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Background: Immunosuppressive regimens with the fewest possible toxic effects are desirable for transplant recipients. This study evaluated the efficacy and relative toxic effects of four immunosuppressive regimens.Methods: We randomly assigned 1645 renal-transplant recipients to receive standard-dose cyclosporine, mycophenolate mofetil, and corticosteroids, or daclizumab induction, mycophenolate mofetil, and corticosteroids in combination with low-dose cyclosporine, low-dose tacrolimus, or low-dose sirolimus. The primary end point was the estimated glomerular filtration rate (GFR), as calculated by the Cockcroft-Gault formula, 12 months after transplantation. Secondary end points included acute rejection and allograft survival.Results: The mean calculated GFR was higher in patients receiving low-dose tacrolimus (65.4 ml per minute) than in the other three groups (range, 56.7 to 59.4 ml per minute). The rate of biopsy-proven acute rejection was lower in patients receiving low-dose tacrolimus (12.3%) than in those receiving standard-dose cyclosporine (25.8%), low-dose cyclosporine (24.0%), or low-dose sirolimus (37.2%). Allograft survival differed significantly among the four groups (P=0.02) and was highest in the low-dose tacrolimus group (94.2%), followed by the low-dose cyclosporine group (93.1%), the standard-dose cyclosporine group (89.3%), and the low-dose sirolimus group (89.3%). Serious adverse events were more common in the low-dose sirolimus group than in the other groups (53.2% vs. a range of 43.4 to 44.3%), although a similar proportion of patients in each group had at least one adverse event during treatment (86.3 to 90.5%).Conclusions: A regimen of daclizumab, mycophenolate mofetil, and corticosteroids in combination with low-dose tacrolimus may be advantageous for renal function, allograft survival, and acute rejection rates, as compared with regimens containing daclizumab induction plus either low-dose cyclosporine or low-dose sirolimus or with standard-dose cyclosporine without induction. (ClinicalTrials.gov number, NCT00231764 [ClinicalTrials.gov].). [ABSTRACT FROM AUTHOR]

