Lung Transplant Rejection Research Articles

Intragraft Hyaluronan Increases in Association With Acute Lung Transplant Rejection

Acute perivascular rejection (AR) is common in lung recipients and increases the risk for chronic lung allograft dysfunction (CLAD). Hyaluronan (HA), an extracellular matrix constituent, accumulates in experimental AR and can act as an innate immune agonist, breaking tolerance and potentiating alloimmunity. We previously demonstrated HA accumulates in CLAD after human-lung transplantation. We sought to determine if HA accumulates in the bronchoalveolar lavage fluid (BALF) concurrent with AR in lung recipients. The cohort consisted of 126 first adult lung recipients at 5 transplant centers with a total of 373 BALF samples collected within the first posttransplant year. All samples were paired with a lung biopsy from the same bronchoscopy. BALF HA (ng/mL) was quantified by ELISA and log-transformed for analysis. Linear-mixed effect models, adjusted for potential confounders, were used to estimate the association between BALF HA concentration and the presence of AR on biopsy. The association between early posttransplant BALF HA levels and the development of CLAD was explored utilizing tertiles of maximum BALF HA level observed within the first 6 months of transplant. In analyses adjusted for potential confounders, BALF HA concentration was significantly increased in association with AR (change in means on log-scale 0.31; 95% CI, 0.01-0.60; P = 0.044). When considered on the original scale (ng/mL), BALF HA concentrations were 1.36 times (36%) higher, on average, among samples with, versus without, AR. The cumulative incidence of CLAD was numerically higher in individuals in the highest tertiles of BALF HA level within the first 6 months after transplant, as compared with those in the lowest tertile; however, this difference was not statistically significant (P = 0.32). These results demonstrate accumulation of HA in clinical AR and suggest a mechanism by which innate and adaptive immune activation might interact in the development of AR and CLAD.

Protective Role of ECMO on Reperfusion Injury and Acute Rejection in Lung Transplantation: A Pathological Analysis

Purpose The routine utilization of intraoperative extracorporeal membrane oxygenation (ECMO) in lung transplantation is recently extensively investigated for its protective role, especially on reperfusion injury. The purpose of this study is to compare pathological features on pre and post-reperfusion biopsies and on 30 and 90-days surveillance trans-bronchial biopsies in lung transplantations performed with or without ECMO support to assess its protective role on the graft Methods 51 patients (13 with ECMO support, 38 without ECMO support) underwent bilateral lung transplantation at our Institution between 2012 and 2019. Only patients on ECMO at time of anesthetic induction or before the first clamping of the pulmonary artery were included. Patients submitted to graft volume reduction before lung reperfusion were excluded from the analysis.Intra-operative pre and post-reperfusion biopsies were collected to analyze edema, leucocyte margination and congestion/extravasation (score 0-3). The presence of acute rejection (grade A0-3, grade B0-2) diffuse alveolar damage (DAD), organizing pneumonia (OP) on 30 and 90-days trans-bronchial surveillance biopsies was also compared in the two groups. Results No significant differences were found between the two groups in terms of edema, leucocyte margination and congestion in pre reperfusion biopsies. On post reperfusion biopsies, leucocyte margination was significantly less frequent in ECMO group (46% vs 76%; p=0.043). A lower frequency of acute rejection (grade A0-3) was also found in the ECMO group on 30-days biopsies (25% vs 58%, p=0.047). Conclusion In conclusion, this analysis does not show significant differences between the two groups except for a lower frequency of leucocyte margination at post-reperfusion biopsies and acute rejection at 30-days biopsies in the ECMO group. Other studies, with a larger population, are needed in order to evaluate whether the positive clinical impact of using ECMO as a prevention of reperfusion edema after lung transplantation can also be explained by a defined pathological picture

Thrombocytosis as a Marker of Allograft Rejection in Lung Transplantation: A Case Series

