Human pulmonary microvascular endothelial cells respond to DAMPs from injured renal tubular cells.

Acute kidney injury (AKI) causes distant organ dysfunction through yet unknown mechanisms, leading to multiorgan failure and death. The lungs are one of the most common extrarenal organs affected by AKI, and combined lung and kidney injury has a mortality as high as 60%-80%. One mechanism that has been implicated in lung injury after AKI involves molecules released from injured kidney cells (DAMPs, or damage-associated molecular patterns) that promote a noninfectious inflammatory response by binding to pattern recognition receptors (PRRs) constitutively expressed on the pulmonary endothelium. To date there are limited data investigating the role of PRRs and DAMPs in the pulmonary endothelial response to AKI. Understanding these mechanisms holds great promise for therapeutics aimed at ameliorating the devastating effects of AKI. In this study, we stimulate primary human microvascular endothelial cells with DAMPs derived from injured primary renal tubular epithelial cells (RTECs) as an ex-vivo model of lung injury following AKI. We show that DAMPs derived from injured RTECs cause activation of Toll-Like Receptor and NOD-Like Receptor signaling pathways as well as increase human primary pulmonary microvascular endothelial cell (HMVEC) cytokine production, cell signaling activation, and permeability. We further show that cytokine production in HMVECs in response to DAMPs derived from RTECs is reduced by the inhibition of NOD1 and NOD2, which may have implications for future therapeutics. This paper adds to our understanding of PRR expression and function in pulmonary HMVECs and provides a foundation for future work aimed at developing therapeutic strategies to prevent lung injury following AKI.

Effects of antineoplastic and immunomodulating agents on postvaccination SARS-CoV-2 breakthrough infections, antibody response, and serological cytokine profile.

BACKGROUND: Despite immunization, patients on antineoplastic and immunomodulating agents have a heightened risk of COVID-19 infection. However, accurately attributing this risk to specific medications remains challenging. METHODS: An observational cohort study from December 11, 2020 to September 22, 2022, within a large healthcare system in San Diego, California, USA was designed to identify medications associated with greatest risk of postimmunization SARS-CoV-2 infection. Adults prescribed WHO Anatomical Therapeutic Chemical (ATC) classified antineoplastic and immunomodulating medications were matched (by age, sex, race, and number of immunizations) with control patients not prescribed these medications yielding a population of 26 724 patients for analysis. From this population, 218 blood samples were collected from an enrolled subset to assess serological response and cytokine profile in relation to immunization. RESULTS: Prescription of WHO ATC classified antineoplastic and immunomodulatory agents was associated with elevated postimmunization SARS-CoV-2 infection risk (HR 1.50, 95% CI 1.38 to 1.63). While multiple immunization doses demonstrated a decreased association with postimmunization SARS-CoV-2 infection risk, antineoplastic and immunomodulatory treated patients with four doses remained at heightened risk (HR 1.23, 95% CI 1.06 to 1.43). Risk variation was identified among medication subclasses, with PD-1/PD-L1 inhibiting monoclonal antibodies, calcineurin inhibitors, and CD20 monoclonal antibody inhibitors identified to associate with increased risk of postimmunization SARS-CoV-2 infection. Antineoplastic and immunomodulatory treated patients also displayed a reduced IgG antibody response to SARS-CoV-2 epitopes alongside a unique serum cytokine profile. CONCLUSIONS: Antineoplastic and immunomodulating medications associate with an elevated risk of postimmunization SARS-CoV-2 infection in a drug-specific manner. This comprehensive, unbiased analysis of all WHO ATC classified antineoplastic and immunomodulating medications identifies medications associated with greatest risk. These findings are crucial in guiding and refining vaccination strategies for patients prescribed these treatments, ensuring optimized protection for this susceptible population in future COVID-19 variant surges and potentially for other RNA immunization targets.

Achieving tolerance modifies cancer susceptibility profiles in liver transplant recipients.

