Selection and Interpretation of Molecular Diagnostics in Heart Transplantation.

The number of heart transplants performed annually in the United States and worldwide continues to increase, but there has been little change in graft longevity and patient survival over the past 2 decades. The reference standard for diagnosis of acute cellular and antibody-mediated rejection includes histologic and immunofluorescence evaluation of endomyocardial biopsy samples, despite invasiveness and high interrater variability for grading histologic rejection. Circulating biomarkers and molecular diagnostics have shown substantial predictive value in rejection monitoring, and emerging data support their use in diagnosing other posttransplant complications. The use of genomic (cell-free DNA), transcriptomic (mRNA and microRNA profiling), and proteomic (protein expression quantitation) methodologies in diagnosis of these posttransplant outcomes has been evaluated with varying levels of evidence. In parallel, growing knowledge about the genetically mediated immune response leading to rejection (immunogenetics) has enhanced understanding of antibody-mediated rejection, associated graft dysfunction, and death. Antibodies to donor human leukocyte antigens and the technology available to evaluate these antibodies continues to evolve. This review aims to provide an overview of biomarker and immunologic tests used to diagnose posttransplant complications. This includes a discussion of pediatric heart transplantation and the disparate rates of rejection and death experienced by Black patients receiving a heart transplant. This review describes diagnostic modalities that are available and used after transplant and the landscape of future investigations needed to enhance patient outcomes after heart transplantation.

The Banff 2022 Kidney Meeting Work Plan: Data-driven refinement of the Banff Classification for renal allografts.

The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell-mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

The impact of IdeS (imlifidase) on allo-specific, xeno-reactive, and protective antibodies in a sensitized rhesus macaque model.

BACKGROUND: Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound. METHODS: IdeS was generated and tested (2 mg/kg, IV) in two naïve and four allosensitized nonhuman primates (NHP). Peripheral blood samples were collected at regular intervals following IdeS administration. Total IgG, total IgM, and anti-CMV antibodies were quantified with ELISA, and donor-specific antibody (DSA) and anti-pig antibodies were evaluated using flow cytometric crossmatch. B cell populations were assessed using flow cytometry. RESULTS: IdeS successfully cleaved rhesus IgG in vitro. In allosensitized NHP, robust reduction of total, DSA, anti-pig, and anti-CMV IgG was observed within one day following IdeS administration. Rapid rebound of all IgG antibody populations was observed, with antibody levels returning to baseline around day 14 post-infusion. Total IgM level was not affected by IdeS. Interestingly, a comparable reduction in antibody populations was observed after the second dose of IdeS. However, we have not observed any significant modulation of B cell subpopulations after IdeS. CONCLUSIONS: This study evaluated efficacy of IdeS in the allosensitized NHP in IgG with various specificities, mirroring antibody kinetics in human patients. The efficacy of IdeS on preexisting anti-pig antibodies may be useful in clinical xenotransplantation. However, given the limitation of IdeS on its durability as a monotherapy, optimization of IdeS with other agents targeting the humoral response is further needed.

The evolving use of biomarkers in heart transplantation: Consensus of an expert panel.

In heart transplantation, the use of biomarkers to detect the risk of rejection has been evolving. In this setting, it is becoming less clear as to what is the most reliable test or combination of tests to detect rejection and assess the state of the alloimmune response. Therefore, a virtual expert panel was organized in heart and kidney transplantation to evaluate emerging diagnostics and how they may be best utilized to monitor and manage transplant patients. This manuscript covers the heart content of the conference and is a work product of the American Society of Transplantation's Thoracic and Critical Care Community of Practice. This paper reviews currently available and emerging diagnostic assays and defines the unmet needs for biomarkers in heart transplantation. Highlights of the in-depth discussions among conference participants that led to development of consensus statements are included. This conference should serve as a platform to further build consensus within the heart transplant community regarding the optimal framework to implement biomarkers into management protocols and to improve biomarker development, validation and clinical utility. Ultimately, these biomarkers and novel diagnostics should improve outcomes and optimize quality of life for our transplant patients.

Sensitization in transplantation: Assessment of Risk 2022 Working Group Meeting Report.

The Sensitization in Transplantation: Assessment of Risk workgroup is a collaborative effort of the American Society of Transplantation and the American Society of Histocompatibility and Immunogenetics that aims at providing recommendations for clinical testing, highlights gaps in current knowledge, and proposes areas for further research to enhance histocompatibility testing in support of solid organ transplantation. This report provides updates on topics discussed by the previous Sensitization in Transplantation: Assessment of Risk working groups and introduces 2 areas of exploration: non-human leukocyte antigen antibodies and utilization of human leukocyte antigen antibody testing measurement to evaluate the efficacy of antibody-removal therapies.