<h3>Introduction</h3> Thrombocytosis (platelet count ≥450 × 10⁹/L) in the early postoperative period after lung transplantation has been correlated with acute cellular rejection (ACR). However, we have also observed significant elevations in platelet counts in the setting of reduced lung function secondary to allograft dysfunction in the late postoperative period. Herein, we describe a case series of three lung transplant recipients with thrombocytosis coincident with episodes of ongoing allograft rejection. <h3>Case Report</h3> Three bilateral lung transplant recipients - a 21-year-old female with trauma-related acute respiratory distress syndrome (patient A), 68-year-old male with interstitial lung disease (patient B), and 60-year-old male with inherited emphysema (patient C) - each presented with few days' history of exertional dyspnea at 4-months, 36-months, and 28-months post-transplant, respectively. Chest computed tomography revealed multifocal bilateral pulmonary consolidations and ground glass changes in patient A, mild upper lobe reticular changes in patient B, and subtle nodular opacities in the upper lobes in patient C. Bronchoscopy with transbronchial lung biopsy revealed influenza virus and grade A2 ACR in patient A. Biopsies in patient B and C showed no evidence of ACR, but revealed focal lung injuries. Marked thrombocytosis was observed on peripheral blood smear in all three patients at the time of admission, and complete blood count showed elevated platelet counts of 525 × 10⁹/L, 626 × 10⁹/L, and 681 × 10⁹/L. Trends of patients' platelet counts and corresponding lung function measures are shown in Figure 1. <h3>Summary</h3> We observed waxing and waning thrombocytosis which corresponded with gradual decline in lung function that ultimately trended towards accelerated chronic lung allograft dysfunction in all three patients. Exploration of this hematologic phenomenon in the setting of late postoperative allograft dysfunction is warranted.

CD26+ Epithelial Cell Clusters as a Novel Biomarker and Therapeutic Target in Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Novel diagnostic and therapeutic strategies for chronic lung allograft dysfunction (CLAD) are pressing priorities. We previously reported that CD26/DPP-4 inhibition attenuated ischemia-reperfusion injury, acute rejection and chronic fibrosis in mouse lung transplantation (LTx), and in our recent clinical study, a CD26/DPP-4 inhibitor improved CLAD-free survival (ISHLT 2020 abstract). To elucidate the mechanism of CD26/DPP-4 inhibitors in LTx, and apply them in the clinic, we investigated CD26 expression in CLAD clinical specimens. <h3>Methods</h3> Among patients who underwent LTx in our institution, we retrieved pathological specimens from re-LTx and autopsy cases. We also included two native lung samples from LTx recipients. We performed hematoxylin-eosin and Elastica-Masson staining and immunohistochemistry using a CD26 antibody. We evaluated the pathological findings and clinical characteristics. <h3>Results</h3> We evaluated 11 patients (6 females, 5 males) with a median age of 43 (8-58) years. The pathological samples were nine allografts and two native lung specimens. Of the allografts, six were obtained at autopsy, performed 38 (12-71) [median (range)] months after LTx, and three were obtained by resection during re-LTx, performed 20 (17-59) months after the initial LTx. Seven patients had diabetes mellitus, but none received CD26/DPP-4 inhibitors. Seven patients (four autopsy and three re-LTx cases) had CLAD with bronchiolitis obliterans (BO) pathology. Immunohistochemistry revealed clusters of CD26+ epithelial cells adjacent to the BO lesions or in the obstructed bronchial lumen in six of the seven CLAD patients. One CLAD patient without CD26 expression died from lung cancer, rather than CLAD, which was diagnosed at autopsy. In the non-CLAD or native lung samples, the CD26+ epithelial cell clusters were not detected. <h3>Conclusion</h3> Clusters of CD26+ epithelial cells were detected in CLAD, but not non-CLAD, samples. CD26 might be a novel biomarker and therapeutic target for CLAD.

Assessment of Carfilzomib Treatment Response in Lung Transplant Recipients With Antibody-mediated Rejection.

All patients treated with CFZ for AMR from 2014 to 2019 were included. The primary outcome was a positive response to CFZ was defined as: (1) loss of DSA C1q-fixing ability after last CFZ dose; (2) clearance of de novo DSA; or (3) decrease in de novo DSA mean fluorescence intensity of >3000. Twenty-eight patients with 31 AMR episodes were treated with CFZ. A positive response was observed in 74.4% of AMR episodes and 82.1% of patients. This response was driven by loss of complement 1q fixation (70.6%), elimination of class I DSAs (78.6%), and reduction in both classes I (median 2815, 79.5% reduction from baseline) and II DSA mean fluorescence intensity (3171, 37.1%). CFZ shows potential for ameliorating AMR; however, additional studies are needed to define optimal time of administration.