Long-term survival of transplant recipients is significantly impacted by malignancy. We aimed to determine whether calcineurin inhibitor (CNI)-treated recipients converted to and weaned off molecular target of rapamycin inhibitor (mTOR-I) therapy have favorable changes in their molecular profiles in regard to malignancy risk. We performed gene expression profiling from liver biopsy and blood (PBMC) specimens followed by network analysis of key dysregulated genes, associated diseases and disorders, molecular and cellular functions using IPA software. Twenty non-immune, non-viremic patients were included, and 8 of them achieved tolerance. Two comparisons were performed: (1) tolerance time point vs tacrolimus monotherapy and (2) tolerance time point vs sirolimus monotherapy. Upon achieving tolerance, IPA predicted significant activation of DNA damage response (p = 5.40e-04) and inhibition of DNA replication (p = 7.56e-03). Conversion from sirolimus to tolerance showed decrease in HCC (p = 1.30e-02), hepatic steatosis (p = 5.60e-02) and liver fibrosis (p = 2.91e-02) associated genes. In conclusion, this longitudinal study of patients eventually achieving tolerance reveals an evolving molecular profile associated with decreased cancer risk and improved hepatic steatosis and liver fibrosis. This provides a biological rationale for attempting conversion to mTOR-I therapy and tolerance following liver transplantation particularly in patients at higher risk of cancer incidence and progression post-transplant.

Renal Function at Discharge Among Kidney Recipients Experiencing Delayed Graft Function and Its Associations With Long-term Outcomes.

Delayed graft function (DGF) after kidney transplantation is associated with higher rates of acute rejection and poor graft survival and outcomes. Current DGF definitions based on posttransplant need for dialysis are not standardized and there are no objective methodologies for quantifying DGF severity. Methods: Using Organ Procurement and Transplantation Network data, we examined DGF, and used recipient serum creatinine at discharge as a correlate of renal function and DGF severity (mild: <2.5 mg/dL; severe: ≥2.5 mg/dL). The associations between donor and recipient factors and DGF severity were quantified using logistic regression. We also examined the associations between DGF severity and long-term recipient outcomes, adjusting for potential confounders. Results: A predictive model using donor and recipient factors had a reasonably good ability to discriminate mild (low creatinine) versus severe (high creatinine) DGF (c-statistic of 0.70). In Cox regression, DGF and creatinine at discharge were both independently associated with long-term outcomes, yet their effects differed depending on the outcome (graft function, death-censored graft function, recipient mortality). Our findings suggest that having DGF, but with relatively good renal function (creatinine <2.5) at discharge, may be less deleterious on graft and recipient survival compared with severe, prolonged DGF, which was associated with a decreased median graft survival of ~2.6 y compared with no DGF with low creatinine at discharge. Conclusions: Our novel DGF severity stratification identified unique factors associated with DGF severity, along with DGF's association with long-term graft and patient survival. The adverse cost and outcome implications of severe DGF warrant additional investigation to improve kidney transplantation practice.

Prediction of Liver Transplant Rejection With a Biologically Relevant Gene Expression Signature.

BACKGROUND: Noninvasive biomarkers distinguishing early immune activation before acute rejection (AR) could more objectively inform immunosuppression management in liver transplant recipients (LTRs). We previously reported a genomic profile distinguishing LTR with AR versus stable graft function. This current study includes key phenotypes with other causes of graft dysfunction and uses a novel random forest approach to augment the specificity of predicting and diagnosing AR. METHODS: Gene expression results in LTRs with AR versus non-AR (combination of other causes of graft dysfunction and normal function) were analyzed from single and multicenter cohorts. A 70:30 approach (61 ARs; 162 non-ARs) was used for training and testing sets. Microarray data were normalized using a LT-specific vector. RESULTS: Random forest modeling on the training set generated a 59-probe classifier distinguishing AR versus non-AR (area under the curve 0.83; accuracy 0.78, sensitivity 0.70, specificity 0.81, positive predictive value 0.54, negative predictive value [NPV] 0.89; F-score 0.61). Using a locked threshold, the classifier performed well on the testing set (accuracy 0.72, sensitivity 0.67, specificity 0.73, positive predictive value 0.48, NPV 0.86; F-score 0.56). Probability scores increased in samples preceding AR versus non-AR, when liver function tests were normal, and decreased following AR treatment (P < 0.001). Ingenuity pathway analysis of the genes revealed a high percentage related to immune responses and liver injury. CONCLUSIONS: We have developed a blood-based biologically relevant biomarker that can be detected before AR-associated graft injury distinct from LTR never developing AR. Given its high NPV ("rule out AR"), the biomarker has the potential to inform precision-guided immunosuppression minimization in LTRs.

