Cardiac Allograft Rejection Research Articles

A phantom for targeted endomyocardial biopsy training.

Endomyocardial biopsy (EMB) of the right ventricular (RV) septal surface during cardiac catheterization is the standard method to assess cardiac allograft rejection, heart failure, or inflammatory cardiomyopathies. We developed methodology using a three-dimensional (3D) printed phantom to provide proof of concept for using biplane overlay technology for targeted EMB. A standard bioptome and steerable sheath were used to discern feasibility of biopsy for seven regions of the RV septum guided by 3D overlay. This novel biopsy phantom can help train operators in biopsy techniques, and biplane overlay technology has the potential to advance targeted EMB in transplant and cardiomyopathy populations.

Microneedle-mediated transdermal delivery of FK506 for treatment of acute cardiac allograft rejection

FK506 has been widely used to prevent acute rejection in heart transplantation (HT). However, its strong hydrophobicity, poor bioavailability, high pharmacokinetic variability, and narrow therapeutic window are obstacles to its application in HT. It has been demonstrated that localized and sustained immunosuppressant delivery may be a suitable route for FK506 administration. Hence, the objective of this study was to evaluate the efficacy of a FK506-loaded microneedle (FK506 MN) in the treatment of acute rejection in HT. The physicochemical properties of FK506 MN were evaluated, and it exhibited constant release over the course of at least 9 days with a 94.6 % cumulative release rate. Also, the FK506 MN treatment could successfully alleviate acute rejection and prolong allograft survival compared with the untreated group (mean survival time, 12.33 versus 7.25 days). Furthermore, T cell and macrophage infiltration as well as secretion of IL-2, IL-6 and IFN-γ were dramatically reduced in the FK506 MN group. As expected, no hepatoxicity and nephrotoxicity were observed after the application of FK506 MN. Taken together, FK506 MN based drug delivery system provides a novelty strategy to deliver FK506 in heart transplantation, which may be also suitable for other organ transplantations in the future.

Total Lymphoid Irradiation in the Treatment of Heart Transplant Rejection: Single Institution Long-Term Results

Acute cardiac allograft rejection is a major cause of morbidity and mortality post-transplant. Total lymphoid irradiation (TLI) has been shown to cause long-lasting alterations in lymphocyte sub-sets, and thereby induces a state of partial response that is useful to prevent organ transplant rejection. In our previous report, treatment with TLI resulted in decreased episodes of acute rejection and steroid dosage requirements for up to 9.1 years post cardiac transplantation. In this study, we report an update on the short- and long-term effects of TLI in the treatment of cardiac transplant rejection. We performed a retrospective analysis of patients with intractable cardiac allograft transplantation evaluated in the department of radiation oncology at our institution over 23 years (1998-2021). Cardiac allograft rejection was defined as multiple or persistent episodes of rejection despite aggressive immunosuppressive therapy. The prescribed dose was 8 Gy in 10 fractions to be delivered over 5 weeks. All episodes of pathologic acute cardiac rejection, duration of immunosuppression, and episodes of acute infection requiring treatment before and after TLI were documented. Overall survival (OS) was analyzed using Kaplan-Meier method. The median time to first cardiac allograft rejection and the median time to first acute rejection was monitored. Of the 29 patients included, 21 (72.4%) had ISHLT grade 1R, 5 (17.2%), grade 2R, and 2(6.9%) grade 3R rejection. Rejection grade was unknown for 1 patient. The mean number of acute rejections after transplant was 14.4 episodes per patient (range 1-44) with median time to first rejection of 0.63 months. The median radiation dose was 8 Gy (range 7.2-8 Gy) delivered over a mean of 31 days. Treatment was stopped early in 2 (6.5%) patients (after 7.2 Gy and 7.9 Gy) for severe bone marrow suppression and drop in CD4 counts, respectively. The number of rejections dropped from 14.2 to 3.97 per patient over a median time of 29.0 months vs 28.1 months pre and post TLI (p = 0.01). The frequency of ISHLT acute grade 3R rejections decreased from 0.58 to 0.06 episodes per patient pre and post TLI (p = 0.02). Immunosuppressant requirements decreased from 2.83 before TLI to 2.69 after TLI (p = 0.71). Infection rates per patient were 0.71 pre TLI and remained at 0.63 post TLI. Treatment was well tolerated with 4 (13.8%) grade 2 or higher radiation toxicity. Median follow up was 76.3 months. Median and 3-year OS were 53.4 months and 60.9%, respectively. TLI is a safe and effective treatment for control of intractable cardiac rejection. The episodes of acute rejection and immunosuppressant requirements are decreased, but with long-term follow up, these patients continue to experience rejection. However, TLI lowers the frequency of ISHLT moderate to severe rejection episodes for up to 6.4 years. There was no increased risk of severe infections and there were only minimal short- and long-term grade 2 or higher radiation toxicities.

