Allograft Injury Research Articles

Combination of everolimus and low-dose tacrolimus controls histological liver allograft injury as sufficiently as high-dose tacrolimus.

The combination of everolimus (EVR) and low-dose tacrolimus (lowTAC) prevents T cell-mediated rejection of liver grafts as sufficiently as high-dose tacrolimus (highTAC) and mycophenolate, but is associated with a preserved kidney function within the first years after orthotopic liver transplantation (OLT). However, none of the available studies assessed the histological pattern of graft injury or fibrosis in surveillance biopsies (svLbx). All svLbx taken under at least one month of stable immunosuppression with either EVR (aim 3-8 ng/ml) combined with lowTAC (aim 3-5 ng/ml) or highTAC (aim 5-8 ng/ml) combined with mycophenolate (500-1500 mg/day) within the first three to four years after OLT at our center were included. Patients who were switched to EVR because of insufficient control of alloreactivity were excluded. Reasons for switches to EVR were mainly malignancies before or after OLT, or chronic kidney injury. We were able to include 20 svLbx with EVR/lowTAC and 49 with highTAC/mycophenolate. Both groups had similar liver enzymes and similar kidney function. The EVR/lowTAC group exhibited lower TAC trough levels at svLbx (4.4 vs. 6.6 ng/ml; p<.001) in comparison to highTAC/mycophenolate. Histological graft injury quantified by the rejection activity index and hepatitis activity index (Ishak), as well as fibrosis were not significantly different between the EVR/lowTAC and highTAC/mycophenolate groups. Likewise, subclinical TCMR, histological criteria justifying immunosuppression minimization, and steatosis had equal prevalence in both regimens. Immunosuppression was adjusted according to the svLbx findings. Immunosuppression regimens had similarly low rates of rejection after immunosuppression reduction, when relevant graft injury was absent in the biopsy. In conclusion, EVR/lowTAC seems to control alloreactivity and histological graft injury as sufficiently as highTAC/mycophenolate within the first 3-4 years after OLT.

Investigative and laboratory assays for allogeneic rejection – A clinical perspective

How to translate basic research on surveillances for post organs transplantation facilitates the development of new laboratory tools for clinical application. Here, we provide the pertinent guide on how to analyze laboratory data in order to help clinicians to accurately determine the status of allograft in the recipient. We begin by discussing the molecular and cellular events that lead to allograft rejection across innate and adaptive immune responses. We focus on the allograft injury events that occur around the time of transplantation as well as molecular activities that ensue long before observable microscopic abnormalities are evident in biopsied samples. Then, we introduce and comment on new technologies involved in rejection detection from a practical point of view. This review outlines the current diagnostic development, describes unmet needs and challenges, and proposes new approaches for future directions.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

(1186) Donor Derived Cell Free Dna after Lung Transplantation: Marker for Acute and Chronic Lung Allograft Injury?

(1186) Donor Derived Cell Free Dna after Lung Transplantation: Marker for Acute and Chronic Lung Allograft Injury?

(152) Metabolomic Profiling During Ex-Vivo Normothermic Perfusion Prior to Heart Transplantation Defines Patterns of Substrate Utilization and Correlates with Markers of Allograft Injury

(152) Metabolomic Profiling During Ex-Vivo Normothermic Perfusion Prior to Heart Transplantation Defines Patterns of Substrate Utilization and Correlates with Markers of Allograft Injury

(1210) Total Cell-Free Dna May Reflect Non-Rejection Causes of Lung Allograft Injury

(1210) Total Cell-Free Dna May Reflect Non-Rejection Causes of Lung Allograft Injury

BAL Fluid Eosinophilia Associates With Chronic Lung Allograft Dysfunction Risk: A Multicenter Study