Background: Plasma donor-derived cell-free DNA (dd-cfDNA) is used to screen for rejection in heart transplants. We launched the Trifecta-Heart study ( ClinicalTrials.gov No. NCT04707872), an investigator-initiated, prospective trial, to examine the correlations between genome-wide molecular changes in endomyocardial biopsies (EMBs) and plasma dd-cfDNA. The present report analyzes the correlation of plasma dd-cfDNA with gene expression in EMBs from 4 vanguard centers and compared these correlations with those in 604 kidney transplant biopsies in the Trifecta-Kidney study ( ClinicalTrials.gov No. NCT04239703)., Methods: We analyzed 137 consecutive dd-cfDNA-EMB pairs from 70 patients. Plasma %dd-cfDNA was measured by the Prospera test (Natera Inc), and gene expression in EMBs was assessed by Molecular Microscope Diagnostic System using machine-learning algorithms to interpret rejection and injury states., Results: Top transcripts correlating with dd-cfDNA were related to genes increased in rejection such as interferon gamma-inducible genes (eg, HLA-DMA ) but also with genes induced by injury and expressed in macrophages (eg, SERPINA1 and HMOX1 ). In gene enrichment analysis, the top dd-cfDNA-correlated genes reflected inflammation and rejection pathways. Dd-cfDNA correlations with rejection genes in EMB were similar to those seen in kidney transplant biopsies, with somewhat stronger correlations for TCMR genes in hearts and ABMR genes in kidneys. However, the correlations with parenchymal injury-induced genes and macrophage genes were much stronger in hearts., Conclusions: In this first analysis of Trifecta-Heart study, dd-cfDNA correlates significantly with molecular rejection but also with injury and macrophage infiltration, reflecting the proinflammatory properties of injured cardiomyocytes. The relationship supports the utility of dd-cfDNA in clinical management of heart transplant recipients., Competing Interests: The Trifecta study is an investigator-initiated study supported by a grant from Natera Inc to Transcriptome Sciences Inc/Alberta Transplant Applied Genomics Centre. 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; all Natera Inc authors are employees and own equity at Natera Inc. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Most kidney transplant patients who undergo biopsies are classified as having no rejection based on consensus thresholds. However, we hypothesized that because these patients have normal adaptive immune systems, T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) may exist as subthreshold activity in some transplants currently classified as no rejection. To examine this question, we studied genome-wide microarray results from 5086 kidney transplant biopsies (from 4170 patients). An updated molecular archetypal analysis designated 56% of biopsies as no rejection. Subthreshold molecular TCMR and/or ABMR activity molecular activity was detectable as elevated classifier scores in many biopsies classified as no rejection, with ABMR activity in many TCMR biopsies and TCMR activity in many ABMR biopsies. In biopsies classified as no rejection histologically and molecularly, molecular TCMR classifier scores correlated with increases in histologic TCMR features and molecular injury, lower estimated glomerular filtration rate, and higher risk of graft loss, and molecular ABMR activity correlated with increased glomerulitis and donor-specific antibody. No rejection biopsies with high subthreshold TCMR or ABMR activity had a higher probability of having TCMR or ABMR, respectively, diagnosed in a future biopsy. We conclude that many kidney transplant recipients have unrecognized subthreshold TCMR or ABMR activity, with significant implications for future problems., Competing Interests: Declaration of competing interest The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. P.F. Halloran holds shares in Transcriptome Sciences Inc., a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between Transcriptome Sciences Inc. and Thermo Fisher Scientific, and by a research grant from Natera, Inc. P.F. Halloran is a consultant to Natera, Inc. and Argenx BV. The other authors have declared no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The first-generation Molecular Microscope (MMDx) system for heart transplant endomyocardial biopsies used expression of rejection-associated transcripts (RATs) to diagnose not only T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) but also acute injury. However, the ideal system should detect rejection without being influenced by injury, to permit analysis of the relationship between rejection and parenchymal injury. To achieve this, we developed a new rejection classification in an expanded cohort of 3230 biopsies: 1641 from INTERHEART (ClinicalTrials.gov NCT02670408), plus 1589 service biopsies added to improve the power of the machine learning algorithms. The new system used 6 rejection classifiers instead of RATs and generated 7 rejection archetypes: No rejection, 48%; Minor, 24%; TCMR1, 2.3%; TCMR2, 2.7%; TCMR/mixed, 2.7%; early-stage ABMR, 3.9%; and fully developed ABMR, 16%. Using rejection classifiers eliminated cross-reactions with acute injury, permitting separate assessment of rejection and injury. TCMR was associated with severe-recent injury and late atrophy-fibrosis and rarely had normal parenchyma. ABMR was better tolerated, seldom producing severe injury, but in later biopsies was often associated with atrophy-fibrosis, indicating long-term risk. Graft survival and left ventricular ejection fraction were reduced not only in hearts with TCMR but also in hearts with severe-recent injury and atrophy-fibrosis, even without rejection., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histologic evaluation of allograft biopsies after lung transplantation has several limitations, suggesting that molecular assessment using tissue transcriptomics could improve biopsy interpretation. This single-center, retrospective cohort study evaluated discrepancies between the histology of transbronchial biopsies (TBBs) with no rejection (NR) and T-cell mediated rejection (TCMR) by molecular diagnosis. The accuracy of diagnosis was assessed based on response to treatment. 54 TBBs from Prague Lung Transplant Program obtained between December 2015 and January 2020 were included. Patients with acute cellular rejection (ACR) grade ≥ 1 by histology received anti-rejection treatment. Response to therapy was defined as an increase in FEV1 of ≥ 10% 4 weeks post-biopsy compared to the pre-biopsy value. Among the 54 analyzed TBBs, 25 (46%) were concordant with histology, while 29 (54%) showed discrepancies. ACR grade 0 was found in 12 TBBs (22%) and grade A1 ≥ 1 in 42 TBBs (78%). Treatment response was present in 14% in the NR group and in 50% in the TCMR group ( p = 0.024). Our findings suggest that low-grade acute cellular rejection is less likely to be associated with molecular TCMR, which might better identify lung transplant recipients who benefit from therapy., Competing Interests: PH 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. PH is a consultant to Natera, Inc. The remaining 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 Zajacova, Mackova, Halloran, Gauthier, Balko, Guney, Rakita, Svorcova, Kolarik, Vachtenheim, Pozniak, Simonek, Fila, Lischke, Halloran and Havlin.)

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.

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.)