DAMPs Released From Injured Renal Tubular Epithelial Cells Activate Innate Immune Signals in Healthy Renal Tubular Epithelial Cells.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) predictably causes acute kidney injury after shock and major cardiovascular procedures in all kidneys procured for transplantation. The earliest events of IRI are triggered by molecules released from injured cells, damage-associated molecular patterns (DAMPs), that bind pattern recognition receptors (PRRs) constitutively expressed on many cells within the kidney. Activation of PRR signaling leads to production of proinflammatory molecules, which incite a cascade of inflammatory events leading to acute kidney injury. Renal tubular epithelial cells (RTECs) are particularly susceptible to ischemic injury, and proximal RTEC injury is pathognomonic of renal IRI. To better understand how injured RTECs contribute to the cycle of deleterious inflammation in the setting of renal IRI, this study asked whether DAMPs released from injured RTECs induced PRR signals in healthy RTECs. METHODS: Human RTECs were necrosed ex vivo to release intracellular DAMPs and resulting necrotic supernatant used to stimulate healthy RTECs, T lymphocytes, and monocytes. RESULTS: DAMPs released from necrosed RTECs upregulated PRRs known to be associated with renal IRI and activated mitogen-activated protein kinase signaling pathways. Proinflammatory cytokines were upregulated in response to necrotic supernatant, and this upregulation was abrogated by MEK-1 inhibition. The RTEC-derived DAMPs were also potent inducers of T-cell activation/proliferation and monocyte migration. CONCLUSIONS: This is the first study to our knowledge to show that endogenous DAMPs released from injured RTECs directly activate PRR signaling in healthy RTECs. These findings provide new insights directed to therapeutics for renal IRI.

Combining Blood Gene Expression and Cellfree DNA to Diagnose Subclinical Rejection in Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: Subclinical acute rejection is associated with poor outcomes in kidney transplant recipients. As an alternative to surveillance biopsies, noninvasive screening has been established with a blood gene expression profile. Donor-derived cellfree DNA (cfDNA) has been used to detect rejection in patients with allograft dysfunction but not tested extensively in stable patients. We hypothesized that we could complement noninvasive diagnostic performance for subclinical rejection by combining a donor-derived cfDNA and a gene expression profile assay. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a post hoc analysis of simultaneous blood gene expression profile and donor-derived cfDNA assays in 428 samples paired with surveillance biopsies from 208 subjects enrolled in an observational clinical trial (Clinical Trials in Organ Transplantation-08). Assay results were analyzed as binary variables, and then, their continuous scores were combined using logistic regression. The performance of each assay alone and in combination was compared. RESULTS: For diagnosing subclinical rejection, the gene expression profile demonstrated a negative predictive value of 82%, a positive predictive value of 47%, a balanced accuracy of 64%, and an area under the receiver operating curve of 0.75. The donor-derived cfDNA assay showed similar negative predictive value (84%), positive predictive value (56%), balanced accuracy (68%), and area under the receiver operating curve (0.72). When both assays were negative, negative predictive value increased to 88%. When both assays were positive, positive predictive value increased to 81%. Combining assays using multivariable logistic regression, area under the receiver operating curve was 0.81, significantly higher than the gene expression profile (P<0.001) or donor-derived cfDNA alone (P=0.006). Notably, when cases were separated on the basis of rejection type, the gene expression profile was significantly better at detecting cellular rejection (area under the receiver operating curve, 0.80 versus 0.62; P=0.001), whereas the donor-derived cfDNA was significantly better at detecting antibody-mediated rejection (area under the receiver operating curve, 0.84 versus 0.71; P=0.003). CONCLUSIONS: A combination of blood-based biomarkers can improve detection and provide less invasive monitoring for subclinical rejection. In this study, the gene expression profile detected more cellular rejection, whereas donor-derived cfDNA detected more antibody-mediated rejection.