Chronic lung allograft dysfunction (CLAD) is the leading cause of death among lung transplant recipients. Eosinophils, effector cells of type 2 immunity, are implicated in the pathobiology of many lung diseases, and prior studies suggest their presence associates with acute rejection or CLAD after lung transplantation. Does histologic allograft injury or respiratory microbiology correlate with the presence of eosinophils in BAL fluid (BALF)? Does early posttransplant BALF eosinophilia associate with future CLAD development, including after adjustment for other known risk factors? We analyzed BALF cell count, microbiology, and biopsy data from a multicenter cohort of 531 lung recipients with 2,592 bronchoscopies over the first posttransplant year. Generalized estimating equation models were used to examine the correlation of allograft histology or BALF microbiology with the presence of BALF eosinophils. Multivariable Cox regression was used to determine the association between≥ 1%BALF eosinophils in the first posttransplant year and definite CLAD. Expression of eosinophil-relevant genes was quantified in CLAD and transplant control tissues. The odds of BALF eosinophils being present was significantly higher at the time of acute rejection and nonrejection lung injury histologies and during pulmonary fungal detection. Early posttransplant≥ 1%BALF eosinophils significantly and independently increased the risk for definite CLAD development (adjusted hazard ratio, 2.04; P= .009). Tissue expression of eotaxins, IL-13-related genes, and the epithelial-derived cytokines IL-33 and thymic stromal lymphoprotein were significantly increased in CLAD. BALF eosinophilia was an independent predictor of future CLAD risk across a multicenter lung recipient cohort. Additionally, type 2 inflammatory signals were induced in established CLAD. These data underscore the need for mechanistic and clinical studies to clarify the role of type 2 pathway-specific interventions in CLAD prevention or treatment.

Donor-Derived Cell-Free DNA for Kidney Allograft Surveillance after Conversion to Belatacept: Prospective Pilot Study

Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m2, p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection-one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA.

Early posttransplant reductions in club cell secretory protein associate with future risk for chronic allograft dysfunction in lung recipients: results from a multicenter study

Chronic lung allograft dysfunction (CLAD) increases morbidity and mortality for lung transplant recipients. Club cell secretory protein (CCSP), produced by airway club cells, is reduced in the bronchoalveolar lavage fluid (BALF) of lung recipients with CLAD. We sought to understand the relationship between BALF CCSP and early posttransplant allograft injury and determine if early posttransplant BALF CCSP reductions indicate later CLAD risk. We quantified CCSP and total protein in 1606 BALF samples collected over the first posttransplant year from 392 adult lung recipients at 5 centers. Generalized estimating equation models were used to examine the correlation of allograft histology or infection events with protein-normalized BALF CCSP. We performed multivariable Cox regression to determine the association between a time-dependent binary indicator of normalized BALF CCSP level below the median in the first posttransplant year and development of probable CLAD. Normalized BALF CCSP concentrations were 19% to 48% lower among samples corresponding to histological allograft injury as compared with healthy samples. Patients who experienced any occurrence of a normalized BALF CCSP level below the median over the first posttransplant year had a significant increase in probable CLAD risk independent of other factors previously linked to CLAD (adjusted hazard ratio 1.95; p=0.035). We discovered a threshold for reduced BALF CCSP to discriminate future CLAD risk; supporting the utility of BALF CCSP as a tool for early posttransplant risk stratification. Additionally, our finding that low CCSP associates with future CLAD underscores a role for club cell injury in CLAD pathobiology.

Clinical features and allograft failure rates of pulmonary antibody-mediated rejection categories.

Pulmonary antibody-mediated rejection (AMR) consensus criteria categorize AMR by diagnostic certainty. This study aims to define the clinical features and associated outcomes of these recently defined AMR categories. Adjudication committees reviewed clinical data of 335 lung transplant recipients to define clinical or subclinical AMR based on the presence of allograft dysfunction, and the primary endpoints, time from transplant to allograft failure, a composite endpoint of chronic lung allograft dysfunction and/or death. Clinical AMR was subcategorized based on diagnostic certainty as definite, probable or possible AMR if 4, 3, or 2 characteristic features were present, respectively. Allograft injury was assessed via plasma donor-derived cell-free DNA (ddcfDNA). Risk of allograft failure and allograft injury was compared for AMR categories using regression models. Over the 38.5 months follow-up, 28.7% of subjects developed clinical AMR (n=96), 18.5% developed subclinical AMR (n=62) or 58.3% were no AMR (n=177). Clinical AMR showed higher risk of allograft failure and ddcfDNA levels compared to subclinical or no AMR. Clinical AMR included definite/probable (n=21) or possible AMR (n=75). These subcategories showed similar clinical characteristics, ddcfDNA levels, and risk of allograft failure. However, definite/probable AMR showed greater measures of AMR severity, including degree of allograft dysfunction and risk of death compared to possible AMR. Clinical AMR showed greater risk of allograft failure than subclinical AMR or no AMR. Subcategorization of clinical AMR based on diagnostic certainty correlated with AMR severity and risk of death, but not with the risk of allograft failure.