79664 Complement Driven Auto-Reactive Antibodies in Lung Transplantation

ABSTRACT IMPACT: Our work unveils a novel mechanism of ischemia repurfusion injury driven by pre-existing autoimmunity following lung transplant and a potential therapeutic strategy for blocking complement-dependent injury thereby reducing risk of lung transplant rejection. OBJECTIVES/GOALS: Our goal was to determine if pre-existing autoimmune autoantibodies, such as those resulting from cigarette smoke (CS), contribute to graft rejection in lung transplantation (LTx) and if autoreactive-mediated graft injury is complement-dependent. METHODS/STUDY POPULATION: For in vivo experiments, we utilized our emphysema mouse model. Briefly, eight-week-old C57BL/6J mice are exposed to 3R4F reference cigarette smoke 5 hours per day, 5 days a week for 6 months. Upon completion, cigarette smoked (CS) mice and control (NS) mice received syngeneic orthotopic left-lung transplant from age-matched C57BL/6J donors. To determine if pre-existing autoreactivity mediated graft injury was complement-dependent we treated CS-LTx mice with a novel, bifunctional complement inhibitor. Autoantibody levels were measured by ELISA and lung injury was assessed by blinded histopathological analyses. Complement inhibition was verified by immunofluorescence. RESULTS/ANTICIPATED RESULTS: We found that CS-exposure leads to production of autoreactive antibodies towards extracellular matrix (ECM) components and contributes to graft injury. Interestingly, LTx into CS exposed mice further increased de-novo ECM autoantibody development. Lastly, treatment with our novel, bifunctional complement inhibitor blocked autoantibody spreading and significantly reduced graft rejection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These data demonstrate that smoking induces pre-LTx autoreactivity to ECM proteins that promotes graft injury following LTx. Furthermore, complement inhibition reduces autoantibody production and protects the graft from injury.

Single-Center Experience of Outcomes and Prescribing Patterns of IV Immunoglobulin Use in Critically Ill Patients.

Retrospective, observational study. Single, academic medical center medical ICU. Adults who received greater than or equal to 1 dose of IV immunoglobulin during their medical ICU admission from 2011 to 2018. Prescribing patterns, level of evidence supporting use, and cost. A total of 389 patients received greater than or equal to 1 dose of IV immunoglobulin for 46 discrete indications and 36.5% of indications had low-quality data supporting use of IV immunoglobulin. The primary indication for IV immunoglobulin was hypogammaglobulinemia (35.5%) followed by antibody-mediated lung transplant rejection (15.4%). Nonsurvivors received lower median dosing (g/kg) and number of doses compared with survivors (0.4 g/kg [0.4-1 g/kg] vs 0.5 g/kg [0.4-1 g/kg] [p = 0.0003] and 1.0 [1-2] vs 2 [1-3] doses [p = 0.0001], respectively). Dosing was based on ideal body weight in 258 patients (66%). High-quality data supported IV immunoglobulin use in 15 patients (4%). The median cost per dose of IV immunoglobulin in nonsurvivors was $4,893 ($4,078-$8,155) versus $5,709 ($4,078-$10,602) in survivors (p = 0.04). IV immunoglobulin is prescribed for many indications in the medical ICU with low-quality evidence supporting its use and dosing regimens are variable. Hospital survivors received a higher dose and greater number of doses of IV immunoglobulin compared with nonsurvivors. National guidelines are needed to help inform IV immunoglobulin utilization and reduce healthcare costs.

THE TISSUE COMMON RESPONSE MODULE (TCRM) SCORE PROVIDES A QUANTITATIVE OBJECTIVE BIOPSY SCORE TO ASSESS THE SEVERITY OF PANCREAS TRANSPLANT REJECTION