Banff Human Organ Transplant Consensus Gene Panel for the Detection of Antibody Mediated Rejection in Heart Allograft Biopsies.

The molecular refinement of the diagnosis of heart allograft rejection based on whole-transcriptome analyses faces several hurdles that greatly limit its widespread clinical application. The targeted Banff Human Organ Transplant gene panel (B-HOT, including 770 genes of interest) has been developed to facilitate reproducible and cost-effective gene expression analysis of solid organ allografts. We aimed to determine in silico the ability of this targeted panel to capture the antibody-mediated rejection (AMR) molecular profile using whole-transcriptome data from 137 heart allograft biopsies (71 biopsies reflecting the entire landscape of histologic AMR, 66 non-AMR control biopsies including cellular rejection and non-rejection cases). Differential gene expression, pathway and network analyses demonstrated that the B-HOT panel captured biologically and clinically relevant genes (IFNG-inducible, NK-cells, injury, monocytes-macrophage, B-cell-related genes), pathways (interleukin and interferon signaling, neutrophil degranulation, immunoregulatory interactions, endothelial activation) and networks reflecting the pathophysiological mechanisms underlying the AMR process previously identified in whole-transcriptome analysis. Our findings support the potential clinical use of the B-HOT-gene panel as a reliable proxy to whole-transcriptome analysis for the gene expression profiling of cardiac allograft rejection.

Donor circadian clock influences the long-term survival of heart transplantation by immunoregulation.

Circadian clocks play important role in immunoregulation. We aimed to investigate cardiac circadian clock specific pathways and compare cardiac grafts procured at different timing on survival after transplantation to explore novel criteria for donor selection. In primate heart, phase set enrichment analysis (PSEA) showed rhythmic transcripts were enriched in antigen processing and presentation during activation of circadian rhythm. Digital sorting of immune cell composition and single-sample gene set enrichment analysis (ssGSEA) in unused donor transcriptomes showed the pathway, positive regulation of circadian rhythm significantly correlates with allograft rejection and antigen presentation pathways as well as with increased compositions of matured dendritic cell, CD4+ T cell, and naive B cell. Single-centre retrospective cohort of 390 adult heart transplants between 1 January 2015 and 31 December 2020 was used to generate a propensity score matching (PSM) cohort. Survival curve differed significantly showing inferior long-term survival when donor hearts were procured at activation group (12 pm to 12 am) compared to repression group (12 am to 12 pm) (6-year survival: 64.2% vs. 75.8%, P = 0.0065). Activation group was also associated with significantly higher rates of in-hospital death, cardiopulmonary resuscitation, and usage of mechanical circulatory support after heart transplantation compared to repression group. Furthermore, tendency for post-transplant free of rejection rates was higher in repression group compared to activation group (acute rejection, Gehan-Breslow P = 0.11 and 0.04; chronic rejection, Log rank P = 0.077 and 0.15, in full and PSM cohorts, respectively). Adjusted Cox regression analysis showed that activation group was associated with 2.20 times increased hazard of death (hazard ratio: 2.20; 95% confidence interval: 1.23-3.95; P = 0.008) compared to repression group. Circadian immunity may represent donor-related risk factors for cardiac allograft rejection through activating genes related to antigen presentation pathway and immune cells oscillation at specific time of day. Molecular circadian clock should be considered during retrieval of cardiac allografts in order to maximize graft durability.