Application of plasma donor-derived cell free DNA for lung allograft rejection diagnosis in lung transplant recipients

BackgroundDonor-derived cell-free DNA (dd-cfDNA) has been applied to monitor acute rejection (AR) in kidney and heart transplantation. This study was aimed to investigate the application of dd-cfDNA levels in the diagnosis of AR and chronic lung allograft dysfunction (CLAD) among the lung transplantation recipients (LTRs).MethodsOne hundred and seventy LTRs were enrolled at the First Affiliated Hospital of Guangzhou Medical University between 1 June 2015 and 30 March 2021. Patients were divided into 4 groups: stable group, AR group, infection group and CLAD group. The level of dd-cfDNA was analyzed using target region sequencing and the performance characteristics of dd-cfDNA for diagnosis of AR and CLAD were determined, respectively.ResultsKruskal–Wallis test showed that there were some significant differences in the level of dd-cfDNA (%) among the 4 groups, with p < 0.001. Among them, the level of dd-cfDNA (%) was highest (median 2.17, IQR [1.40–3.82]) in AR group, and higher in CLAD group (median 1.07, IQR [0.98–1.31]), but lower in infection group (median 0.71, IQR [0.57–1.07]) and lowest in stable group (median 0.71, IQR [0.61–0.84]). AUC-ROC curve analysis showed that the threshold of dd-cfDNA for AR was 1.17%, with sensitivity being 89.19% and specificity being 86.47%, and the optimal threshold of 0.89% was determined of CLAD, with sensitivity being 95.00% and specificity of 76.99%.ConclusionsPlasma dd-cfDNA could be a useful tool for the assessment of lung allograft rejection, including AR and CLAD, and holds promise as a noninvasive biomarker for “allograft injury” in both acute and chronic rejection following lung transplantation.

Donor and recipient human leukocyte antigen-G polymorphisms modulate the risk of adverse immunologic events following lung transplantation

The long-term benefits of lung transplantation (LTx) are limited by pathogenic alloimmune responses that drive injury, inflammation, and chronic dysfunction. Human leukocyte antigen-G (HLA-G) plays a key role in the modulation of these pathways. This study assesses the impact of the HLA-G genotype on immunologic risk and survival following LTx.This retrospective cohort study included 289 bilateral LTx. Recipient and donor HLA-G genotypes were analyzed to identify associations with de novo donor-specific antibodies, acute rejection, chronic lung allograft dysfunction, and allograft survival. We further assessed these associations, both individually and in paired analysis, based on a grouped haplotype classification of HLA-G expression.Donor HLA-G single nucleotide polymorphisms were associated with allograft injury, the onset of chronic lung allograft dysfunction following injury, and allograft survival. Recipient HLA-G single nucleotide polymorphisms were associated with allograft injury, cellular rejection, and donor-specific antibody formation. “Low HLA-G expression” donor haplotypes were associated with impaired allograft survival, as were “low HLA-G expression” donor-recipient haplotype pairs.This study provides compelling evidence for the role of HLA-G in modulating immunologic risk after LTx. Our results highlight the importance of both donor and recipient HLA-G genotypes on the overall risk profile and underscore the lasting influence of donor genotype on lung transplant outcomes.

Diarrhea after kidney transplantation: A study of risk factors and outcomes.

Diarrhea in kidney transplant recipients (KTRs) can be associated with significant morbidity. We evaluated 198 KTRs for a history of diarrhea post-kidney transplant at a tertiary care center in western India over 1 year. A protocol-based evaluation of diarrhea was done with respect to clinical features, diagnostic evaluation, associated acute allograft dysfunction, and its impact on long-term allograft function. Primary outcomes of interest were: chronic allograft injury (CAI) and the need for mycophenolate mofetil (MMF) withdrawal. We also assessed the effect of MMF withdrawal on the risk of the development of CAI. Eighty-five of 198 (42.5%) recipients experienced diarrhea and a total of 140 diarrheal episodes were evaluated. The mean age of these 85 recipients was 38 ± 12 years and 72 (84.7%) were males. 73 of 85 recipients were on MMF at the time of diarrhea and in 35 (48%) of them MMF withdrawal was needed for chronic and persistent symptoms. Diarrhea was attributed to infective etiologies in 90 of 140 (64.2%) cases. Among the microbiologically confirmed infective diarrheal episodes, giardia and cryptosporidium were the common pathogens in 11/28 (39%) and 6/28 (21.4%) episodes respectively. One hundred and twenty-eight episodes out of 140 (91.4%) episodes were complicated by acute allograft dysfunction. Forty-one of 85 recipients (48.2%) developed chronic allograft injury and 12 (14.1%) developed allograft rejection (acute and/or chronic). Probability of chronic allograft injury was higher in those with MMF withdrawal. Diarrhea post-kidney transplant adversely affects graft function, especially after MMF withdrawal.