Introduction: Several studies have used gene expression profiling to identify key immunologic and inflammatory markers in the blood and tissue of solid-organ transplant recipients experiencing rejection (AR), but none of these have included samples from pancreas transplant (PT) recipients. Gene expression analysis of public transcriptional data from kidney, heart, liver, and lung transplant rejection led to the tissue Common Response Module (tCRM) score based on 11 genes (BASP1, ISG20, PSMB9, RUNX3, TAP1, NKG7, LCK, INPP5D, CXCL9, CD6, CXCL10; Khatri et al, 2013) which can diagnose the severity of tissue rejection, but this has not been validated in PT. In this study, we aimed to evaluate the locked tCRM score and identify additional markers of PT-AR. Materials and Methods: We performed a retrospective study applying gene expression analysis to RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue from 51 pancreas biopsies, grouped by histopathologic grade of acute cellular rejection (Grade 1 (n=14), Grade 2 (n=14), and Grade 3 (n=9)) versus normal (n=14). Gene expression analysis was performed using the Nanostring Cancer Immune v1.1 oligonucleotide set, which includes 40 set housekeeping (HK) genes. Appropriate HK genes were selected, data was log transformed and normalized. Differential gene expression (DE) analysis involved a negative binomial model, unsupervised hierarchical clustering, and calculation of the tCRM score with removal of Nanostring designated HK genes confounded by rejection. P-value was considered significant at <0.05 (Benjamini-Yekutieli, Mann-Whitney, Graph-Pad PRISM). Pathway analysis (PA) was performed to evaluate biological significance. Results: Some Nanostring designated HK genes showed significant differences with AR and hence a custom set of HK genes was used for normalization of log transformed data. Significant transcriptional differences were seen with higher grades of rejection in PT (Fig.1). The tCRM score, run on a sub-component of 11 genes, captures the same message with a scaled score that increases across rejection grades (Fig.2). Of the 22 genes differentially expressed in Grade 3, 18 were also significant in Grade 2. Specifically, within the 11 tCRM genes diagnostic of rejection in other solid organ transplants, 9 of 11 were significant for PT rejection, with the last two tCRM gene (CXCL9 and CXCL10) approaching significance at p=0.055 and 0.08 respectively. Conclusion: This study shows that a common hub of immune mediated genes, highly significant in transplant rejection (tCRM), and not confounded by tissue source (kidney, heart, liver and lung), were also highly informative for diagnosis and quantification of AR injury in PT. The bulk of the AR signal in PT, similar to other solid organs, is attributable to infiltration of activated leukocytes in the tissue, with background tissue injury specific transcriptional changes. The clinical and molecular drivers for Grade 1 PT rejection deserve additional analysis.Fig. 1.

Acute Lung Rejection in Lung Transplantation

Acute Lung Rejection in Lung Transplantation

Screening and identification of key regulatory connections and immune cell infiltration characteristics for lung transplant rejection using mucosal biopsies

This study aimed to explore key regulatory connections underlying lung transplant rejection. The differentially expressed genes (DEGs) between rejection and stable lung transplantation (LTx) samples were screened using R package limma, followed by functional enrichment analysis and protein–protein interaction network construction. Subsequently, a global triple network, including miRNAs, mRNAs, and transcription factors (TFs), was constructed. Furthermore, immune cell infiltration characteristics were analyzed to investigate the molecular immunology of lung transplant rejection. Finally, potential drug–target interactions were generated. In brief, 739 DEGs were found between rejection and stable LTx samples. PTPRC, IL-6, ITGAM, CD86, TLR8, TYROBP, CXCL10, ITGB2, and CCR5 were defined as hub genes. Eight TFs, including STAT1, SPIB, NFKB1, SPI1, STAT5A, RUNX1, VENTX, and BATF, and five miRNAs, including miR-335-5p, miR-26b-5p, miR-124-3p, miR-1-3p, and miR-155-5p, were involved in regulating hub genes. The immune cell infiltration analysis revealed higher proportions of activated memory CD4 T cells, follicular helper T cells, γδ T cells, monocytes, M1 and M2 macrophages, and eosinophils in rejection samples, besides lower proportions of resting memory CD4 T cells, regulatory T cells, activated NK cells, M0 macrophages, and resting mast cells. This study provided a comprehensive perspective of the molecular co-regulatory network underlying lung transplant rejection.

LPS-induced Airway-centered Inflammation Leading to BOS-like Airway Remodeling Distinct From RAS-like Fibrosis in Rat Lung Transplantation.