There is a major unmet need for improved accuracy and precision in the assessment of transplant rejection and tissue injury. Diagnoses relying on histologic and visual assessments demonstrate significant variation between expert observers (as represented by low kappa values) and have limited ability to assess many biological processes that produce little histologic changes, for example, acute injury. Consensus rules and guidelines for histologic diagnosis are useful but may have errors. Risks of over- or under-treatment can be serious: many therapies for transplant rejection or primary diseases are expensive and carry risk for significant adverse effects. Improved diagnostic methods could alleviate healthcare costs by reducing treatment errors, increase treatment efficacy, and serve as useful endpoints for clinical trials of new agents that can improve outcomes. Molecular diagnostic assessments using microarrays combined with machine learning algorithms for interpretation have shown promise for increasing diagnostic precision via probabilistic assessments, recalibrating standard of care diagnostic methods, clarifying ambiguous cases, and identifying potentially missed cases of rejection. This review describes the development and application of the Molecular Microscope® Diagnostic System (MMDx), and discusses the history and reasoning behind many common methods, statistical practices, and computational decisions employed to ensure that MMDx scores are as accurate and precise as possible. MMDx provides insights on disease processes and highly reproducible results from a comparatively small amount of tissue and constitutes a general approach that is useful in many areas of medicine, including kidney, heart, lung, and liver transplants, with the possibility of extrapolating lessons for understanding native organ disease states., (© 2024 The Author(s).)

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: We explored the changes in gene expression correlating with dysfunction and graft failure in endomyocardial biopsies., Methods: Genome-wide microarrays (19,462 genes) were used to define mRNA changes correlating with dysfunction (left ventricular ejection fraction [LVEF] ≤ 55) and risk of graft loss within 3 years postbiopsy. LVEF data was available for 1,013 biopsies and survival data for 779 patients (74 losses). Molecular classifiers were built for predicting dysfunction (LVEF ≤ 55) and postbiopsy 3-year survival., Results: Dysfunction is correlated with dedifferentiation-decreased expression of normal heart transcripts, for example, solute carriers, along with increased expression of inflammation genes. Many genes with reduced expression in dysfunction were matrix genes such as fibulin 1 and decorin. Gene ontology (GO) categories suggested matrix remodeling and inflammation, not rejection. Genes associated with the risk of failure postbiopsy overlapped dysfunction genes but also included genes affecting microcirculation, for example, arginase 2, which reduces NO production, and endothelin 1. GO terms also reflected increased glycolysis and response to hypoxia, but decreased VEGF and angiogenesis pathways. T cell-mediated rejection was associated with reduced survival and antibody-mediated rejection with relatively good survival, but the main determinants of survival were features of parenchymal injury. Both dysfunction and graft loss were correlated with increased biopsy expression of BNP (gene NPPB). Survival probability classifiers divided hearts into risk quintiles, with actuarial 3-year postbiopsy survival >95% for the highest versus 50% for the lowest., Conclusions: Dysfunction in transplanted hearts reflects dedifferentiation, decreased matrix genes, injury, and inflammation. The risk of short-term loss includes these changes but is also associated with microcirculation abnormalities, glycolysis, and response to hypoxia., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

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.)

