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Background: Microvascular inflammation (MVI) is a key feature of antibody-mediated rejection (AMR) among patients with HLA donor-specific antibody (DSA), but MVI at AMR thresholds (Banff glomerulitis [g] + peritubular capillaritis [ptc] score ≥ 2) without DSA has been increasingly recognized. We aimed to determine the incidence of MVI among highly sensitized kidney transplant recipients without DSA., Methods: We performed a single-center, retrospective, matched cohort study comparing outcomes of kidney transplant recipients with cPRA ≥90% with preexisting DSA (n = 49), cPRA ≥90% without preexisting DSA (n = 47), and matched controls with cPRA = 0 without preexisting DSA (n = 49). Controls were matched by age, donor type, and transplant date. Indication and surveillance biopsies combined with annual de novo DSA screening were obtained., Results: Kidney transplant recipients with a cPRA ≥90% and no evidence of preexisting or de novo DSA had a higher incidence of MVI (glomerulitis + peritubular capillaritis ≥ 2) than patients with cPRA = 0 [35% (17/49) versus 12% (6/49), P  = 0.0003] over a median (interquartile range) follow-up of 5 (4-6) y posttransplant. Among this cPRA ≥90% group without DSA, MVI persisted in 54% of cases on follow-up biopsy (7/13), and 24% (4/13) of cases developed transplant glomerulopathy (Banff cg score > 0)., Conclusions: Highly sensitized transplant recipients have a high incidence of persistent and progressive MVI, even without DSA. The mechanisms underlying these histologic features needs to be elucidated, but this information is important to consider when making decisions about transplantation among highly sensitized individuals., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: The aim of this study is to investigate the clinical validity of donor-derived cell-free DNA (dd-cfDNA) in comparison with that of donor specific anti-HLA antibody (DSA) for predicting biopsy-proven rejection (BPR)and severe microvascular inflammation (severe MVI) in kidney transplant recipients (KTRs)., Methods: In this prospective observational investigation, 64 KTRs who underwent the indicated biopsies were included. Blood samples collected prior to biopsy were tested for dd-cfDNA and DSA. Biopsy specimens were classified by a renal pathologist according to the Banff classification. The predictive performance of dd-cfDNA and DSA for histological allograft diagnosis was assessed., Results: KTRs were categorized into the high and low dd-cfDNA groups based on a level of 0.4%. Eighteen patients (28.1%) had positive DSA at biopsy, exhibiting higher dd-cfDNA levels than the DSA-negative patients. BPR and severe MVI incidences were elevated in the high dd-cfDNA group (BPR: 42.9% vs. 3.4%, P <0.001; severe MVI: 37.1% vs. 3.4%, P = 0.001). Also, elevated glomerulitis and MVI scores were observed in the high dd-cfDNA group. DSA showed the highest predictive value for BPR (AUC = 0.880), whereas dd-cfDNA alone excelled in predicting severe MVI (AUC = 0.855). Combination of DSA and dd-cfDNA (>0.4%) yielded sensitivities of 80.0% and 50.0% with specificities of 90.7% and 88.0% for antibody-mediated rejection and severe MVI detection, respectively., Conclusion: The dd-cfDNA test is a predictive tool for BPR and severe MVI, and it can improve the performance, especially when combined with DSA for BPR., 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 Kim, Bae, Kang, Lee, Eum, Yang, Choi, Chung and Oh.)

Background: The Banff Foundation produces recommendations for classifying various lesions in renal allografts. Experts gather to update the classification every other year based on new scientific and clinical evidence., Objectives: This article presents the most important changes incorporated into the new recommendations after the last Banff conference., Materials and Methods: The author of this article personally took part in the Banff conference and the subsequent survey, reported on the activities of a Banff working group (peritubular capillaritis) on-site, and contributed to drafting the recently published meeting report., Results: Lesions of antibody-mediated kidney allograft rejection (AMR), especially microvascular inflammation, have been part of the diagnostic algorithm for over 20 years. Experts advocated for a simplified AMR algorithm and mindful inclusion of molecular pathological data in the clinicopathological reflection regarding therapeutic decision. A new, more descriptive diagnostic entity-microvascular inflammation, C4d negative and DSA negative-has been introduced into the AMR category to acknowledge this histological constellation and motivate research into this pathophysiologically and immunologically probably different phenotype., Conclusions: The Banff classification provides a structure for diagnosing kidney transplant pathology. Regular updates serve to adapt to ever-growing knowledge about alloimmunity. Particular challenges are capturing the complexity of various immunological scenarios and ensuring an understandable representation of these in a pathology report., (© 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.)

