Development and clinical validity of a novel blood‐based molecular biomarker for subclinical acute rejection following kidney transplant

Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead to chronic rejection and graft loss. We conducted a multicenter study to develop a blood‐based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%‐88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%‐61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy‐proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor‐specific antibodies. We also found that <50% showed histologic improvement of subAR on follow‐up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood‐based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.     

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus‐based immunosuppression with basiliximab induction

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non‐rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy‐proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL‐2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first‐week peripheral blood Treg frequency correlates with AR after LT under tacrolimus‐based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.     

Sinusoidal obstruction syndrome as a manifestation of acute antibody-mediated rejection after liver transplantation

Antibody-mediated rejection (AMR) after liver transplantation is uncommon but, when present, manifests as graft dysfunction. We report the case of a 54-year-old woman who developed portal hypertension with pleural effusion and ascites secondary to sinusoidal obstruction syndrome (SOS) due to acute AMR following an ABO-matched liver transplantation for autoimmune cirrhosis and hepatocellular carcinoma. Initial immunosuppression comprised basiliximab, decreasing prednisone, tacrolimus, and mycophenolate mofetil. After 1 month, she presented with the massive pleural effusion, slight ascites, and normal liver tests. After excluding common causes of pleural effusion, we performed a liver biopsy that showed atypical rejection with the involvement of large centrilobular veins partially occluded by marked endotheliitis and lax fibrosis suggestive of SOS. Direct immunofluorescence study of C4d showed diffuse endothelial sinusoidal staining, and de novo donor-specific anti-human leukocyte antigen antibodies were detected in his blood. Thus, we diagnosed AMR focused on centrilobular veins and initiated treatment with defibrotide, steroid pulses, and diuretics. However, this was ineffective, and the pleural effusion only resolved when plasmapheresis and intravenous immunoglobulin were started. This case shows that AMR can cause SOS with portal hypertension and present with a pleural effusion, and as such, it should be suspected after excluding other more common causes of effusion.     

Outcomes following liver transplantation in young infants: Data from the SPLIT registry

Liver transplantation (LT) in young patients is being performed with greater frequency. We hypothesized that objective analysis of pre-, intra-, and postoperative events would help understand contributors to successful outcomes and guide transplant decision processes. We queried SPLIT registry for pediatric transplants between 2011 and 2018. Outcomes were compared for age groups: 0-<3, 3-<6, 6-<12 months, and 1-<3 years (Groups A, B, C, D respectively) and by weight categories: <5, 5-10, >10 kg; 1033 patients were available for analysis. Cholestatic disease and fulminant failure were highest in group A and those <5 kg; and biliary atresia in group C (72.8%). Group A had significantly higher life support dependence (34.6%; P < .001), listing as United Network for Organ Sharing status 1a/1b (70.4%; P < .001), and shortest wait times (P < .001). The median (interquartile range) for international normalized ratio and bilirubin were highest in group A (3.0 [2.1-3.9] and 16.7 [6.8-29.7] mg/dL) and those <5 kg (2.6 [1.8-3.4] and 13.5 [3.0-28.4] mg/dL). A pediatric end -stage liver disease score ≥40, postoperative hospital stays, rejection, and nonanastomotic biliary strictures were highest in group A with lowest survival at 93.1%. Infants 0 to <3 months and those <5 kg need more intensive care with lower survival and higher complications. Importantly, potential LT before reaching status 1a/1b and aggressive postoperative management may positively influence their outcomes.     

Angiotensin II type I receptor agonistic autoantibodies are associated with poor allograft survival in liver retransplantation

Angiotensin II type I receptor (AT1R) agonistic autoantibodies (AT1R‐AA) are detrimental to kidney transplantation. Early studies suggested a similar negative effect in primary liver transplantation. Here, we studied AT1R‐AA in a retrospective cohort of 94 patients who received a second liver transplant to determine their prevalence and effects. The concentrations of preformed AT1R‐AA before transplantation were higher (P = .019) in the 48 patients who lost their liver grafts than in the 46 patients whose grafts survived. About half (48/94, 51.1%) of the patients were positive for AT1R‐AA >17 U/mL before the second liver transplantation. In 22 (23.4%) patients, strong positive AT1R‐AA (defined as >40 U/mL) were detected, of whom 16 (72.7%) patients lost their grafts. Based on Kaplan‐Meier analysis, patients with strong positive AT1R‐AA had significantly worse graft survival than those with AT1R‐AA <40 U/mL (P = .035). In multivariate Cox models that included confounders such as sex and age, either AT1R‐AA >40 U/mL (HR = 1.999 [1.085‐3.682], P = .026) or increased concentrations of AT1R‐AA (HR = 1.003 [1.001‐1.006] per incremental U/mL, P = .019) were significantly associated with elevated risk for graft loss. In conclusion, our data indicate that there is a high prevalence of AT1R‐AA in candidates for second liver transplantation and that their presence is associated with inferior long‐term outcomes of the second graft.     

Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: A scoping review

Indefinite allograft acceptance after immunosuppression withdrawal (ISW), also known as operational tolerance (OT), can occur spontaneously after liver transplantation (LT), but reliable and reproducible prognosis of OT versus non-OT outcomes remains elusive. To prime this, systematic extraction of OT-predictive factors from the literature is crucial. We provide the first comprehensive identification and synthesis of clinical parameters and biomarkers predicting spontaneous OT in non-autoimmune/non-replicative viral LT recipients selected for ISW. We searched Embase, Medline, the Cochrane Central Register of Controlled Trials, clinicaltrials.gov, and the World Health Organization International Clinical Trials Registry Platform for articles, conference abstracts, and ongoing trials. We contacted principal investigators of stand-alone abstracts and ongoing trials for unpublished data and screened citations and references of eligible articles. Twenty-three articles reporting on 11 completed ISW studies, 13 abstracts, and five trial registry entries were included. Longer time between LT and ISW was the only clinical parameter that may increase the incidence of OT. Prognostic biomarkers conspicuously differed between pediatric and adult ISW candidates. These included allograft gene expression patterns and peripheral blood immune exhaustion markers for adults, and histological allograft scores for children. Our results will foster cross-validation efforts to facilitate safe and harmonized candidate selection for successful ISW.     

Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent “top‐down” proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight–based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high‐resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum‐rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.     

Donor‐Specific HLA Antibodies in Living Versus Deceased Donor Liver Transplant Recipients

With less ischemia, improved donor selection and controlled procedures, living donor liver transplantation (LDLT) might lead to less HLA donor‐specific antibody (DSA) formation or fewer adverse outcomes than deceased donor liver transplantation (DDLT). Using the multicenter A2ALL (Adult‐to‐Adult Living Donor Liver Transplantation Cohort Study) biorepository, we compared the incidence and outcomes of preformed and de novo DSAs between LDLT and DDLT. In total, 129 LDLT and 66 DDLT recipients were identified as having serial samples. The prevalence of preformed and de novo DSAs was not different between DDLT and LDLT recipients (p = 0.93). There was no association between patient survival and the timing (preformed vs. de novo), class (I vs. II) and relative levels of DSA between the groups; however, preformed DSA was associated with higher graft failure only in DDLT recipients (p = 0.01). De novo DSA was associated with graft failure regardless of liver transplant type (p = 0.005) but with rejection only in DDLT (p = 0.0001). On multivariate analysis, DSA was an independent risk factor for graft failure regardless of liver transplant type (p = 0.017, preformed; p = 0.002, de novo). In conclusion, although similar in prevalence, DSA may have more impact in DDLT than LDLT recipients. Although our findings need further validation, future research should more robustly test the effect of donor type and strategies to mitigate the impact of DSA.     

Outcomes of liver transplantation for acute fatty liver disease of pregnancy

Acute fatty liver of pregnancy (AFLP) often resolves after pregnancy delivery but can progress to acute liver failure necessitating liver transplantation. We performed a retrospective review of the national Scientific Registry of Transplant Recipients (SRTR) data to identify all women in the United States undergoing liver transplantation (LT) for acute liver failure (ALF) from AFLP from 1991 to 2015, and compared to outcomes in women of childbearing age undergoing transplant for ALF from acetaminophen and ALF from other etiologies. Women with AFLP were likely to be on life support at time of LT and had high rates of renal dysfunction (median Cr 2.1, IQR 1.2‐2.3), and hyperbilirubinemia (median bilirubin 17.1, IQR 11.0, 19.9). Although their early and late LT survival outcomes were comparable to the other indications for LT, cumulative 5‐year graft survival was numerically lower among AFLP patients (54%, 95% CI, 27‐76) compared to APAP (70%, 95% CI, 63‐77) and “Other ALF” (76%, 95% CI, 72‐80) groups. In conclusion, although AFLP is a rare indication for LT, AFLP patients were as sick or sicker than other women of childbearing age undergoing LT for ALF. Worsened graft survival may be related to higher rates of rejection in the AFLP group.     