Today we know that both the humoral and the cellular arm of the immune system are engaged in severe immunological challenges. A close interaction between B and T cells can be observed in most "natural" challenges, including infections, malignancies, and autoimmune diseases. The importance and power of humoral immunity are impressively demonstrated by the current coronavirus disease 2019 pandemic. Organ transplant rejection is a normal immune response to a completely "artificial" challenge. It took a long time before the multifaceted action of different immunological forces was recognized and a unified, generally accepted opinion could be formed. Here, we address prominent paradigms and paradigm shifts in the field of transplantation immunology. We identify several instances in which the transplant community missed a timely paradigm shift because essential, available knowledge was ignored. Moreover, we discuss key findings that critically contributed to our understanding of transplant immunology but sometimes developed with delay and in a roundabout way, as was the case with antibody-mediated rejection-a main focus of this article. These include the discovery of the molecular principles of histocompatibility, the recognition of the microcirculation as a key interface of immune damage, the refinement of alloantibody detection, the description of C4d as a footmark of endothelium-bound antibody, and last but not least, the developments in biopsy-based diagnostics beyond conventional morphology, which only now give us a glimpse of the enormous complexity and pathogenetic diversity of rejection., Competing Interests: P.F.H. holds shares in Transcriptome Sciences Inc., a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between Transcriptome Sciences Inc. 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 Wolters Kluwer Health, 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: Trifecta (ClinicalTrials.gov #NCT04239703) is a prospective trial defining relationships between donor-derived cell-free DNA (dd-cfDNA), donor-specific antibody (DSA), and molecular findings in kidney transplant biopsies. Previous analyses of double results showed dd-cfDNA was strongly associated with rejection-associated molecules in the biopsy. The present study analyzed the triple results in 280 biopsies, focusing on the question of dd-cfDNA levels in DSA-negative antibody-mediated rejection (AMR)., Methods: Molecular Microscope Diagnostic System biopsy testing was performed at Alberta Transplant Applied Genomics Centre, dd-cfDNA testing at Natera, Inc, and central HLA antibody testing at One Lambda Inc. Local DSA and histologic diagnoses were assigned per center standard-of-care., Results: DSA was frequently negative in both molecular (56%) and histologic (51%) AMR. DSA-negative AMR had slightly less molecular AMR activity and histologic peritubular capillaritis than DSA-positive AMR. However, all AMRs-DSA-positive or -negative-showed elevated %dd-cfDNA. There was no association between dd-cfDNA and DSA in biopsies without rejection. In AMR, %dd-cfDNA ≥1.0 was more frequent (75%) than DSA positivity (44%). In logistic regression, dd-cfDNA percent (area under the curve [AUC] 0.85) or quantity (AUC 0.86) predicted molecular AMR better than DSA (AUC 0.66). However, the best predictions incorporated both dd-cfDNA and DSA, plus time posttransplant (AUC 0.88)., Conclusions: DSA-negative AMR has moderately decreased mean molecular and histologic AMR-associated features compared with DSA-positive AMR, though similarly elevated dd-cfDNA levels. In predicting AMR at the time of indication biopsies in this population, dd-cfDNA is superior to DSA, reflecting the prevalence of DSA-negative AMR, but the optimal predictions incorporated both dd-cfDNA and DSA., 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., Z.D., P.G., and P.B. are all employees of Natera Inc., with stocks or options to buy stocks in the company. C.L. and D.L. are employees of One Lambda Inc. L.G.H. is a consultant for AdaptImmune Therapeutics. All remaining authors declare no conflicts of interest and have nothing to disclose., (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.)

This review describes the development of the Molecular Microscope Diagnostic System (MMDx) for heart transplant endomyocardial biopsies (EMBs). MMDx-Heart uses microarrays to measure biopsy-based gene expression and ensembles of machine learning algorithms to interpret the results and compare each new biopsy to a large reference set of earlier biopsies. MMDx assesses T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), recent parenchymal injury, and atrophy-fibrosis, continually "learning" from new biopsies. Rejection-associated transcripts mapped in kidney transplants and experimental systems were used to identify TCMR, AMR, and recent injury-induced inflammation. Rejection and injury emerged as gradients of intensity, rather than binary classes. AMR was one-third donor-specific antibody (DSA)-negative, and many EMBs first considered to have no rejection displayed minor AMR-like changes, with increased probability of DSA positivity and subtle inflammation. Rejection-associated transcript-based algorithms now classify EMBs as "Normal," "Minor AMR changes," "AMR," "possible AMR," "TCMR," "possible TCMR," and "recent injury." Additionally, MMDx uses injury-associated transcript sets to assess the degree of parenchymal injury and atrophy-fibrosis in every biopsy and study the effect of rejection on the parenchyma. TCMR directly injures the parenchyma whereas AMR usually induces microcirculation stress but relatively little initial parenchymal damage, although slowly inducing parenchymal atrophy-fibrosis. Function (left ventricular ejection fraction) and short-term risk of failure are strongly determined by parenchymal injury. These discoveries can guide molecular diagnostic applications, either as a central MMDx system or adapted to other platforms. MMDx can also help calibrate noninvasive blood-based biomarkers to avoid unnecessary biopsies and monitor response to therapy., 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, and by a research grant from Natera. P.F.H. is a consultant to Natera. The other author declares no conflict of interest. H. is a consultant to Natera. The other author declares no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Many lung transplants fail due to chronic lung allograft dysfunction (CLAD). We recently showed that transbronchial biopsies (TBBs) from CLAD patients manifest severe parenchymal injury and dedifferentiation, distinct from time-dependent changes. The present study explored time-selective and CLAD-selective transcripts in mucosal biopsies from the third bronchial bifurcation (3BMBs), compared to those in TBBs., Methods: We used genome-wide microarray measurements in 324 3BMBs to identify CLAD-selective changes as well as time-dependent changes and develop a CLAD classifier. CLAD-selective transcripts were identified with linear models for microarray data (limma) and were used to build an ensemble of 12 classifiers to predict CLAD. Hazard models and random forests were then used to predict the risk of graft loss using the CLAD classifier, transcript sets associated with rejection, injury, and time., Results: T cell-mediated rejection and donor-specific antibody were increased in CLAD 3BMBs but most had no rejection. Like TBBs, 3BMBs showed a time-dependent increase in transcripts expressed in inflammatory cells that was not associated with CLAD or survival. Also like TBBs, the CLAD-selective transcripts in 3BMBs reflected severe parenchymal injury and dedifferentiation, not inflammation or rejection. While 3BMBs and TBBs did not overlap in their top 20 CLAD-selective transcripts, many CLAD-selective transcripts were significantly increased in both for example LOXL1, an enzyme controlling matrix remodeling. In Cox models for one-year survival, the 3BMB CLAD-selective transcripts and CLAD classifier predicted graft loss and correlated with CLAD stage. Many 3BMB CLAD-selective transcripts were also increased by injury in kidney transplants and correlated with decreased kidney survival, including LOXL1., Conclusions: Mucosal and transbronchial biopsies from CLAD patients reveal a diffuse molecular injury and dedifferentiation state that impacts prognosis and correlates with the physiologic disturbances. CLAD state in lung transplants shares features with failing kidney transplants, indicating elements shared by the injury responses of distressed organs., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