Development of donor-specific antibodies is associated with reduced allograft survival in renal transplantation. Recent clinical studies highlight the prevalence of human leukocyte antigen (HLA)-DQ antibodies amongst de novo donor-specific antibodies (DSAs), yet the specific contribution of these DSAs to rejection has not been examined. Antibody-mediated rejection primarily targets the microvasculature, so this study explored how patient HLA-DQ alloantibodies can modulate endothelial activation and so immunoregulation. HLA-DQ antibodies phosphorylated Akt and S6 kinase in microvascular endothelial cells. This activation prior to culture with alloreactive lymphocytes increased IL-6 and RANTES secretion. The antibody-mediated upregulation of IL-6 was indeed Akt-dependent. The binding of HLA-DQ antibodies to endothelial cells selectively reduced T cell alloproliferation and FoxP3 high Treg differentiation. In clinical studies, detection of HLA-DQ DSAs with other DSAs is associated with worse graft survival than either alone. Endothelial cells stimulated with HLA-DR and HLA-DQ antibodies showed a synergistic increase in pro-inflammatory cytokine secretion and a decrease in Treg expansion. HLA-DQ antibodies strongly promote pro-inflammatory responses in isolation and in combination with other HLA antibodies. Thus, our data give new insights into the pathogenicity of HLA-DQ DSAs., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Limited information exists about outcomes of HLA donor-specific antibody (DSA) negative (DSA-) microvascular inflammation (MVI)., Methods: We report our experience with 25 DSA- patients with MVI compared to 155 DSA+ patients who met Banff 2013 criteria for antibody-mediated rejection (AMR). We also compared outcomes to 228 DSA+ patients whose biopsies were negative for rejection and served as a negative control., Results: There were no significant differences in the baseline characteristics between the DSA- MVI and DSA+ AMR groups. At the time of diagnosis, both groups had similar graft function. The DSA- group had higher MVI scores but lower C4d scores. At last follow-up, renal function was similar between the groups. There were 12 (48%) graft failures in the DSA- group and 59 (38%) graft failures in the DSA+ group, which was not statistically different. Similar results were found after matching for the MVI scores, C4d, and treatment between 2 groups. We also found similar outcomes between DSA- and DSA+ patients when only including those who would have met Banff 2017 criteria for AMR. In univariate Cox regression analyses, estimated glomerular filtration rate at time of biopsy, glomerulitis, rituximab, diabetes, v score, allograft glomerulopathy, fibrous intimal thickening, tubular atrophy, and interstitial fibrosis scores were associated with graft failure. In multivariate analysis, only estimated glomerular filtration rate was protective. Both groups had significantly worse outcomes than the DSA+-negative controls without AMR., Conclusions: Our findings suggest that outcomes and response to treatment with HLA DSA- MVI patients are similarly poor to those with DSA+ MVI patients, supporting a critical role for MVI in the diagnosis of AMR.

Independent of the initial cause of kidney disease, microvascular injury to the peritubular capillary network appears to play a central role in the development of interstitial fibrosis in both native and transplanted kidney disease. This association is explained by mechanisms such as the upregulation of profibrotic genes and epigenetic changes induced by hypoxia, capillary leakage, endothelial and pericyte transition to interstitial fibroblasts, as well as modifications in the secretome of endothelial cells. Alloimmune injury due to antibody-mediated rejection and ischemia-reperfusion injury are the two main etiologies of microvascular damage in kidney transplant recipients. The presence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies, histological findings, such as diffuse C4d staining in peritubular capillaries, and the extent and severity of peritubular capillaritis, are commonly used clinically to provide both diagnostic and prognostic information. Complement-dependent assays, circulating non-HLA antibodies, or evaluation of the microvasculature with novel imaging techniques are the subject of ongoing studies.