Validation of the clinical utility of microRNA as noninvasive biomarkers of cardiac allograft rejection: A prospective longitudinal multicenter study

While studies have shown an association between microRNAs and cardiac rejection, the clinical relevance of a pre-identified miRNA signature as a non-invasive biomarker has never been assessed in prospective multicentric unselected cohorts. To address this unmet need, we designed a prospective study (NCT02672683) including recipients from 11 centers between 08/2016 to 03/2018. The objective was to validate the association between three previously identified circulating microRNA (10a, 92a, 155) and the histopathological diagnosis of rejection. Both relative and absolute (sensitivity analysis) quantifications of microRNAs were performed. Overall, 461 patients were included (831 biopsies, 79 rejections). A per-protocol interim analysis (258 biopsies, 49 rejections) did not find any association between microRNA and rejection (microRNA10-a: OR=1.05, 95%CI=0.87-1.27, p=0.61; 92-a: OR=0.98, 95%CI=0.87-1.10, p=0.68; 155: OR=0.91, 95%CI=0.76-1.10, p=0.33). These results were confirmed in the sensitivity analysis. The analysis of the remaining sera was stopped for futility. This study shows no clinical utility of circulating microRNAs 10a, 92a and 155 monitoring in heart allograft recipients.

Noninvasive quantification of granzyme B in cardiac allograft rejection using targeted ultrasound imaging.

Endomyocardial biopsy is the gold standard method for the diagnosis of cardiac allograft rejection. However, it causes damage to the heart. In this study, we developed a noninvasive method for quantification of granzyme B (GzB) in vivo by targeted ultrasound imaging, which detects and provides quantitative information for specific molecules, for acute rejection assessment in a murine cardiac transplantation model. Microbubbles bearing anti-GzB antibodies (MBGzb) or isotype antibodies (MBcon) were prepared. Hearts were transplanted from C57BL/6J (allogeneic) or C3H (syngeneic) donors to C3H recipients. Target ultrasound imaging was performed on Days 2 and 5 post-transplantations. A pathologic assessment was performed. The expression of granzyme B and IL-6 in the heart was detected by Western blotting. After MB injection, we observed and collected data at 3 and 6min before and after the flash pulse. Quantitative analysis revealed that the reduction in peak intensity was significantly higher in the allogeneic MBGzb group than in the allogeneic MBcon group and the isogeneic MBcon group at PODs 2 and 5. In the allogeneic groups, granzyme B and IL-6 expression levels were higher than those in the isogeneic group. In addition, more CD8 T cells and neutrophils were observed in the allogeneic groups. Ultrasound molecular imaging of granzyme B can be used as a noninvasive method for acute rejection detection after cardiac transplantation.

MicroRNA let-7a mediates posttranscriptional inhibition of Nr4A1 and exacerbates cardiac allograft rejection

BackgroundAcute allograft rejection remains a major obstacle after heart transplantation, and CD4+ T cells play a crucial role in allograft rejection. Upregulation of Nr4A1 could regulate CD4+ T-cell function and alleviate allograft rejection. However, the regulatory mechanism of Nr4A1 in allograft rejection remains elusive. MethodsBALB/c mouse hearts were transplanted into WT C57BL/6 mice, and dynamic detection of the changes in Nr4A1 expression revealed that Nr4A1 was regulated posttranscriptionally after heart transplantation. Potential upstream miRNAs of Nr4A1 were screened, and the transfection of cells with these miRNA mimics/inhibitors and dual-luciferase reporter experiments were performed to clarify the regulatory mechanism of miRNAs on Nr4A1 expression. The miRNA agomiR/antagomiR was applied in vivo to validate the role of the corresponding miRNA in heart transplantation. Finally, Nr4A1 knockout mice and an adoptive T-cell cotransfer model were used to confirm the specific effects of miRNA. ResultsThe expression of Nr4A1 protein (rather than mRNA) exhibited a trend of initially increasing and then decreasing rapidly, and this phenomenon could not be reversed by lysosomal or proteasomal inhibitors. The miRNA let-7a directly binds to the Nr4A1 3’UTR and posttranscriptionally regulates Nr4A1 expression. The let-7a antagomiR prolonged allograft survival and regulated CD4+ T-cell function by upregulating Nr4A1 protein expression in CD4+ T cells. ConclusionsThis study confirmed that let-7a is a potential target for interfering with Nr4A1 expression in CD4+ T cells and preventing the pathological progression of cardiac allograft rejection.

