression in the I + T group overlapped significantly with previously described pathogenesis-based transcript sets associated with cytotoxic and effector T cell transcripts, and with T cell-mediated rejection (TCMR). MMDx classified 25/32 (78%) ISO-T biopsies and 12/19 (63%) I + T biopsies as no-rejection. ISO-T had significantly lower MMDx scores for interstitial inflammation (p = 0.014), tubulitis (p = 0.035) and TCMR (p = 0.016) compared to I + T. Fewer molecular signals of inflammation in isolated tubulitis suggest that this is also a benign phenotype on a molecular level.

Prior studies on belatacept conversion from calcineurin inhibitor (CNI) have been limited by an absence of postconversion surveillance biopsies that could underestimate subclinical rejection, or a case-controlled design. A total of 53 adult patients with allograft dysfunction underwent belatacept conversion (median: 6 months) post-transplant. At a median follow-up = 2.5 years, patient survival was 94% with a death-censored graft survival of 85%. Seven (13%) patients had acute rejection (including 3 subclinical) at median 6 months postconversion. Overall, eGFR improved (P = <0.001) from baseline = 31±15 to 40.2 ± 17.6 ml/min/1.73m 2 by 6 months postconversion, but then stayed stable. This improvement was also observed (P < 0.001) in comparison with a propensity matched control cohort on CNI, where eGFR stayed stable (mean ~ 32ml/min/1.72m 2 ) over 2-year follow-up. Patients converted < 6 months post-transplant were more likely to have a long-term improvement in kidney function. Paired gene expression analysis of 30 (of 53) consecutive pre- and postconversion surveillance biopsies did not reveal changes in inflammation/acute injury; although atrophy-fibrosis score worsened (mean = 0.28 to 0.44; P = 0.005). Thus, improvement in renal function with belatacept conversion occurred early and then sustained in comparison with controls where renal function remained unchanged overtime. We were unable to show molecular signals that could be related to CNI administration and regressed after withdrawal., (© 2020 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Molecular diagnosis of rejection is emerging in kidney, heart, and lung transplant biopsies and could offer insights for liver transplant biopsies. We measured gene expression by microarrays in 235 liver transplant biopsies from 10 centers. Unsupervised archetypal analysis based on expression of previously annotated rejection-related transcripts identified 4 groups: normal "R1 normal " (N = 129), T cell-mediated rejection (TCMR) "R2 TCMR " (N = 37), early injury "R3 injury " (N = 61), and fibrosis "R4 late " (N = 8). Groups differed in median time posttransplant, for example, R3 injury 99 days vs R4 late 3117 days. R2 TCMR biopsies expressed typical TCMR-related transcripts, for example, intense IFNG-induced effects. R3 injury displayed increased expression of parenchymal injury transcripts (eg, hypoxia-inducible factor EGLN1). R4 late biopsies showed immunoglobulin transcripts and injury-related transcripts. R2 TCMR correlated with histologic rejection although with many discrepancies, and R4 late with fibrosis. R2 TCMR , R3 injury , and R4 late correlated with liver function abnormalities. Supervised classifiers trained on histologic rejection showed less agreement with histology than unsupervised R2 TCMR scores. No confirmed cases of clinical antibody-mediated rejection (ABMR) were present in the population, and strategies that previously revealed ABMR in kidney and heart transplants failed to reveal a liver ABMR phenotype. In conclusion, molecular analysis of liver transplant biopsies detects rejection, has the potential to resolve ambiguities, and could assist with immunosuppressive management., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Discrepancy analysis comparing two diagnostic platforms offers potential insights into both without assuming either is always correct. Having optimized the Molecular Microscope Diagnostic System (MMDx) in renal transplant biopsies, we studied discrepancies within MMDx (reports and sign-out comments) and between MMDx and histology. Interpathologist discrepancies have been documented previously and were not assessed. Discrepancy cases were classified as "clear" (eg, antibody-mediated rejection [ABMR] vs T cell-mediated rejection [TCMR]), "boundary" (eg, ABMR vs possible ABMR), or "mixed" (eg, Mixed vs ABMR). MMDx report scores showed 99% correlations; sign-out interpretations showed 7% variation between observers, all located around boundaries. Histology disagreed with MMDx in 37% of biopsies, including 315 clear discrepancies, all with implications for therapy. Discrepancies were distributed widely in all histology diagnoses but increased in some scenarios; for example, histology TCMR contained 14% MMDx ABMR and 20% MMDx no rejection. MMDx usually gave unambiguous diagnoses in cases with ambiguous histology, for example, borderline and transplant glomerulopathy. Histology lesions or features associated with more frequent discrepancies (eg, tubulitis, arteritis, and polyomavirus nephropathy) were not associated with increased MMDx uncertainty, indicating that MMDx can clarify biopsies with histologic ambiguity. The patterns of histology-MMDx discrepancies highlight specific histology diagnoses in which MMDx assessment should be considered for guiding therapy., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