Background: Antibody-mediated rejection (ABMR) is the leading cause of kidney graft loss worldwide. Criteria for acute humoral rejection (currently labeled active humoral rejection) established by the 2007 Banff classification are highly specific but lack sensitivity. Modifications to the Banff classification were introduced for its 2013 and 2017 versions in order to identify more cases of this entity., Purpose: We intend to demonstrate that, compared to its 2007 version, the 2017 Banff classification bears an improved capacity for graft loss prediction when histologic criteria for active ABMR are met., Patients and Methods: Single-center retrospective cohort study. A random sample of 201 kidney recipients who underwent a graft biopsy since January 2004 was analyzed. Patients were classified as ever developing histologic characteristics of acute ABMR (2007 Banff) or not and renal survival between groups was compared. The same patients were then classified as ever developing histologic characteristics of active ABMR (2017 Banff) or not and renal survival was again compared. Presence of circulating donor-specific antibodies (DSA) was not taken into consideration., Results: Patients were followed for a median 13.9 ± 7.9 years, during which grafts were biopsied on 537 occasions (2.7 ± 1.6 biopsies per graft). Baseline eGFR was 73.26 ± 17.6 ml/min and baseline creatinine 1.14 ± 0.25 mg/dl. Graft loss occurred in 38 recipients (18.9%) mainly due to ABMR (60.5%). Acute ABMR (2007 Banff) was identified in 11 recipients (5.5%) and graft survival did not differ between groups with and without active ABMR occurrence (log-rank p = 0.939). Active ABMR (2017 Banff) was found in 59 recipients (29%) and graft survival was better from the second post-transplant year onward in the group of patients without active ABMR occurrence (log-rank p = 0.001). Moderate microvascular inflammation was present in 89.6% of the 48 additional patients with active ABMR., Conclusion: The 2017 Banff classification identifies more patients who develop active ABMR and stratifies graft loss risk better than the 2007 version., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: In ABO-incompatible kidney transplantation, the diagnostic criteria for antibody-mediated rejection remain controversial because C4d deposition is commonly observed. Thus, we investigated microvascular inflammation (MVI score ≥ 2) within 1 year as a predictor of graft outcome., Methods: A total of 148 recipients without preformed or de novo donor-specific anti-HLA antibody were stratified based on MVI score less than 2 (n = 117) and MVI score of 2 or greater (n = 31)., Results: We found that 5-year graft survival was significantly lower (P = 0.0129) in patients with MVI (89.8%) than in patients without MVI (97.0%). Graft function, as characterized by serum estimated glomerular filtration rate, was also significantly worse for patients with MVI than it was for patients without MVI, between 3 months and 10 years after transplantation (P = 0.048). Multivariate analysis indicated that HLA class II mismatch (P = 0.0085) was an independent marker of MVI., Conclusions: Microvascular inflammation score of 2 or greater is significantly associated with poor graft outcome after ABO-incompatible kidney transplantation. We suggest that MVI score of 2 or greater in ABOi transplantation be used as a basis to diagnose antibody-mediated rejection.

Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key initial events include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these early changes in the microvasculature lead to abnormal blood flow patterns, local tissue hypoxia, and an associated overexpression of HIF-1α-inducible genes, including vascular endothelial growth factor. We also discuss how cell intrinsic regulators of mTOR-mediated signaling within EC are of critical importance in microvascular stability and may thus have a role in the inhibition of chronic rejection. Finally, we discuss recent findings indicating that miRNAs may regulate EC stability, and we review their potential as novel non-invasive biomarkers of allograft rejection. Overall, this review provides insights into molecular events, genes, and signals that promote chronic rejection and their potential as biomarkers that serve to support the future development of interruption therapeutics.

Aim: Chronic active antibody-mediated rejection (chronic ABMR) is one important cause of late-stage renal allograft loss. However, few reports have used protocol biopsy to observe changes over time in cases that develop chronic ABMR. The aim of this study was to use protocol biopsy to clarify the histological features of cases that develop chronic ABMR., Methods: We recruited 379 ABO compatible patients who underwent protocol biopsy at our hospital from 2010 to 2014. Seventeen of these patients were diagnosed with chronic ABMR (chronic ABMR group), and 12 patients were class 2 donor-specific antibody (DSA) positive and were not diagnosed with chronic ABMR (class 2 DSA-positive group). With the addition of a control group consisting of 30 DSA negative patients, these three groups were compared for Banff factors in protocol biopsies taken 3 months, 6 months, 1 year, 3 years, and 5 years after the transplant., Results: Three months post transplant, the chronic ABMR group had a significantly higher number of patients exhibiting g + ptc > 0 than that in the control group (P = 0.01). At 1, 3, and 5 years post transplant, significantly more subjects in the chronic ABMR and class 2 DSA-positive groups compared with the control group exhibited g + ptc > 0 (P < 0.03). Five years post transplant, the chronic ABMR group exhibited a significantly higher mean c4d score than that in the control group (P = 0.02). The only significant difference observed between the chronic ABMR group and the class 2 DSA-positive group was in cg scores at 5 years post transplant, which were significantly higher in the chronic ABMR group (P = 0.03)., Conclusions: These results suggest that cases exhibiting microvascular inflammation in the early post-transplant period may develop chronic ABMR, and it would be highly beneficial to perform focused electron microscope surveillance of these cases., (© 2015 Asian Pacific Society of Nephrology.)