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.

STXBP3 and GOT2 predict immunological activity in acute allograft rejection.

Acute allograft rejection (AR) following renal transplantation contributes to chronic rejection and allograft dysfunction. The current diagnosis of AR remains dependent on renal allograft biopsy which cannot immediately detect renal allograft injury in the presence of AR. In this study, sensitive biomarkers for AR diagnosis were investigated and developed to protect renal function. We analyzed pre- and postoperative data from five databases combined with our own data to identify the key differently expressed genes (DEGs). Furthermore, we performed a bioinformatics analysis to determine the immune characteristics of DEGs. The expression of key DEGs was further confirmed using the real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemical (IHC) staining in patients with AR. ROC curves analysis was used to estimate the performance of key DEGs in the early diagnosis of AR. We identified glutamic-oxaloacetic transaminase 2 (GOT2) and syntaxin binding protein 3 (STXBP3) as key DEGs. The higher expression of STXBP3 and GOT2 in patients with AR was confirmed using RT-qPCR, ELISA, and IHC staining. ROC curve analysis also showed favorable values of STXBP3 and GOT2 for the diagnosis of early stage AR. STXBP3 and GOT2 could reflect the immunological status of patients with AR and have strong potential for the diagnosis of early-stage AR.

Has the Time Come for Widespread MHC Class I MICA Genotyping in Solid Organ Transplantation?

Has the Time Come for Widespread MHC Class I MICA Genotyping in Solid Organ Transplantation?

Antibiotic ampicillin induces immune tolerance in renal transplantation by regulating the proportion of intestinal flora in mice.

There are significant differences in the composition of intestinal flora in renal transplant recipients before and after an operation, which has a great impact on the prognosis of renal transplantation. The purpose of this project is to study the effect of intestinal flora imbalance on renal transplantation. The animal model of renal transplantation was established after intestinal flora imbalance (mice pretreated with compound antibiotics), or the animal model of renal transplantation was established after being pretreated with single antibiotics. HE, PAS, and Masson staining was used to detecting the histopathological changes of transplanted renal. The expression of inflammatory factors and infiltration of inflammatory cells of renal tissue were respectively been detected by ELISA kit and flow cytometry. Antibiotic pretreatment restored weight loss, and decreased serum creatinine level in mice after renal transplantation. The tissue staining, ELISA assay, and flow cytometry data showed that antibiotic pretreatment alleviated injury of the renal allograft, inhibited the inflammatory factors levels, and reduced inflammatory cell infiltration in mice after renal transplantation. Furthermore, single antibiotic, especially ampicillin pretreatment can also play the same role as compound antibiotics, such as restoring weight loss, decreasing serum creatinine level, alleviating renal allograft injury, inhibiting inflammatory factors levels, and reducing inflammatory cell infiltration in mice after renal transplantation. Antibiotic ampicillin may inhibit inflammatory cell infiltration after renal transplantation by regulating the proportion of intestinal flora in mice, to reduce renal injury and play a role in renal protection.

Assessment of dd-cfDNA Levels in Clinically Stable Lung Allograft Recipients Beyond the Initial 2 y Posttransplant.