UNOS/OPTN Data-guided Assessment of Focal Segmental Glomerulosclerosis After Kidney Transplantation and Evaluation of Immunosuppressive Protocols in a Steroid-free Center.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a common recurrent glomerulopathy associated with graft loss and patient survival after kidney transplantation (KT). However, its natural history, clinical predictors, and treatment response are still poorly understood. Steroid withdrawal regimens in KT have been associated with improvements in cardiovascular risk and patient outcomes. The Scripps Center for Organ Transplantation (SCOT) uses a rapid low-dose steroid withdrawal immunosuppression (IS) protocol for KT maintenance. METHODS: We assessed the impact of our protocol on FSGS disease recurrence over a 10-y period to reassess our steroid and IS protocols and to evaluate if our patient outcomes diverge from published data. We compared 4 groups: steroids always, steroid free, steroid switch on, and steroid weaned off. We used IS and induction-matched retrospective data from United Network for Organ Sharing (UNOS) to investigate patient and graft survival for FSGS at SCOT. RESULTS: Our analysis results differ from earlier studies showing that FSGS was associated with a higher risk of graft loss, perhaps because of selection of a UNOS data set filtered to match the SCOT IS protocol for making direct comparisons. Overall outcomes of graft failure and recipient death did not differ between SCOT patients and steroid-free transplant patient data from the UNOS data for FSGS. SCOT recurrence rate for FSGS was 7.5%, which was lower than in most published single-center studies. CONCLUSIONS: Based on our results, we believe that it is safe to continue the steroid avoidance protocols at SCOT and the steroid-free protocol may not be detrimental when the adverse effects and toxicities associated with steroid use are considered.

Mitigation of radiation exposure during surgical hepatectomy after yttrium-90 radioembolization.

Yttrium-90 (Y-90) radioembolization for the treatment of hepatocellular carcinoma can present safety challenges when transplanting recently treated Y-90 patients. To reduce surgeons' contact with radioactive tissue and remain within occupational dose limits, current guidelines recommend delaying transplants at least 14 days, if possible. We wanted to determine the level of radiation exposure to the transplant surgeon when explanting an irradiated liver before the recommended decay period. Anex-vivoradiation exposure analysis was conducted on the explanted liver of a patient who received Y-90 therapy 46 h prior to orthotopic liver transplant. To estimate exposure to the surgeon's hands, radiation dosimeter rings were placed inside three different surgical glove configurations and exposed to the explanted liver. Estimated radiation doses corrected for Y-90 decay were calculated. Radiation safety gloves performed best, with an average radiation exposure rate of 5.36 mSV h-1in the static hand position, an 83% reduction in exposure over controls with no glove (31.31 mSv h-1). Interestingly, non-radiation safety gloves also demonstrated reduced exposure rates, well below occupational regulation limits. Handling of Y-90 radiated organs within the immediate post-treatment period can be done safely and does not exceed federal occupational dose limits if appropriate gloves and necessary precautions are exercised.

Data-Driven Kidney Transplant Phenotyping as a Histology-Independent Framework for Biomarker Discovery.

BACKGROUND: In transplant medicine, clinical decision making largely relies on histology of biopsy specimens. However, histology suffers from low specificity, sensitivity, and reproducibility, leading to suboptimal stratification of patients. We developed a histology-independent immune framework of kidney graft homeostasis and rejection. METHODS: We applied tailored RNA deconvolution for leukocyte enumeration and coregulated gene network analysis to published bulk human kidney transplant RNA transcriptomes as input for unsupervised, high-dimensional phenotype clustering. We used framework-based graft survival analysis to identify a biomarker that was subsequently characterized in independent transplant biopsy specimens. RESULTS: We found seven immune phenotypes that confirm known rejection types and uncovered novel signatures. The molecular phenotypes allow for improved graft survival analysis compared with histology, and identify a high-risk group in nonrejecting transplants. Two fibrosis-related phenotypes with distinct immune features emerged with reduced graft survival. We identified lysyl oxidase-like 2 (LOXL2)-expressing peritubular CD68+ macrophages as a framework-derived biomarker of impaired allograft function. These cells precede graft fibrosis, as demonstrated in longitudinal biopsy specimens, and may be clinically useful as a biomarker for early fibrogenesis. CONCLUSIONS: This study provides a comprehensive, data-driven atlas of human kidney transplant phenotypes and demonstrates its utility to identify novel clinical biomarkers.