Extracorporeal photopheresis in the treatment of cardiac allograft rejection: A single-centre experience

Despite novel immunosuppressive (IS) protocols, adverse effects of IS drugs continue to have notable negative impact on patient and cardiac allograft survival after heart transplantation (HTx). Therefore, IS regimens with less toxic side effects are sorely needed. We aimed to evaluate the efficacy of extracorporeal photopheresis (ECP) in combination with tacrolimus-based maintenance IS therapy in the treatment of allograft rejection in adult HTx recipients. Indications for ECP included acute moderate-to-severe or persistent mild cellular rejection, or mixed rejection. Twenty-two patients underwent a median of 22(2–44) ECP treatments after HTx. Median duration of ECP course was 173.5(2–466) days. No relevant adverse effects of ECP were noted. Reduction of methylprednisolone doses was safe throughout the ECP course. ECP, used in conjunction with pharmacological anti-rejection therapy, had a successful reversal of cardiac allograft rejection, decreased the rates of subsequential rejection episodes and normalized the allograft function in patients completing the ECP course. Short- and long-term survivals were excellent (91% at 1 and 5 years post-ECP) and comparable to International Society for Heart and Lung Transplantation registry data on HTx recipient overall survival. In conclusion, ECP can be safely used for the treatment and prevention of cardiac allograft rejection in conjunction with traditional IS regimen.

Unsupervised mRNA-seq classification of heart transplant endomyocardial biopsies.

Endomyocardial biopsy (EMB) is currently considered the gold standard for diagnosing cardiac allograft rejection. However, significant limitations related to histological interpretation variability are well-recognized. We sought to develop a methodology to evaluate EMB solely based on gene expression, without relying on histology interpretation. Sixty-four EMBs were obtained from 47 post-heart transplant recipients, who were evaluated for allograft rejection. EMBs were subjected to mRNA sequencing, in which an unsupervised classification algorithm was used to identify the molecular signatures that best classified the EMBs. Cytokine and natriuretic peptide peripheral blood profiling was also performed. Subsequently, we performed gene network analysis to identify the gene modules and gene ontology to understand their biological relevance. We correlated our findings with the unsupervised and histological classifications. Our algorithm classifies EMBs into three categories based solely on clusters of gene expression: unsupervised classes 1, 2, and 3. Unsupervised and histological classifications were closely related, with stronger gene module-phenotype correlations for the unsupervised classes. Gene ontology enrichment analysis revealed processes impacting on the regulation of cardiac and mitochondrial function, immune response, and tissue injury response. Significant levels of cytokines and natriuretic peptides were detected following the unsupervised classification. We have developed an unsupervised algorithm that classifies EMBs into three distinct categories, without relying on histology interpretation. These categories were highly correlated with mitochondrial, immune, and tissue injury response. Significant cytokine and natriuretic peptide levels were detected within the unsupervised classification. If further validated, the unsupervised classification could offer a more objective EMB evaluation.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Allograft TLR9 is required for endogenous donor-reactive memory CD8 T cell activation to mediate CTLA-4Ig resistant rejection of high-risk cardiac allografts