We performed a prospective, single-center, observational study to identify plasma dd-cfDNA levels in clinically stable lung allograft recipients >2 y posttransplant. Fifty-one subjects were enrolled and ≥3 baseline dd-cfDNA measurements were acquired during a median of 252 d. The median baseline percent dd-cfDNA level in our cohort was 0.45% (interquartile range [IQR], 0.26-0.69). There were statistically significant differences in dd-cfDNA based on posttransplant duration (≤5 y posttransplant median 0.41% [IQR, 0.21-0.64] versus >5 y posttransplant median 0.50% [IQR, 0.33-0.76]; P < 0.02). However, the clinical significance of this small change in dd-cfDNA is uncertain because this magnitude of change is within the biologic test variation of 73%. This study is the first to define levels of dd-cfDNA in clinically stable patients who are >2 y post-lung transplant. These findings lay the groundwork for the study of dd-cfDNA as a possible biomarker for CLAD.

Proteomic study on the lymphocytes from pregnant Wistar rat females treated with immunosuppressive regimen.

Kidney transplantation remains the therapeutic option for patients with end-stage kidney disease. Current immunosuppressive regimens are efficient in combating acute kidney rejection. However, insights into chronic kidney allograft injury remains limited. Simultaneously, pregnancy is more common after kidney transplantation than during dialysis treatment. Due to ethical issues, comprehensive studies on the impact of immunosuppressive regimens on pregnancy are challenging. The study aimed to investigate the proteomic status of lymphocytes obtained from pregnant female rats under immunosuppressive treatment. The experiment involved a group of 10 female, pregnant Wistar rats, five of which were treated with tacrolimus, mofetil mycophenolate, and glucocorticosteroids; five were used as control. The lymphocytes were obtained and analyzed with mass spectrometry. Measurements were processed by a database search in the ProteinPilot software with a cutoff of 1% false discovery rate. The outcomes were verified statistically by a t-test (p value < 0.05) regarding proteins up- and downregulation. A total of 2082 proteins were identified in all experiments. Eight hundred five proteins were quantified in an absolute manner in a data-independent acquisition-total protein approach analysis. Ninety-five proteins were recognized as present at different concentrations in analyzed groups and were annotated to intracellular pathways. The proteins involved in nonsense-mediated decay and L13a-mediated translational silencing of ceruloplasmin expression were recognized as downregulated. The set of proteins clinically identified as acute phase proteins was upregulated. Despite the blockade of adaptive cellular immunity, the lymphocytes in the analyzed group reveal sustained proinflammatory status with decreased ability to regulate translation. This potentially affects pregnancy and immunity.

Abstract 9800: Racial Differences in Donor-Derived and Mitochondrial Cell-Free Dna Could Explain Inferior Outcomes in Black Heart Transplant Patients, on Behalf of the Graft Investigators

Introduction: Black heart transplant patients are at higher risk of acute rejection (AR) and death than White patients. Our group has shown that %donor-derived cell-free DNA (%ddcfDNA) is a sensitive and specific marker of AR in heart transplantation. Cell-free mitochondrial DNA (cfmtDNA) has been linked to allograft dysfunction and may mechanistically implicated in inferior outcomes for Black heart transplant recipients. Hypothesis: Higher levels of allograft injury when quantitated using %ddcfDNA and total cfmtDNA will be seen in Black heart transplant patients as compared to White patients. Methods: The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter, prospective, longitudinal cohort study of heart transplant patients transplanted between 2015 and 2021. We included Black and White patients and shotgun sequencing was used to quantitate %ddcfDNA and polymerase chain reaction (PCR) to quantitate total cfmtDNA levels. AR was defined as grade ≥2R cellular rejection or pAMR ≥1 antibody-mediated rejection (AMR). Results: We included 151 patients (73 Blacks and 78 Whites), the median age was 56y (29% Female sex). Patients of both racial groups had comparable baseline characteristics. There were a ~1100 ddcfDNA and cfmtDNA measures included in the analysis. At 18-months, the incidence of AR was 2.8% in White patients and 9.2% in Black patients (p&lt;0.01). The diagnostic accuracy of %ddcfDNA to detect AR was comparable between Black and White patients (0.90 vs. 0.83, p=0.19). The %ddcfDNA and cfmtDNA was higher at serial time points after transplant in Black compared to White patients (Figure). CfmtDNA levels were also elevated with AMR which occurred almost exclusively in Black patients (No rejection: 146,345 copies/ul, AMR: 242,238 copies/ul; p=0.004). Conclusions: Elevated levels of %ddcfDNA and cfmtDNA after heart transplant may explain the higher incidence of AR, AMR, and inferior outcomes in Black heart transplant recipients.