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.)

Mesenchymal stem cell (MSCs) therapy has already been studied in kidney transplant recipients (KTRs), and the available data showed that it is safe and well tolerated. The aim of this study was to evaluate the safety and efficacy of autologous MSCs in combination with standard therapy in KTRs with biopsy-proven chronic active antibody-mediated rejection (AMR). Patients with biopsy-proven chronic active AMR received treatment with autologous bone marrow-derived MSCs (3 × 10 6 cells/kg iv) after completion of standard therapy and were followed for up to 12 months. The primary endpoints were safety by assessment of adverse events. Secondary endpoints included assessment of kidney graft function, immunological and histological changes related to AMR activity and chronicity assessed by conventional microscopy and molecular transcripts. A total of 3 patients were enrolled in the study before it was terminated prematurely because of adverse events. We found that AMR did not improve in any of the patients after treatment with MSCs. In addition, serious adverse events were observed in one case when autologous MSCs therapy was administered in the late phase after kidney transplantation, which requires further elucidation., Competing Interests: Authors MK, AB, and LG were employed by the company Educell d.o.o Cell Therapy Service. The remaining 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 Večerić-Haler, Sever, Kojc, Halloran, Boštjančič, Mlinšek, Oblak, Poženel, Švajger, Hartman, Kneževič, Barlič, Girandon, Aleš Rigler, Zver, Buturović Ponikvar and Arnol.)

Targeting interleukin-6 (IL-6) was shown to counteract donor-specific antibody production and antibody-mediated rejection (AMR) activity. It is not known whether, or to what extent, IL-6 antagonism modulates biomarkers indicative of tissue damage (donor-derived cell-free DNA [dd-cfDNA]) and parenchymal inflammation (C-X-C motif chemokine ligand [CXCL] 10)., Methods: We report a secondary endpoint analysis of a phase 2 trial of anti-IL-6 antibody clazakizumab in late AMR (ClinicalTrials.gov, NCT03444103). Twenty kidney transplant recipients were randomized to treatment with clazakizumab or placebo over 12 wk (part A), followed by an extension in which all recipients received clazakizumab through week 52 (part B). Biomarkers were evaluated at day 0 and after 12 and 52 wk, respectively., Results: Fractional dd-cfDNA (dd-cfDNA[%]) did not significantly change under clazakizumab, with no differences between study arms (clazakizumab versus placebo) at week 12 (1.65% [median; interquartile range: 0.91%-2.78%] versus 0.97% [0.56%-2.30%]; P  = 0.25) and no significant decrease from weeks 12 to 52 (1.15% [0.70%-2.38%] versus 1.0% [0.61%-1.70%]; P  = 0.25). Similarly, urine CXCL10 was not different between groups at week 12 (55.7 [41.0-91.4] versus 60.2 [48.8-208.7.0] pg/mg creatinine; P  = 0.44) and did not change over part B (CXCL10 [pg/mg creatinine]: from 58 [46.3-93.1] to 67.4 [41.5-132.0] pg/mL creatinine; P  = 0.95). Similar results were obtained for serum CXCL10. There was no association between biomarker levels and resolution of molecular and morphologic AMR activity., Conclusions: Our results suggest that IL-6 blockade does not significantly affect levels of dd-cfDNA[%] and CXCL10. Subtle responses to this therapeutic principle may be overlooked by early biomarker surveillance., Competing Interests: T.V. and S.C. are employed by CareDx Inc., Brisbane, San Francisco, CA. G.A.B. is 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). The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Transplantation Direct. 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).)