Abstract Organ cold ischemic storage (CIS) is inherent to deceased-donor solid organ transplantation and CIS time correlates with poorer transplant outcomes. Prolonged CIS enhances ischemia-reperfusion injury (IRI) following graft revascularization, generating an inflammatory environment that promotes endogenous donor-reactive memory T cell activation leading to CTLA-4Ig costimulatory blockade-resistant rejection. Using a murine heterotopic heart transplant model, we tested the impact of prolonged (8hr) versus minimal (0.5hr) CIS on cell-free mitochondrial DNA (cf-mtDNA) release and activation of the cf-mtDNA sensor TLR9 to understand their contributions to early post-transplant donor-reactive endogenous memory T cell activation and CTLA-4Ig resistant rejection. Plasma cf-mtDNA was significantly elevated in 8hr versus 0.5hr CIS allograft recipients as early as 12hr post-transplant. Flow cytometry analysis of graft infiltrating leukocytes showed reduced memory CD4 and CD8 T cell proliferation on D2 following 8hr CIS in TLR9KO compared to WT allografts. In contrast, graft TLR9KO did not impact the de novo donor-reactive T cell response. 8hr CIS TLR9KO and WT allografts had similar survival in untreated recipients (MST 7.5 vs 7 days) and there was no difference in IFN-γ producing donor-reactive T cells in recipient spleens on D7. When recipients of allografts subjected to 8hr CIS were conditioned with CTLA-4Ig to inhibit de novo T cell priming, graft TLR9KO significantly prolonged graft survival compared to WT (MST 59 vs 28 days). These findings indicate that IRI-induced cf-mtDNA release and TLR9 activation are novel targets for abrogating CTLA-4Ig resistant donor-reactive memory T cell activation within high-risk allografts. This work was supported by NIH grants R01AI40459, T32AI089474, and T32GM007250.

Deleting Runx3 in CD4+ T Cells Disrupts Effector Functions and Facilitates Tolerance Induction in Solid Organ Transplantation

Abstract Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.

(1012) The Role of High-Sensitive Troponin in Identifying Patients with Cardiac Allograft Rejection

(1012) The Role of High-Sensitive Troponin in Identifying Patients with Cardiac Allograft Rejection

(999) Validation of the Clinical Utility of MicroRNA as Non-Invasive Biomarkers of Cardiac Allograft Rejection Monitoring: A Prospective Longitudinal Multicenter Study

(999) Validation of the Clinical Utility of MicroRNA as Non-Invasive Biomarkers of Cardiac Allograft Rejection Monitoring: A Prospective Longitudinal Multicenter Study

(553) Cardiac Allograft Rejection Surveillance with Donor Derived Cell Free Dna is a Reliable Non-Invasive Alternative to Endomyocardial Biopsy

(553) Cardiac Allograft Rejection Surveillance with Donor Derived Cell Free Dna is a Reliable Non-Invasive Alternative to Endomyocardial Biopsy

Non-invasive detection of cardiac allograft rejection among heart transplant recipients using an electrocardiogram based deep learning model.

Current non-invasive screening methods for cardiac allograft rejection have shown limited discrimination and are yet to be broadly integrated into heart transplant care. Given electrocardiogram (ECG) changes have been reported with severe cardiac allograft rejection, this study aimed to develop a deep-learning model, a form of artificial intelligence, to detect allograft rejection using the 12-lead ECG (AI-ECG). Heart transplant recipients were identified across three Mayo Clinic sites between 1998 and 2021. Twelve-lead digital ECG data and endomyocardial biopsy results were extracted from medical records. Allograft rejection was defined as moderate or severe acute cellular rejection (ACR) based on International Society for Heart and Lung Transplantation guidelines. The extracted data (7590 unique ECG-biopsy pairs, belonging to 1427 patients) was partitioned into training (80%), validation (10%), and test sets (10%) such that each patient was included in only one partition. Model performance metrics were based on the test set (n = 140 patients; 758 ECG-biopsy pairs). The AI-ECG detected ACR with an area under the receiver operating curve (AUC) of 0.84 [95% confidence interval (CI): 0.78-0.90] and 95% (19/20; 95% CI: 75-100%) sensitivity. A prospective proof-of-concept screening study (n = 56; 97 ECG-biopsy pairs) showed the AI-ECG detected ACR with AUC = 0.78 (95% CI: 0.61-0.96) and 100% (2/2; 95% CI: 16-100%) sensitivity. An AI-ECG model is effective for detection of moderate-to-severe ACR in heart transplant recipients. Our findings could improve transplant care by providing a rapid, non-invasive, and potentially remote screening option for cardiac allograft function.