Subclinical biliary strictures as a cause of long-term allograft dysfunction in children who underwent liver transplantation

We aimed to evaluate the role of liver biopsy to predict subclinical biliary strictures (BS) and assess the impact of BS on long-term allograft dysfunction following liver transplantation in children (LT). We reviewed all liver biopsies performed from 2012-2018. Percutaneous transhepatic cholangiography (PTC) was performed in patients presenting cholangiolar proliferation on cytokeratin-7 stained sections. We performed 271 biopsies in 161 children (86% with a left lateral segment); 44/161 (27%) presented with diffuse or multifocal cholangiolar proliferation. Among them, a tight BS was confirmed in 38/44 (86%, 24% of total) and it was managed by balloon dilatation. Cholangiolar proliferation showed a positive predictive value (PPV) for BS of 86.4%. Levels of alkaline phosphatase >325 IU/L predicted BS (P = .007). Dilatation of intrahepatic bile ducts on ultrasound was found only in 44% of patients with BS. Following a median follow-up of 9.2 years, only 15/38 (39%) patients resolved the BS. In conclusion subclinical BS is very common and probably underdiagnosed in these patients. Histological evidence of cholangiolar proliferation detectable by cytokeratin-7 immunostain should be preferred to liver function tests and ultrasound to suspect BS. BS in this setting should be regarded as a main cause of long-term allograft dysfunction.     

The first case of ischemia‐free organ transplantation in humans: A proof of concept

Ischemia and reperfusion injury (IRI) is an inevitable event in conventional organ transplant procedure and is associated with significant mortality and morbidity post‐transplantation. We hypothesize that IRI is avoidable if the blood supply for the organ is not stopped, thus resulting in optimal transplant outcomes. Here we described the first case of a novel procedure called ischemia‐free organ transplantation (IFOT) for patients with end‐stage liver disease. The liver graft with severe macrovesicular steatosis was donated from a 25‐year‐old man. The recipient was a 51‐year‐old man with decompensated liver cirrhosis and hepatocellular carcinoma. The graft was procured, preserved, and implanted under continuous normothermic machine perfusion. The recipient did not suffer post‐reperfusion syndrome or vasoplegia after revascularization of the allograft. The liver function test and histological study revealed minimal hepatocyte, biliary epithelium and vascular endothelium injury during preservation and post‐transplantation. The inflammatory cytokine levels were much lower in IFOT than those in conventional procedure. Key pathways involved in IRI were not activated after allograft revascularization. No rejection, or vascular or biliary complications occurred. The patient was discharged on day 18 post‐transplantation. This marks the first case of IFOT in humans, offering opportunities to optimize transplant outcomes and maximize donor organ utilization.     

Circulating T follicular helper cells are a biomarker of humoral alloreactivity and predict donor‐specific antibody formation after transplantation

Donor‐specific antibodies (DSAs) contribute to renal allograft loss. However, biomarkers to guide clinical management of DSA posttransplant or detect humoral alloimmune responses before alloantibodies develop are not available. Circulating T follicular helper (cTfh) cells are CD4⁺CXCR5⁺ Tfh‐like cells in the blood that have been associated with alloantibodies in transplant recipients, but whether they precede antibody formation for their evaluation as a predictive biomarker in transplant is unknown. To evaluate the ability of cTfh cells to predict DSA, we used murine transplant models to determine the temporal relationship between cTfh cells, germinal center formation, and DSA development. We observed that donor‐reactive CD4⁺CXCR5⁺ cTfh cells expand after allotransplant. These cTfh cells were equivalent to graft‐draining lymph node‐derived Tfh cells in their ability to provide B cell help for antibody production. cTfh cell expansion and differentiation into ICOS⁺PD‐1⁺ cells temporally correlated with germinal center alloreactivity and preceded the generation of DSAs in instances of modified and unmodified alloantibody formation. Importantly, delayed costimulation blockade initiated after the detection of ICOS⁺PD‐1⁺ cTfh cells prevented DSAs. These findings suggest that cTfh cells could serve as a biomarker for humoral alloreactivity before the detection of alloantibodies and inform therapeutic approaches to prevent DSAs.     