Background: This study investigated the mechanisms involved in development of donor-specific antibody (DSA) and/or C4d-negative transplant glomerulopathy (TGP) by allograft gene expression profiles using microarrays., Design, Setting, Participants, & Measurements: This cohort study was conducted in kidney transplant recipients. Patients were eligible for inclusion if they required a clinically indicated biopsy at any time point after their transplant. They were then classified according to their histopathology findings and DSA and C4d results. Eighteen chronic antibody-mediated rejection (CAMR), 14 DSA+/C4d- TGP, 25 DSA-/C4d- TGP, and 47 nonspecific interstitial fibrosis/tubular atrophy (IFTA) biopsy specimens were identified. In a subset of patients from the study population, biopsy specimens in each group and normal transplant kidney specimens were analyzed with Affymetrix Human Gene 1.0 ST Arrays., Results: The mean sum score of glomerulitis and peritubular capillaritis increased from 0.28±0.78 in IFTA specimens to 0.75±0.85 in DSA-/C4d- TGP specimens, 1.71±1.49 in DSA+/C4d-/TGP specimens, and 2.11±1.74 in CAMR specimens (P<0.001). During a median follow-up time of 2 (interquartile range, 1.4-2.8) years after biopsy, graft loss was highest in CAMR specimens (27.8%) compared to IFTA specimens (8.5%), DSA+/C4d- TGP specimens (14.3%), and DSA-/C4d- TGP specimens (16%) (P=0.01). With use of microarrays, comparison of the gene expression profiles of DSA-/C4d- TGP specimens with glomerulitis + peritubular capillaritis scores > 0 to normal and IFTA biopsy specimens revealed higher expression of quantitative cytotoxic T cell-associated transcripts (QCAT). However, both CAMR and DSA+/C4d- TGP specimens had higher expression of not only QCAT but also IFN-γ and rejection-induced, constitutive macrophage-associated, natural killer cell-associated, and DSA-selective transcripts. Endothelial cell-associated transcript expression was upregulated only in CAMR biopsy specimens., Conclusions: These results suggested that DSA+/C4d- TGP biopsy specimens may be classified as CAMR. In contrast, DSA-/C4d- TGP specimens showed increased cytotoxic T cell-associated transcripts, suggesting T cell activation as a mechanism of injury.

Purpose of Review: In the last decade, there has been a growing recognition of alloantibody responses in organ transplantation, but phenotypes related to antibody-mediated rejection (ABMR) remain incompletely defined. This article reviews recent molecular studies in kidney allograft tissues that decipher molecular burden and mechanisms of ABMR, leading to discovery of a new phenotype: 'C4d-negative ABMR'., Recent Findings: High endothelial gene expression in kidney transplant biopsies with anti-human leukocyte antigen alloantibody indicates active antibody-mediated damage and poor graft survival, defining a previously unknown group of C4d-negative ABMR. C4d-negative ABMR is characterized by high intragraft endothelial gene expression, alloantibodies, histology of chronic ABMR (less frequently acute ABMR), and poor outcomes. Thus, endothelial molecular phenotype in biopsies with circulating antibody detects degree of active graft injury, and many of these transcripts reflect endothelial activation. C4d-negative ABMR is twice as common as C4d-positive ABMR. Recognition of this new phenotype reveals ABMR (C4d positive or negative) as the most common cause of late kidney transplant loss., Summary: C4d staining, although very useful, is insensitive for detecting ABMR. Measuring endothelial gene expression in biopsies from kidneys with alloantibody is a sensitive and specific method to diagnose ABMR and predict graft outcomes.