Addressing sex-based disparities in solid organ transplantation in the United States - a conference report.

Solid organ transplantation provides the best treatment for end-stage organ failure, but significant sex-based disparities in transplant access exist. On June 25, 2021, a virtual multidisciplinary conference was convened to address sex-based disparities in transplantation. Common themes contributing to sex-based disparities were noted across kidney, liver, heart, and lung transplantation, specifically the existence of barriers to referral and wait listing for women, the pitfalls of using serum creatinine, the issue of donor/recipient size mismatch, approaches to frailty and a higher prevalence of allosensitization among women. In addition, actionable solutions to improve access to transplantation were identified, including alterations to the current allocation system, surgical interventions on donor organs, and the incorporation of objective frailty metrics into the evaluation process. Key knowledge gaps and high-priority areas for future investigation were also discussed.

Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Toward Personalized Management.

BACKGROUND AND AIMS: Tolerance is transplantation's holy grail, as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance. APPROACH AND RESULTS: We conducted a multicenter, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2), and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyltransferase exceeded 100 U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95% confidence interval [CI] 27.4%, 48.5%) were operationally tolerant, 16 were nontolerant by histology (met biochemical but failed histological criteria), and 39 were nontolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n = 32) occurred at ≤32% of the trial-entry immunosuppression dose and was treated with corticosteroids (n = 32) and/or tacrolimus (n = 38) with resolution (liver tests within 1.5 times the baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or nontolerant subjects. CONCLUSIONS: Immunosuppression withdrawal showed that 37.5% of selected pediatric liver-transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or nontolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.

A 2020 Banff Antibody-mediatedInjury Working Group examination of international practices for diagnosing antibody-mediated rejection in kidney transplantation - a cohort study.

The Banff antibody-mediated rejection (ABMR) classification is vulnerable to misinterpretation, but the reasons are unclear. To better understand this vulnerability, we evaluated how ABMR is diagnosed in practice. To do this, the Banff Antibody-Mediated Injury Workgroup electronically surveyed an international cohort of nephrologists/surgeons (n = 133) and renal pathologists (n = 99). Most providers (97%) responded that they use the Banff ABMR classification at least sometimes, but DSA information is often not readily available. Only 41.1% (55/133) of nephrologists/surgeons and 19.2% (19/99) of pathologists reported that they always have DSA results when the biopsy is available. Additionally, only 19.6% (26/133) of nephrologists/surgeons responded that non-HLA antibody or molecular transcripts are obtained when ABMR histologic features are present but DSA is undetected. Several respondents agreed that histologic features concerning for ABMR in the absence of DSA and/or C4d are not well accounted for in the current classification [31.3% (31/99) pathologists and 37.6% (50/133) nephrologist/surgeons]. The Banff ABMR classification appears widely accepted, but efforts to improve the accessibility of DSA information for the multidisciplinary care team are needed. Further clarity is also needed in Banff ABMR nomenclature to account for the spectrum of ABMR and for histologic features suspicious for ABMR when DSA is absent.

IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.

The impact of donor-specific HLA antibody (DSA) following liver transplantation remains controversial. We hypothesized DSA IgG subclass characteristics, compared to total DSA IgG, might correlate with specific histopathological phenotype(s) of subclinical graft injury. We therefore studied 129 stable, arguably "clinically ideal," pediatric liver recipients at the time of a screening biopsy to enter an immunosuppression withdrawal trial. Sixty-five (50%) subjects tested positive for class II DSA. IgG subclass profile was characterized by mean fluorescence intensity (MFI) and normalized subclass composition (>5%). A prominent IgG4 DSA profile was strongly correlated with greater HLA mismatch, a histopathological phenotype characterized by the presence of interface activity (with variable degrees of fibrosis), and a transcriptional profile of attenuated T cell-mediated rejection. Specifically, compared to those without class II DSA, those with IgG4 class II DSA MFI sum >2000 exhibited an odds ratio (OR) of 20.79 (95% confidence interval [CI] 4.38-98.69) and IgG4 subclass composition >5% exhibited an OR of 8.99 (95% CI 2.70-29.9). Our data suggest that IgG4 DSA may serve as a useful biomarker to identify, among clinically and biochemically stable liver transplant recipients, a subset with histological and transcriptional features indicative of an active, suboptimally controlled alloimmune response.

Tailored use of belatacept in adolescent kidney transplantation.