We previously reported a system for assessing rejection in kidney transplant biopsies using microarray-based gene expression data, the Molecular Microscope ® Diagnostic System (MMDx). The present study was designed to optimize the accuracy and stability of MMDx diagnoses by replacing single machine learning classifiers with ensembles of diverse classifier methods. We also examined the use of automated report sign-outs and the agreement between multiple human interpreters of the molecular results. Ensembles generated diagnoses that were both more accurate than the best individual classifiers, and nearly as stable as the best, consistent with expectations from the machine learning literature. Human experts had ≈93% agreement (balanced accuracy) signing out the reports, and random forest-based automated sign-outs showed similar levels of agreement with the human experts (92% and 94% for predicting the expert MMDx sign-outs for T cell-mediated (TCMR) and antibody-mediated rejection (ABMR), respectively). In most cases disagreements, whether between experts or between experts and automated sign-outs, were in biopsies near diagnostic thresholds. Considerable disagreement with histology persisted. The balanced accuracies of MMDx sign-outs for histology diagnoses of TCMR and ABMR were 73% and 78%, respectively. Disagreement with histology is largely due to the known noise in histology assessments (ClinicalTrials.gov NCT01299168)., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Late antibody-mediated rejection (ABMR) is a cardinal cause of kidney allograft failure, manifesting as a continuous and, in contrast with early rejection, often clinically silent alloimmune process. While significant progress has been made towards an improved understanding of its molecular mechanisms and the definition of diagnostic criteria, there is still no approved effective treatment. In recent small randomized controlled trials, therapeutic strategies with promising results in observational studies, such as proteasome inhibitor bortezomib, anti-C5 antibody eculizumab, or high dose intravenous immunoglobulin plus rituximab, had no significant impact in late and/or chronic ABMR. Such disappointing results reinforce a need of new innovative treatment strategies. Potential candidates may be the interference with interleukin-6 to modulate B cell alloimmunity, or innovative compounds that specifically target antibody-producing plasma cells, such as antibodies against CD38. Given the phenotypic heterogeneity of ABMR, the design of adequate systematic trials to assess the safety and efficiency of such therapies, however, is challenging. Several trials are currently being conducted, and new developments will hopefully provide us with effective ways to counteract the deleterious impact of antibody-mediated graft injury. Meanwhile, the weight of evidence would suggest that, when approaching using existing treatments for established antibody-mediated rejection, "less may be more"., (© 2019 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

Anti-angiotensin II type 1 receptor (AT1R) antibodies have been associated with allograft rejection. We hypothesized that circulating AT1R antibodies might identify kidney transplant recipients at increased risk of allograft rejection and loss who are not identified by the HLA system. We prospectively enrolled 1845 kidney transplant recipients from two centers. Donor-specific HLA antibodies (DSAs) and AT1R antibodies were measured at the time of the first acute rejection episode or at 1 year post-transplant. Allograft biopsy was performed to evaluate the rejection phenotype and to assess for endothelial activation. Overall, 371 (20.1%) participants had AT1R antibodies, 334 (18.1%) had DSAs, and 133 (7.2%) had both. AT1R antibodies were associated with an increased risk of allograft loss (adjusted HR 1.49, 95% CI 1.07-2.06 for AT1R antibodies alone and 2.26, 95% CI 1.52-3.36 for AT1R antibodies and DSAs). Participants with AT1R antibodies had a higher incidence of antibody-mediated rejection (AMR) compared with participants without AT1R antibodies (25.0% vs. 12.9%). Among 77 participants with histological features of AMR but without DSAs, 51 (66.2%) had AT1R antibodies. Compared to participants with prototypical DSA-mediated rejection, those with AT1R antibody-associated rejection had a higher prevalence of hypertension, more vascular rejection with arterial inflammation, higher levels of endothelial-associated transcripts, and lack of complement deposition in allograft capillaries. Thus, AT1R antibodies may identify kidney transplant recipients at high risk of allograft rejection and loss, independent of the HLA system. Recognition of complement-independent AT1R antibody-mediated vascular rejection could lead to the development of new treatment strategies to improve allograft survival., (Copyright © 2019. Published by Elsevier Inc.)