All transplanted kidneys are subjected to some degree of injury as a result of the donation-implantation process and various post-transplant stresses such as rejection. Because transplants are frequently biopsied, they present an opportunity to explore the full spectrum of kidney response-to-wounding from all causes. Defining parenchymal damage in transplanted organs is important for clinical management because it determines function and survival. In this study, we classified the scenarios associated with parenchymal injury in genome-wide microarray results from 1,526 kidney transplant indication biopsies collected during the INTERCOMEX study. We defined injury groups by using archetypal analysis (AA) of scores for gene sets and classifiers previously identified in various injury states. Six groups and their characteristics were defined in this population: No injury, minor injury, two classes of acute kidney injury ("AKI," AKI1, and AKI2), chronic kidney disease (CKD), and CKD combined with AKI. We compared the two classes of AKI, namely, AKI1 and AKI2. AKI1 had a poor function and increased parenchymal dedifferentiation but minimal response-to-injury and inflammation, instead having increased expression of PARD3, a gene previously characterized as being related to epithelial polarity and adherens junctions. In contrast, AKI2 had a poor function and increased response-to-injury, significant inflammation, and increased macrophage activity. In random forest analysis, the most important predictors of function (estimated glomerular filtration rate) and graft loss were injury-based molecular scores, not rejection scores. AKI1 and AKI2 differed in 3-year graft survival, with better survival in the AKI2 group. Thus, injury archetype analysis of injury-induced gene expression shows new heterogeneity in kidney response-to-wounding, revealing AKI1, a class of early transplants with a poor function but minimal inflammation or response to injury, a deviant response characterized as PC3, and an increased risk of failure. Given the relationship between parenchymal injury and kidney survival, further characterization of the injury phenotypes in kidney transplants will be important for an improved understanding that could have implications for understanding native kidney diseases (ClinicalTrials.gov #NCT01299168)., Competing Interests: PH is a consultant to Natera, 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, and by a research grant from Natera. The remaining 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 Halloran, Böhmig, Bromberg, Einecke, Eskandary, Gupta, Myslak, Viklicky, Perkowska-Ptasinska, Madill-Thomsen and the INTERCOMEX Investigators.)

Transplanted lungs suffer worse outcomes than other organ transplants with many developing chronic lung allograft dysfunction (CLAD), diagnosed by physiologic changes. Histology of transbronchial biopsies (TBB) yields little insight, and the molecular basis of CLAD is not defined. We hypothesized that gene expression in TBBs would reveal the nature of CLAD and distinguish CLAD from changes due simply to time posttransplant. Whole-genome mRNA profiling was performed with microarrays in 498 prospectively collected TBBs from the INTERLUNG study, 90 diagnosed as CLAD. Time was associated with increased expression of inflammation genes, for example, CD1E and immunoglobulins. After correcting for time, CLAD manifested not as inflammation but as parenchymal response-to-wounding, with increased expression of genes such as HIF1A, SERPINE2, and IGF1 that are increased in many injury and disease states and cancers, associated with development, angiogenesis, and epithelial response-to-wounding in pathway analysis. Fibrillar collagen genes were increased in CLAD, indicating matrix changes, and normal transcripts were decreased-dedifferentiation. Gene-based classifiers predicted CLAD with AUC 0.70 (no time-correction) and 0.87 (time-corrected). CLAD related gene sets and classifiers were strongly prognostic for graft failure and correlated with CLAD stage. Thus, in TBBs, molecular changes indicate that CLAD primarily reflects severe parenchymal injury-induced changes and dedifferentiation., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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.)