Electrospun polymer fibers modified with FK506 for the long-term treatment of acute cardiac allograft rejection in a heart transplantation model.

FK506, a first-line immunosuppressant, is routinely administered orally and intravenously following heart transplantation. However, frequent administration can result in a substantial psychological burden to patients, resulting in non-adherence to medication. The purpose of our study is to overcome the disadvantages of systemic drug administration by developing a polymer-based delivery system that is tunable and biodegradable and that can release highly hydrophobic FK506 over extended periods to treat or prevent acute cardiac allograft rejection. Using an electrospinning method, long-acting microfibers were prepared, and FK506 appeared to be continuously released for up to 14 days based on the in vitro release profiles. After implanting the microfiber subcutaneously into the abdominals of transplanted rats, it was found that the infiltration of T cells and macrophages and the secretion of interleukin-2 (IL-2) and IL-1β were significantly reduced compared with those of the free FK506 groups. More importantly, the mean survival time (MST) of the PCL-FK506 group was significantly extended in comparison with that of untreated control recipients and free FK506 (MST of untreated control recipients, free FK506, and PCL-FK506 was 8, 26.1, and 37, respectively). In conclusion, we propose that this drug delivery approach would be suitable for developing long-lasting immunomodulatory agents that prolong cardiac graft survival safely and effectively.

Combination prophylaxis in CMV high risk heart transplant recipients: A single center experience.

Cytomegalovirus (CMV) is the most clinically relevant infectious agent following heart transplantation (HTX). Data on the beneficial effects of prophylactic use of CMV immunoglobulins (CMVIG) are scarce. In this single-center, retrospective study, we reported patient outcomes following cardiac transplantation using prophylactic CMV treatment, including CMVIG. Distinct clinically relevant outcomes were compared across different CMV risk groups (CMV D-/R-, CMV D-/R+, CMV D+/R+, and CMV D+/R- or CMV high risk group). We included 272 heart transplant procedures, performed between 1/1/2009 and 1/11/2020. Sixty-one (22%) procedures belonged to the CMV high risk group, while 96 (35%), 50 (18%), and 65 (24%) were CMV D-/R-, CMV D-/R+, and CMV D+/R+, respectively. Baseline donor and recipient characteristics (sex, age, body mass index, cause of death, indication for HTX), ischemia times and baseline immunosuppressive regimens were similar across the different CMV risk groups, yet fewer patients were bridged with a mechanical circulatory support in the CMV D+/R- group. CMV disease following cardiac transplantation was more common in the CMV D+/R- risk group (n=40 or 66.7%; p<.001), yet mortality and re-transplantation rates, cardiac allograft vasculopathy (CAV) severity, rejection episodes, and development of donor-specific antibodies (DSA), post-transplant lymphoproliferative diseases (PTLD), and EBV infections were similar across all four CMV risk groups. High risk CMV D+/R- patients had a similar survival compared to low and intermediate CMV risk groups using a prophylactic strategy combining CMVIG and viral DNA polymerase inhibitors. This may be related to a number of factors unrelated to prophylaxis strategy as two out of three CMV D+/R- recipients developed CMV primary infection after prophylaxis was discontinued.

Targeting IL-6 to prevent cardiac allograft rejection

Outcomes following heart transplantation remain suboptimal with acute and chronic rejection being major contributors to poor long-term survival. IL-6 is increasingly recognized as a critical pro-inflammatory cytokine involved in allograft injury and has been shown to play a key role in regulating the inflammatory and alloimmune responses following heart transplantation. Therapies that inhibit IL-6 signaling have emerged as promising strategies to prevent allograft rejection. Here, we review experimental and pre-clinical evidence that supports the potential use of IL-6 signaling blockade to improve outcomes in heart transplant recipients.