Adolescent transplant recipients are at risk for nonadherence, development of de novo donor-specific antibody (dnDSA), and allograft loss. Belatacept, a selective T cell costimulatory blocker, is associated with reduced dnDSA, improved renal function, and prolonged allograft survival when compared to calcineurin inhibitor-based regimens in adults; however, its use in children is scant. Three adolescents were initiated on belatacept between August 2017 and September 2018 at the time of kidney transplantation. Selection criteria included age ≥ 14 and EBV IgG + serostatus. Intraoperative alemtuzumab and methylprednisolone were given as induction therapy. Tailored maintenance therapy included steroid-free belatacept and sirolimus for two patients. One patient was initially maintained steroid-free on belatacept and belimumab, an inhibitor of B cell activating factor to treat concurrent systemic lupus erythematous; steroids were added subsequently. Renal function, biopsy-proven rejection, dnDSA, allograft survival, infection, nonadherence, and proteinuria were monitored. Renal function was 86, 73, 52 mL/min/1.73 m2 at 20, 20, and 8 months, respectively. There was 100% adherence to therapy and no development of dnDSA. All patients had treatable infections. One developed steroid-responsive acute cellular rejection. Belatacept-based regimens can be tailored for adolescent recipients with good short-term clinical outcomes.

The impact of belatacept on third-party HLA alloantibodies in highly sensitized kidney transplant recipients.

Recent evidence suggests that belatacept reduces the durability of preexisting antibodies to class I and class II human leukocyte antigens (HLAs). In this case series of 163 highly sensitized kidney transplant candidates whose calculated panel-reactive antibody (cPRA) activity was ≥98% to 100%, the impact of belatacept on preexisting HLA antibodies was assessed. Of the 163 candidates, 72 underwent transplantation between December 4, 2014 and April 15, 2017; 60 of these transplanted patients remained on belatacept consecutively for at least 6 months. We observed a decrease in the breadth and/or strength of HLA class I antibodies as assessed by FlowPRA in belatacept-treated patients compared to controls who did not receive belatacept. Specifically, significant HLA antibody reduction was evident for class I (P < .0009). Posttransplant belatacept-treated patients also had a clinically significant reduction in their cPRA compared to controls (P < .01). Collectively, these findings suggest belatacept can reduce HLA class I antibodies in a significant proportion of highly sensitized recipients and could be an option to improve pretransplant compatibility with organ donors.

B cells in transplant tolerance and rejection: friends or foes?

Our understanding of the role of B cells in organ transplantation remains incomplete and continues to grow. The majority of research has focused on the detrimental role of antibodies that drive the development of pathogenesis of the transplanted organ. However, it has been shown that not all donor-specific antibodies are harmful and in some circumstances can even promote tolerance through the mechanism of accommodation. Furthermore, B cells can have effects on transplanted organs through their interaction with T cells, namely antigen presentation, cytokine production, and costimulation. More recently, the role and importance of Bregs was introduced to the field of transplantation. Due to this functional and ontogenetic heterogeneity, targeting B cells in transplantation may bring undesired immunologic side effects including increased rejection. Therefore, the selective control of B cells that contribute to the humoral response against donor antigens will continue to be an important and challenging area of research and potentially lead to improved long-term transplant outcomes.

The role of non-HLA antibodies in solid organ transplantation: a complex deliberation.

PURPOSE OF REVIEW: There is tremendous interest in understanding when, if, and how non-HLA antibodies contribute to allograft injury. Numerous non-HLA target antigens have been identified and sensitization to these targets have been associated with delayed allograft function, rejection, and allograft failure. This review focuses on the clinical utility of HLA antibody testing, highlighting the strengths and limitations of current clinical studies, and the need for defining characteristics to inform non-HLA antibody pathogenicity. RECENT FINDINGS: Clinical studies continue to show associations between non-HLA antibodies and rejection and reduced allograft survival across multiple transplanted organ types. The worst clinical outcomes continue to be observed among recipients testing positive for both non-HLA and donor-specific HLA antibodies. Mechanistic insights from both animal and clinical studies support a model in which tissue injury accompanied by an inflammatory environment influence non-HLA antibody formation and pathogenicity. SUMMARY: Immune triggers that lead to non-HLA antibody formation and pathogenicity are complex and poorly understood. The ability of non-HLA antibodies to mediate allograft injury may depend upon their affinity and strength (titer), target specificity, density of the target antigen, and synergy with donor-specific HLA antibodies.

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

BACKGROUND & AIMS: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. METHODS: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. RESULTS: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. CONCLUSION: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Microvascular inflammation in renal allograft biopsies assessed by endothelial and leukocyte co-immunostain: a retrospective study on reproducibility and clinical/prognostic correlates.