Background: Improved understanding of lung transplant disease states is essential because failure rates are high, often due to chronic lung allograft dysfunction. However, histologic assessment of lung transplant transbronchial biopsies (TBBs) is difficult and often uninterpretable even with 10 pieces., Methods: We prospectively studied whether microarray assessment of single TBB pieces could identify disease states and reduce the amount of tissue required for diagnosis. By following strategies successful for heart transplants, we used expression of rejection-associated transcripts (annotated in kidney transplant biopsies) in unsupervised machine learning to identify disease states., Results: All 242 single-piece TBBs produced reliable transcript measurements. Paired TBB pieces available from 12 patients showed significant similarity but also showed some sampling variance. Alveolar content, as estimated by surfactant transcript expression, was a source of sampling variance. To offset sampling variation, for analysis, we selected 152 single-piece TBBs with high surfactant transcripts. Unsupervised archetypal analysis identified 4 idealized phenotypes (archetypes) and scored biopsies for their similarity to each: normal; T-cell‒mediated rejection (TCMR; T-cell transcripts); antibody-mediated rejection (ABMR)-like (endothelial transcripts); and injury (macrophage transcripts). Molecular TCMR correlated with histologic TCMR. The relationship of molecular scores to histologic ABMR could not be assessed because of the paucity of ABMR in this population., Conclusions: Molecular assessment of single-piece TBBs can be used to classify lung transplant biopsies and correlated with rejection histology. Two or 3 pieces for each TBB will probably be needed to offset sampling variance., (Copyright © 2019 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

In kidney transplant biopsies, inflammation in areas of atrophy-fibrosis (i-IFTA) is associated with increased risk of failure, presumably because inflammation is evoked by recent parenchymal injury from rejection or other insults, but some cases also have rejection. The present study explored the frequency of rejection in i-IFTA, by using histology Banff 2015 and a microarray-based molecular diagnostic system (MMDx). In unselected indication biopsies (108 i-IFTA, 73 uninflamed IFTA [i0-IFTA], and 53 no IFTA), i-IFTA biopsies occurred later, showed more scarring, and had more antibody-mediated rejection (ABMR) based on histology (28%) and MMDx (45%). T cell-mediated rejection (TCMR) was infrequent in i-IFTA based on histology (8%) and MMDx (16%). Twelve i-IFTA biopsies (11%) had molecular TCMR not diagnosed by histology, although 6 were called borderline and almost all had histologic TCMR lesions. The prominent feature of i-IFTA biopsies was molecular injury (eg, acute kidney injury [AKI] transcripts). In multivariate analysis of biopsies >1 year posttransplant, the strongest associations with graft loss were AKI transcripts and histologic atrophy-scarring; i-IFTA was not significant when molecular AKI was included. We conclude that i-IFTA in indication biopsies reflects recent/ongoing parenchymal injury, often with concomitant ABMR but few with TCMR. Thus, the application of Banff i-IFTA in the population of late biopsies needs to be reconsidered., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Late antibody-mediated rejection (ABMR) triggered by donor-specific antibodies (DSA) is a cardinal cause of kidney allograft dysfunction and loss. Diagnostic criteria for this rejection type are well established, but effective treatment remains a major challenge. Recent randomized controlled trials (RCT) have failed to demonstrate the efficacy of widely used therapies, such as rituximab plus intravenous immunoglobulin or proteasome inhibition (bortezomib), reinforcing a great need for new therapeutic concepts. One promising target in this context may be interleukin-6 (IL-6), a pleiotropic cytokine known to play an important role in inflammation and adaptive immunity., Methods: This investigator-driven RCT was designed to assess the safety and efficacy of clazakizumab, a genetically engineered humanized monoclonal antibody directed against IL-6. The study will include 20 DSA-positive kidney allograft recipients diagnosed with ABMR ≥ 365 days after transplantation. Participants will be recruited at two study sites in Austria and Germany (Medical University of Vienna; Charité University Medicine Berlin). First, patients will enter a three-month double-blind RCT (1,1 randomization, stratification according to ABMR phenotype and study site) and will receive either clazakizumab (subcutaneous administration of 25 mg in monthly intervals) or placebo. In a second open-label part of the trial (months 4-12), all