The Advantage of Multiple Listing Continues in the Kidney Allocation System Era.

BACKGROUND: Transplant candidates can be listed at multiple transplant centers to increase the probability of receiving an organ. We evaluated the association between multilisting (ML) status and access to a deceased donor kidney transplant (DDKT) to determine if ML provides a long-term advantage regarding wait-list mortality and recipient outcomes. MATERIALS AND METHODS: Candidates between January 2010 and October 2017 were identified as either singly or multiply listed using Organ Procurement and Transplantation Network data and cohorts before and after implementation of the Kidney Allocation System (KAS). Cross-sectional logistic regression was used to assess relationships between candidate factors and ML prevalence (5.4%). RESULTS: Factors associated with ML pre-KAS included having blood type B (reference, type O; odds ratio [OR], 1.20; P < .001), having private insurance (OR, 1.5; P < .001), wait time (OR, 1.28; P < .001), and increasing calculated panel-reactive antibody (cPRA) (reference, cPRA 0-100; OR for cPRA 80-98, 2.83; OR for cPRA 99, 3.47; OR for cPRA 100, 5.18; P < .001). Transplant rates were double for multilisted vs singly listed recipients (adjusted hazard ratio [aHR], 2.16; P < .001). Extra-donor service area ML candidates received transplants 2.5 years quicker than single-listing (SL) candidates, conferring a 42% wait-list advantage. Recipient death (aHR, 0.94; P = .122) and graft failure (aHR, 0.91; P = .006) rates were also lower for ML recipients. CONCLUSIONS: In the KAS era, ML continues to increase the likelihood of receiving a DDKT and lower the incidence of wait-list mortality, and it confers a survival advantages over SL.

Healthcare worker seroconversion for SARS-CoV-2 at two large health systems in San Diego.

Coronavirus Disease 2019 infections among healthcare workers were widely reported in China and Europe as the pandemic expanded to the United States. In order to examine the infection rate among these essential workers, we combined results of SARS-CoV-2 serology testing offered free to healthcare workers at two large San Diego health systems when the antibody assays first became available.

Systems biology approaches in solid organ transplantation.

PURPOSE OF REVIEW: Organ transplantation research has led to the discovery of several interesting individual mechanistic pathways, molecules and potential drug targets but there are still no comprehensive studies that have addressed how these varied mechanisms work in unison to regulate the posttransplant immune response that drives kidney rejection and dysfunction. RECENT FINDINGS: Systems biology is a rapidly expanding field that aims to integrate existing knowledge of molecular concepts and large-scale genomic and clinical datasets into networks that can be used in cutting edge computational models to define disease mechanisms in a holistic manner. Systems biology approaches have brought a paradigm shift from a reductionist view of biology to a wider agnostic assessment of disease from several lines of evidence. Although the complex nature of the posttransplant immune response makes it difficult to pinpoint mechanisms, systems biology is enabling discovery of unknown biological interactions using the cumulative power of genomic data sets, clinical data and endpoints, and improved computational methods for the systematic deconvolution of this response. SUMMARY: An integrative systems biology approach that leverages genomic data from varied technologies, such as DNA sequencing, copy number variation, RNA sequencing, and methylation profiles along with long-term clinical follow-up data has the potential to define a framework that can be mined to provide novel insights for developing therapeutic interventions in organ transplantation.

Safety and Efficacy of a Steroid Avoidance Immunosuppression Regimen in Renal Transplant Patients With De Novo or Preformed Donor-Specific Antibodies: A Single-Center Study.