The most prominent histologic lesion in antibody-mediated rejection is microvascular inflammation (MVI); however, its recognition and scoring can be challenging and poorly reproducible between pathologists. We developed a dual immunohistochemical (IHC)-stain (anti-CD34/anti-CD45 for endothelium/leukocytes) as ancillary tool to improve on the semi-quantitative Banff scores and allow quantification of MVI. We examined the relationship between CD34-CD45 IHC-based quantitative MVI score (the inflamed peritubular capillary ratio, iptcr) and renal-graft failure or donor-specific antibodies (DSA) strength at the time of biopsy. Quantitative iptcr score was significantly associated with renal graft failure (hazard ratio 1.81, per 1 SD-unit [0.13 points] of iptcr-increase; P = 0.026) and predicted the presence and strength of DSA (ordinal odds ratio: 2.42; P = 0.005; 75 biopsies/60 kidney transplant recipients; 30 HLA- and/or ABO-incompatible). Next, we assessed inter-pathologist agreement for ptc score and ptc extent (focal/diffuse) using CD34-CD45 IHC as compared to conventional stain. Compared to conventional stain, CD34-CD45 IHC significantly increased inter-pathologist agreement on ptc score severity and extent (κ-coefficient from 0.52-0.80 and 0.46-0.68, respectively, P < 0.001). Our findings show that CD34-CD45 IHC improves reproducibility of MVI scoring and facilitates MVI quantification and introduction of a dual anti-CD34/CD45 has the potential to improve recognition of MVI ahead of DSA results.

Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: A systematic review and meta-analysis.

BACKGROUND: Anti-human leukocyte antigen donor-specific antibodies (anti-HLA DSAs) are recognized as a major barrier to patients' access to organ transplantation and the major cause of graft failure. The capacity of circulating anti-HLA DSAs to activate complement has been suggested as a potential biomarker for optimizing graft allocation and improving the rate of successful transplantations. METHODS AND FINDINGS: To address the clinical relevance of complement-activating anti-HLA DSAs across all solid organ transplant patients, we performed a meta-analysis of their association with transplant outcome through a systematic review, from inception to January 31, 2018. The primary outcome was allograft loss, and the secondary outcome was allograft rejection. A comprehensive search strategy was conducted through several databases (Medline, Embase, Cochrane, and Scopus). A total of 5,861 eligible citations were identified. A total of 37 studies were included in the meta-analysis. Studies reported on 7,936 patients, including kidney (n = 5,991), liver (n = 1,459), heart (n = 370), and lung recipients (n = 116). Solid organ transplant recipients with circulating complement-activating anti-HLA DSAs experienced an increased risk of allograft loss (pooled HR 3.09; 95% CI 2.55-3.74, P = 0.001; I2 = 29.3%), and allograft rejection (pooled HR 3.75; 95% CI: 2.05-6.87, P = 0.001; I2 = 69.8%) compared to patients without complement-activating anti-HLA DSAs. The association between circulating complement-activating anti-HLA DSAs and allograft failure was consistent across all subgroups and sensitivity analyses. Limitations of the study are the observational and retrospective design of almost all included studies, the higher proportion of kidney recipients compared to other solid organ transplant recipients, and the inclusion of fewer studies investigating allograft rejection. CONCLUSIONS: In this study, we found that circulating complement-activating anti-HLA DSAs had a significant deleterious impact on solid organ transplant survival and risk of rejection. The detection of complement-activating anti-HLA DSAs may add value at an individual patient level for noninvasive biomarker-guided risk stratification. TRIAL REGISTRATION: National Clinical Trial protocol ID: NCT03438058.

Correction: Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: A systematic review and meta-analysis.

[This corrects the article DOI: 10.1371/journal.pmed.1002572.].

Meeting report of the STAR-Sensitization in Transplantation Assessment of Risk: Naïve Abdominal Transplant Organ subgroup focus on kidney transplantation.

The development of de novo donor-specific HLA antibody (dnDSA) is a critical feature contributing to late allograft failure. The complexity of the issue is further complicated by organ-specific differences, detection techniques, reliance of tissue histopathology and changing diagnostic criteria, ineffective therapies, and lack of consensus. To tackle these issues, the Sensitization in Transplantation Assessment of Risk (STAR) 2017 was initiated as a collaboration of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics consisting of 8 working groups with the goal to provide guidelines on how to assess risk and risk stratify patients based on their potential alloimmune and DSA status. Herein is a summary of discussions by the Naïve Abdominal Working Group, highlighting currently available data and identifying gaps in our knowledge on the development and impact of dnDSA following kidney transplantation.