patients will receive clazakizumab at 25 mg every month. The primary endpoint is safety and tolerability. Secondary endpoints are the pharmacokinetics and pharmacodynamics of clazakizumab, its effect on drug metabolism in the liver, DSA characteristics, morphological ABMR lesions and molecular gene expression patterns in three- and 12-month protocol biopsies, serum/urinary biomarkers of inflammation and endothelial activation/injury, Torque Teno viral load as a measure of overall immunosuppression, kidney function, urinary protein excretion, as well as transplant and patient survival., Discussion: Currently, there is no treatment proven to be effective in halting the progression of late ABMR. Based on the hypothesis that antagonizing the effects of IL-6 improves the outcome of DSA-positive late ABMR by counteracting DSA-triggered inflammation and B cell/plasma cell-driven alloimmunity, we suggest that our trial has the potential to provide proof of concept of a novel treatment of this type of rejection., Trial Registration: ClinicalTrials.gov, NCT03444103 . Registered on 23 February 2018 (retrospective registration).

Fc-dependent effector mechanisms may contribute to antibody-mediated rejection (ABMR), and distinct gene polymorphisms modifying the function of Fc gamma receptors (FcγRs) may influence the capability of donor-specific antibodies (DSAs) to trigger inflammation. To evaluate the relevance of functional FcγR variants in late ABMR, 85 DSA-positive kidney allograft recipients, who were recruited upon antibody screening of 741 prevalent patients, were genotyped for polymorphisms in FcγRIIA (FCGR2A-H/R 131 ; rs1801274), FcγRIIIA (FCGR3A-V/F 158 ; rs396991), and FcγRIIIB (FCGR3B-neutrophil antigen 1 ([NA1]/NA2; rs35139848). Individuals with high-affinity FCGR3A-V 158 alleles (V/V 158 or V/F 158 ) showed a higher rate (and extent) of peritubular capillaritis (ptc) in protocol biopsies than homozygous carriers of the lower-affinity allele (ptc score ≥1: 53.6% vs 25.9%; P = .018). Associations were independent of C1q-binding to DSA or capillary C4d. In parallel, there was a trend toward increased macrophage- and injury-repair response-associated transcript subsets. Kidney function over 24 months, however, was not different. In support of a functional role of FcγRIIIA polymorphism, NK92 cells expressing FCGR3A-V 158 produced >2 times as much interferon gamma upon incubation with HLA antibody-coated cells as those expressing FCGR3A-F 158 . FcγRIIA and FcγRIIIB polymorphisms were not associated with allograft morphology. Our data suggest that the presence of high-affinity FcγRIIIA variants may favor DSA-triggered microcirculation inflammation., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Calcineurin inhibitor immunosuppressive drugs induce changes such as arteriolar hyalinosis (ah) in kidney transplants, raising the possibility that molecular changes in biopsies related to histologic ah can provide information about drug exposure. We hypothesized that molecular changes associated with less-than-expected hyalinosis might highlight a subpopulation of patients with under-immunosuppression/nonadherence at intermediate times of biopsy posttransplant (TxBx). Using gene expression data from 562 indication biopsies, we developed a molecular classifier for predicting the expected ah lesions (M ah ) at a particular TxBx. M ah -scores increased linearly with log(TxBx), but some biopsies had lower scores than expected for TxBx. The deviation of individual M ah -scores below the predicted regression line of M ah -scores vs TxBx is defined as "low hyalinosis index." Low hyalinosis indices were frequent in biopsies between 3 months and 3 years posttransplant, particularly among biopsies lacking histologic hyalinosis (ah0), and were associated with T cell-mediated rejection and a subset of recent-onset antibody-mediated rejection without glomerular double contours. In patients with medical records available for review, low hyalinosis indices were frequently associated with physician-recorded concerns about nonadherence (suspected or proven). We conclude that the M ah classifier and hyalinosis index identify indication biopsies with rejection for which the possibility of patient nonadherence should be considered., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection [BORTEJECT] Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m 2 intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib ( n =21) or placebo ( n =23). The 0.5-ml/min per 1.73 m 2 per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant ( P =0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m 2 ; P =0.31), 2-year graft survival (81% versus 96%; P =0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR., (Copyright © 2018 by the American Society of Nephrology.)