The use of induction therapy in liver transplantation is highly variable and is associated with posttransplant outcomes

The use of induction immunosuppression in liver transplantation (LT) remains controversial. This was a retrospective cohort study of adult, first‐time liver‐alone recipients (N = 69 349) at 114 US centers between 2005 and 2018 using data from the United Network for Organ Sharing. The comparative effectiveness of nondepleting and depleting induction (NDI and DI) was assessed. Overall, 27% of recipients received induction with 65.7% of the variance in the receipt of induction being attributed to transplant center alone. NDI and DI were associated with a lower risk of death/graft failure compared to no induction (adjusted hazard ratio 0.90 [95% confidence interval (CI): 0.86‐0.95] and 0.91 [95% CI: 0.85‐0.97], respectively; P < .001). In nondialysis recipients at the mean transplant estimated glomerular filtration rate (eGFR), NDI was associated with an adjusted gain in eGFR by 6 months of +3.8 mL/min per 1.73 m² and DI of +3.33 mL/min per 1.73 m² compared to no induction (P < .001). Recipients with lower eGFR at LT had greater predicted improvement in eGFR (interaction P < .001). Only NDI was associated with a reduced risk of acute rejection in the first year post‐LT (odds ratio 0.87, 95% CI: 0.8‐0.94). Significant variability in induction practices exists, with center being a major determinant. The absolute incremental benefits of NDI and DI over no induction were small.     

On the impact of hepatitis C virus and heterologous immunity on alloimmune responses following liver transplantation

Virus-induced heterologous immunity is considered a barrier to transplantation tolerance. Yet, hepatitis C (HCV)-infected liver transplant (LT) patients occasionally achieve operational tolerance. We investigated the mechanisms through which HCV infection modulates donor-specific T cell responses following LT and the influence of HCV eradication. We generated T cell lines from HCV-infected LT and non-LT patients before and after HCV eradication and quantified alloreactive responses using cell lines expressing single-HLA class-I antigens in the presence/absence of PD-1/CTLA-4 blockade. HCV-specific CD8⁺ T cells cross-reacted with allogeneic class-I HLA molecules. HCV-positive LT recipients exhibited a higher proportion of CD8⁺ T cells coexpressing inhibitory receptors (PD-1/CTLA4) than HCV-negative LT, and their expression correlated with CXCL10 plasma levels. This resulted in decreased antidonor and third-party proliferative responses, which were significantly reversed by HCV eradication. PD-1/CTLA-4 blockade increased the proportion of HCV-specific CD8⁺ T cells reacting against donor only before viral clearance. In conclusion, HCV infection results in the generation of HCV-specific CD8⁺ T cells capable of reacting against allogeneic HLA molecules. Following LT, this results in a PD-1/CTLA4-dependent decrease in alloimmune responses. Our findings challenge the notion that heterologous immunity is necessarily detrimental in LT and provide an explanation for the association between HCV eradication and immune-mediated allograft damage.     

Evidence for a rebalanced hemostatic system in pediatric liver transplantation: A prospective cohort study

In adults with end‐stage liver disease concurrent changes in pro‐ and antihemostatic pathways result in a rebalanced hemostasis. Children though, have a developing hemostatic system, different disease etiologies, and increased risk of thrombosis. This study aimed to assess the hemostatic state of children during and after liver transplantation. Serial blood samples were obtained from 20 children (≤16 years) undergoing primary liver transplantation (September 2017‐October 2018). Routine hemostasis tests, thrombomodulin‐modified thrombin generation, clot lysis times, and hemostatic proteins were measured. Reference values were established using an age‐matched control group of 30 children. Thrombocytopenia was present in study patients. Von Willebrand factors were doubled and ADAMTS13 levels decreased during and after transplantation up until day 30, when platelet count had normalized. Whereas prothrombin time and activated partial thromboplastin time were prolonged during transplantation, thrombin generation was within normal ranges, except during perioperative heparin administration. Fibrinogen, factor VIII levels, and clot lysis time were elevated up until day 30. In conclusion, children with end‐stage liver disease are in tight hemostatic balance. During transplantation a temporary heparin‐dependent hypocoagulable state is present, which rapidly converts to a hemostatic balance with distinct hypercoagulable features that persist until at least day 30. This hypercoagulable state may contribute to the risk of posttransplant thrombosis.     

Liver transplantation for hepatitis C virus (HCV) non‐viremic recipients with HCV viremic donors

In the context of organ shortage, the opioid epidemic, and effective direct‐acting antiviral (DAA) therapy for hepatitis C virus (HCV), more HCV‐infected donor organs may be used for liver transplantation. Current data regarding outcomes after donor‐derived HCV in previously non‐viremic liver transplant recipients are limited. Clinical data for adult liver transplant recipients with donor‐derived HCV infection from March 2017 to January 2018 at our institution were extracted from the medical record. Ten patients received livers from donors known to be infected with HCV based on positive nucleic acid testing. Seven had a prior diagnosis of HCV and were treated before liver transplantation. All recipients were non‐viremic at the time of transplantation. All 10 recipients derived hepatitis C infection from their donor and achieved sustained virologic response at 12 weeks posttreatment with DAA‐based regimens, with a median time from transplant to treatment initiation of 43 days (IQR 20–59). There have been no instances of graft loss or death, with median follow‐up of 380 days (IQR 263–434) posttransplant. Transplantation of HCV‐viremic livers into non‐viremic recipients results in acceptable short‐term outcomes. Such strategies may be used to expand the donor pool and increase access to liver transplantation.     