Although interest in the role of donor-specific antibodies (DSAs) in kidney transplant rejection, graft survival, and histopathological outcomes is increasing, their impact on steroid avoidance or minimization in renal transplant populations is poorly understood. Primary outcomes of graft survival, rejection, and histopathological findings were assessed in 188 patients who received transplants between 2012 and 2015 at the Scripps Center for Organ Transplantation, which follows a steroid avoidance protocol. Analyses were performed using data from the United Network for Organ Sharing. Cohorts included kidney transplant recipients with de novo DSAs (dnDSAs; n = 27), preformed DSAs (pfDSAs; n = 15), and no DSAs (nDSAs; n = 146). Median time to dnDSA development (classes I and II) was shorter (102 days) than in previous studies. Rejection of any type was associated with DSAs to class I HLA (P < .05) and class II HLA (P < .01) but not with graft loss. Although mean fluorescence intensity (MFI) independently showed no association with rejection, an MFI >5000 showed a trend toward more antibody-mediated rejection (P < .06), though graft loss was not independently associated. Banff chronic allograft nephropathy scores and a modified chronic injury score were increased in the dnDSA cohort at 6 months, but not at 2 years (P < .001 and P < .08, respectively). Our data suggest that dnDSAs and pfDSAs impact short-term rejection rates but do not negatively impact graft survival or histopathological outcomes at 2 years. Periodic protocol post-transplant DSA monitoring may preemptively identify patients who develop dnDSAs who are at a higher risk for rejection.

Immune and gene expression profiling during tacrolimus to everolimus conversion early after liver transplantation.

Early elimination of tacrolimus in favor of everolimus can improve renal function in liver transplant recipients. However, as this approach increases the risk of acute rejection, it may benefit from predictive biomarkers guiding weaning. We enrolled 20 recipients on stable tacrolimus + everolimus to undergo tacrolimus withdrawal early post-liver transplant. Blood samples were collected at month 3 (withdrawal initiation), 4 (withdrawal completion), 4.5 and 6 (both everolimus alone). 15 patients did not reject and 5 had mild rejection responding to tacrolimus resumption. Before tacrolimus withdrawal, eventual rejecters had higher percentages of CD56+ NK cells and CD19+CD27+CD24+ memory B cells, and lower levels of T cells expressing the exhaustion marker PD-1. Over time, memory B cells, Ki-67+CD3+ (proliferating) cells and CD4+CD127-CD25HIGH FOXP3+ Tregs increased in rejecters. Tregs also increased in non-rejecters over time. The number of differentially expressed genes progressively increased in rejecters, particularly in mTOR, Eukaryotic Initiation Factor 2, and Neuroinflammation signaling pathways. There was no difference in anti-HLA antibodies between the groups. In summary, blood mononuclear cell and gene expression may predict successful vs. failed early tacrolimus withdrawal in liver transplant recipients. While needing validation, these preliminary findings highlight the potential for cellular and molecular biomarkers to guide decision-making during tacrolimus weaning.

Feasibility and Comparison Study of Fecal Sample Collection Methods in Healthy Volunteers and Solid Organ Transplant Recipients Using 16S rRNA and Metagenomics Approaches.

The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.

Discovery and validation of a novel blood-based molecular biomarker of rejection following liver transplantation.

Noninvasive biomarker profiles of acute rejection (AR) could affect the management of liver transplant (LT) recipients. Peripheral blood was collected following LT for discovery (Northwestern University [NU]) and validation (National Institute of Allergy and Infectious Diseases Clinical Trials in Organ Transplantation [CTOT]-14 study). Blood gene profiling was paired with biopsies showing AR or ADNR (acute dysfunction no rejection) as well as stable graft function samples (Transplant eXcellent-TX). CTOT-14 subjects had serial collections prior to AR, ADNR, TX, and after AR treatment. NU cohort gene expression (46 AR, 45 TX) was analyzed using random forest models to generate a classifier training set (36 gene probe) distinguishing AR vs TX (area under the curve 0.92). The algorithm and threshold were locked and tested on the CTOT-14 validation cohort (14 AR, 50 TX), yielding an accuracy of 0.77, sensitivity 0.57, specificity 0.82, positive predictive value (PPV) 0.47, and negative predictive value (NPV) 0.87 for AR vs TX. The probability score line slopes were positive preceding AR, and negative preceding TX and non-AR (TX + ADNR) (P ≤ .001) and following AR treatment. In conclusion, we have developed a blood biomarker diagnostic for AR that can be detected prior to AR-associated graft injury as well a normal graft function (non-AR). Further studies are needed to evaluate its utility in precision-guided immunosuppression optimization following LT.

Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus.

BACKGROUND AND AIMS: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal. APPROACH AND RESULTS: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group. CONCLUSIONS: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.