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i-IFTA) and its relationship to T cell-mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody-mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i-IFTA is associated with reduced graft survival. Furthermore, these groups presented that i-IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i-IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor-specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next-generation clinical trials., (© 2017 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Human organ allograft rejection depends on effector lymphocytes: NK cells in antibody-mediated rejection (ABMR) and effector T cells in T cell-mediated rejection (TCMR). We hypothesized that NK cell CD16a stimulation and CD8 T cell TCR/CD3 stimulation represent highly similar effector systems, and should lead to shared molecular changes between ABMR and TCMR. We studied similarity between soluble proteins and the transcripts induced in CD16a stimulated NK cells and TCR/CD3-stimulated T cells in vitro. Of 30 soluble mediators tested, CD16a-activated NK cells and CD3/TCR activated T cells produced the same limited set of five mediators-CCL3, CCL4, CSF2, IFNG, and TNF-and failed to produce 25 others. Many transcripts increased in stimulated NK cells were also increased in CD3-stimulated CD8 T cells (FDR < 0.05), including IFNG, CSF2, CCL3, CCL4, and XCL1. We hypothesized that shared transcripts not produced by other cell types should be expressed both in ABMR and TCMR kidney transplant biopsies. CD160, XCL1, TNFRSF9, and IFNG were selective for TCR/CD3-activated T cells and CD16a-NK cells and all were strongly increased in ABMR and TCMR. The molecules such as CD160 and XCL1 shared between NK cells in ABMR and effector T cells in TCMR may hold insights into important rejection mechanisms., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The authors conducted a prospective trial to assess the feasibility of real time central molecular assessment of kidney transplant biopsy samples from 10 North American or European centers. Biopsy samples taken 1 day to 34 years posttransplantation were stabilized in RNAlater, sent via courier overnight at ambient temperature to the central laboratory, and processed (29 h workflow) using microarrays to assess T cell- and antibody-mediated rejection (TCMR and ABMR, respectively). Of 538 biopsy samples submitted, 519 (96%) were sufficient for microarray analysis (average length, 3 mm). Automated reports were generated without knowledge of histology and HLA antibody, with diagnoses assigned based on Molecular Microscope Diagnostic System (MMDx) classifier algorithms and signed out by one observer. Agreement between MMDx and histology (balanced accuracy) was 77% for TCMR, 77% for ABMR, and 76% for no rejection. A classification tree derived to provide automated sign-outs predicted the observer sign-outs with >90% accuracy. In 451 biopsy samples where feedback was obtained, clinicians indicated that MMDx more frequently agreed with clinical judgment (87%) than did histology (80%) (p = 0.0042). In 81% of feedback forms, clinicians reported that MMDx increased confidence in management compared with conventional assessment alone. The authors conclude that real time central molecular assessment is feasible and offers a useful new dimension in biopsy interpretation. ClinicalTrials.gov NCT#01299168., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Histologic assessment of kidney transplant biopsies relies on cortex rather than medulla, but for microarray studies, the proportion cortex in a biopsy is typically unknown and could affect the molecular readings. The present study aimed to develop a molecular estimate of proportion cortex in biopsies and examine its effect on molecular diagnoses. Microarrays from 26 kidney transplant biopsies divided into cortex and medulla components and processed separately showed that many of the most significant differences were in glomerular genes (e.g. NPHS2, NPHS1, CLIC5, PTPRO, PLA2R1, PLCE1, PODXL, and REN). Using NPHS2 (podocin) to estimate proportion cortex, we examined whether proportion cortex influenced molecular assessment in the molecular microscope diagnostic system. In 1190 