Renal Function in De Novo Liver Transplant Recipients Receiving Different Prolonged‐Release Tacrolimus Regimens—The DIAMOND Study

DIAMOND: multicenter, 24‐week, randomized trial investigating the effect of different once‐daily, prolonged‐release tacrolimus dosing regimens on renal function after de novo liver transplantation. Arm 1: prolonged‐release tacrolimus (initial dose 0.2mg/kg/day); Arm 2: prolonged‐release tacrolimus (0.15–0.175mg/kg/day) plus basiliximab; Arm 3: prolonged‐release tacrolimus (0.2mg/kg/day delayed until Day 5) plus basiliximab. All patients received MMF plus a bolus of corticosteroid (no maintenance steroids). Primary endpoint: eGFR (MDRD4) at Week 24. Secondary endpoints: composite efficacy failure, BCAR and AEs. Baseline characteristics were comparable. Tacrolimus trough levels were readily achieved posttransplant; initially lower in Arm 2 versus 1 with delayed initiation in Arm 3. eGFR (MDRD4) was higher in Arms 2 and 3 versus 1 (p = 0.001, p = 0.047). Kaplan–Meier estimates of composite efficacy failure‐free survival were 72.0%, 77.6%, 73.9% in Arms 1–3. BCAR incidence was significantly lower in Arm 2 versus 1 and 3 (p = 0.016, p = 0.039). AEs were comparable. Prolonged‐release tacrolimus (0.15–0.175mg/kg/day) immediately posttransplant plus basiliximab and MMF (without maintenance corticosteroids) was associated with lower tacrolimus exposure, and significantly reduced renal function impairment and BCAR incidence versus prolonged‐release tacrolimus (0.2mg/kg/day) administered immediately posttransplant. Delayed higher‐dose prolonged‐release tacrolimus initiation significantly reduced renal function impairment compared with immediate posttransplant administration, but BCAR incidence was comparable.     

Absolute quantification of donor‐derived cell‐free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study

Donor‐derived cell‐free DNA (dd‐cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd‐cfDNA quantification of copies/mL plasma (dd‐cfDNA[cp/mL]) was compared to dd‐cfDNA fraction (dd‐cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy‐proven rejection (BPR), median dd‐cfDNA(cp/mL) was 3.3‐fold and median dd‐cfDNA(%) 2.0‐fold higher (82 cp/mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy‐proven rejection (BPR). dd‐cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd‐cfDNA(cp/mL) (AUC = 0.83) compared to dd‐cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd‐cfDNA(cp/mL), and 6.02 for dd‐cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd‐cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd‐cfDNA(cp/mL) with lower tacrolimus levels (<8 μg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd‐cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd‐cfDNA(cp/mL) allowed for better discrimination than dd‐cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.     

Immune checkpoint CD47 molecule engineered islets mitigate instant blood‐mediated inflammatory reaction and show improved engraftment following intraportal transplantation

Instant blood‐mediated inflammatory reaction (IBMIR) causes significant destruction of islets transplanted intraportally. Myeloid cells are a major culprit of IBMIR. Given the critical role of CD47 as a negative checkpoint for myeloid cells, we hypothesized that the presence of CD47 on islets will minimize graft loss by mitigating IBMIR. We herein report the generation of a chimeric construct, SA‐CD47, encompassing the extracellular domain of CD47 modified to include core streptavidin (SA). SA‐CD47 protein was expressed in insect cells and efficiently displayed on biotin‐modified mouse islet surface without a negative impact on their viability and function. Rat cells engineered with SA‐CD47 were refractory to phagocytosis by mouse macrophages. SA‐CD47‐engineered islets showed intact structure and minimal infiltration by CD11b⁺ granulocytes/macrophages as compared with SA‐engineered controls in an in vitro loop assay mitigating IBMIR. In a syngeneic marginal mass model of intraportal transplantation, SA‐CD47‐engineered islets showed better engraftment and function as compared with the SA‐control group (87.5% vs 14.3%). Engraftment was associated with low levels of intrahepatic inflammatory cells and mediators of islet destruction, including high‐mobility group box‐1, tissue factor, and IL‐1β. These findings support the use of CD47 as an innate immune checkpoint to mitigate IBMIR for enhanced islet engraftment with translational potential.