unselected kidney transplant indication biopsies (Clinicaltrials.govNCT01299168), only 11% had <50% cortex. Molecular scores for antibody-mediated rejection, T cell-mediated rejection, and injury were independent of proportion cortex. Rejection was diagnosed in many biopsies that were mostly or all medulla. Agreement in molecular diagnoses in paired cortex/medulla samples (23/26) was similar to biological replicates (32/37). We conclude that NPHS2 expression can estimate proportion cortex; that proportion cortex has little influence on molecular diagnosis of rejection; and that, although histology cannot assess medulla, rejection does occur in medulla as well as cortex., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

We annotated the top transcripts associated with kidney transplant rejection by p-value, either universal for all rejection or selective for T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR; ClinicalTrials.gov NCT01299168). We used eight class-comparison algorithms to interrogate microarray results from 703 biopsies, 205 with rejection. The positive comparators were all rejection, TCMR, or ABMR; the negative comparators varied from normal biopsies to all nonrejecting biopsies, including other diseases. The universal algorithm, rejection versus all nonrejection, identified transcripts mainly inducible by interferon γ. Selectivity for ABMR or TCMR required the other rejection class as well as nonrejection biopsies in the comparator to avoid selecting universal transcripts. Direct comparison of ABMR versus TCMR yielded only transcripts related to TCMR, the stronger signal. Transcripts highly associated with rejection were never completely specific for rejection: Many were increased in biopsies without rejection, reflecting sharing between rejection and injury-induced innate immunity. Union of the top 200 transcripts from universal and selective algorithms yielded 454 transcripts that permitted unsupervised analysis of biopsies in principal component analysis: PC1 was rejection, and PC2 was separation of TCMR from ABMR. Appreciating rejection-associated molecular changes requires a diverse case mix, accurate histologic classification (including C4d-negative ABMR), and both selective and universal algorithms., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Antibody-mediated rejection (ABMR) can occur in patients with preexisting anti-HLA donor-specific antibodies (DSA) or in patients who develop de novo DSA. However, how these processes compare in terms of allograft injury and outcome has not been addressed. From a cohort of 771 kidney biopsy specimens from two North American and five European centers, we performed a systematic assessment of clinical and biologic parameters, histopathology, circulating DSA, and allograft gene expression for all patients with ABMR ( n =205). Overall, 103 (50%) patients had preexisting DSA and 102 (50%) had de novo DSA. Compared with patients with preexisting DSA ABMR, patients with de novo DSA ABMR displayed increased proteinuria, more transplant glomerulopathy lesions, and lower glomerulitis, but similar levels of peritubular capillaritis and C4d deposition. De novo DSA ABMR was characterized by increased expression of IFN γ -inducible, natural killer cell, and T cell transcripts, but less expression of AKI transcripts compared with preexisting DSA ABMR. The preexisting DSA ABMR had superior graft survival compared with the de novo DSA ABMR (63% versus 34% at 8 years after rejection, respectively; P <0.001). After adjusting for clinical, histologic, and immunologic characteristics and treatment, we identified de novo DSA ABMR (hazard ratio [HR], 1.82 compared with preexisting DSA ABMR; 95% confidence interval [95% CI], 1.07 to 3.08; P =0.03); low eGFR (<30 ml/min per 1.73 m 2 ) at diagnosis (HR, 3.27; 95% CI, 1.48 to 7.23; P <0.001); ≥0.30 g/g urine protein-to-creatinine ratio (HR, 2.44; 95% CI, 1.47 to 4.09; P <0.001); and presence of cg lesions (HR, 2.25; 95% CI, 1.34 to 3.79; P =0.002) as the main independent determinants of allograft loss. Our findings support the transplant of kidneys into highly sensitized patients and should encourage efforts to monitor patients for de novo DSA., (Copyright © 2017 by the American Society of Nephrology.)