Localization of inflammatory stimuli may direct lung allografts to different phenotypes of chronic dysfunction, such as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). We hypothesized that airway stimulation with lipopolysaccharide (LPS) in rats leads to airway-centered inflammation similar to human BOS. Rat left lung transplantation was conducted (donor: Brown Norway, recipient: Lewis). Allotransplant recipients received cyclosporine A (CsA) until postoperative day 56 with airway instillation of LPS (Allo-LPS, n = 8), phosphate buffered saline (Allo-PBS, n = 5) from days 35 to 46 (3 times a wk), or no further treatment (n = 4). Some allotransplant recipients received CsA until day 14 and were immunosuppression free after day 15 until day 56. Bronchial and pleural fibrosis were semiquantified; alveolar fibrosis was evaluated with a histological scale. The Allo-LPS group had significantly increased International Society for Heart and Lung Transplantation rejection grades (grade A, P = 0.005; grade B, P = 0.004), bronchial obstructive proportion (0.34 ± 0.04% [Allo-LPS] versus 0.11 ± 0.04% [Allo-PBS], P = 0.006), and airway resistance (3.05 ± 1.78 cm H2O·s/mL [Allo-LPS] versus 0.83 ± 0.58 cm H2O·s/mL [Allo-PBS], P = 0.007) compared with other groups. Allotransplant recipients that underwent a short course of CsA developed RAS-like fibrosis involving the airways, alveoli, and pleura. Airway instillation of LPS in allografts under immunosuppression resulted in BOS-like airway-centered inflammation and fibrosis distinct from RAS-like diffuse fibrosis, which was induced by a shortened course of immunosuppression. We propose novel animal models for BOS and RAS after lung transplantation.

Differences in airway microbiome and metabolome of single lung transplant recipients

BackgroundRecent studies suggest that alterations in lung microbiome are associated with occurrence of chronic lung diseases and transplant rejection. To investigate the host-microbiome interactions, we characterized the airway microbiome and metabolome of the allograft (transplanted lung) and native lung of single lung transplant recipients.MethodsBAL was collected from the allograft and native lungs of SLTs and healthy controls. 16S rRNA microbiome analysis was performed on BAL bacterial pellets and supernatant used for metabolome, cytokines and acetylated proline-glycine-proline (Ac-PGP) measurement by liquid chromatography-high-resolution mass spectrometry.ResultsIn our cohort, the allograft airway microbiome was distinct with a significantly higher bacterial burden and relative abundance of genera Acinetobacter & Pseudomonas. Likewise, the expression of the pro-inflammatory cytokine VEGF and the neutrophil chemoattractant matrikine Ac-PGP in the allograft was significantly higher. Airway metabolome distinguished the native lung from the allografts and an increased concentration of sphingosine-like metabolites that negatively correlated with abundance of bacteria from phyla Proteobacteria.ConclusionsAllograft lungs have a distinct microbiome signature, a higher bacterial biomass and an increased Ac-PGP compared to the native lungs in SLTs compared to the native lungs in SLTs. Airway metabolome distinguishes the allografts from native lungs and is associated with distinct microbial communities, suggesting a functional relationship between the local microbiome and metabolome.

Apoptotic cells secrete metabolites to regulate immune homeostasis.

Apoptotic cells secrete metabolites to regulate immune homeostasis.

Surveillance Bronchoscopy in Lung Transplantation Recipients: A Single Center Experience Analysis

Bronchoscopy with bronchoalveolar lavage and transbronchial biopsy is the gold standard for the diagnosis of infection or acute cellular rejection in lung transplantation (LTx) recipients, but there is some controversy to perform it in asymptomatic patients. We conducted a retrospective analysis of medical reports of LTx recipients who survived in the first year after transplant during the period of August 2003 to February 2018 to evaluate the applicability of this procedure in the management of asymptomatic acute cellular rejection in our center. We assessed 1252 bronchoscopies of 247 patients during this period, and, facing the histopathological results, we defined our management that included conservative or intervention therapy. In our service the information obtained by surveillance bronchoscopy was sufficient to modify the management mainly in the first 2 surveillance bronchoscopies (second and sixth week post LTx). This effect seems to dilute after the second month, making its applicability more questionable.

Pseudomonas Aeruginosa Infection of Lung Transplants Breaks Established Tolerance through B Cell-Intrinsic Innate Immune Signaling That Promotes Intragraft Plasma Cell Generation and Donor Specific Antibody Accumulation

Antibody mediated rejection (AMR) has become an increasingly recognized form of lung transplant rejection. Our group recently identified Pseudomonas aeruginosa infection as a risk factor for clinical AMR but whether infection is sufficient for donor specific antibody (DSA) production and AMR remains unknown. Here we ask in a mouse model of orthotopic lung allograft transplantation whether airway infection with P. aeruginosa promotes DSA generation and AMR. Balb/c lungs were transplanted into the following recipients: B6 WT (wildtype), uMT KO (No B cells), AID/uS KO (Have B cells but are unable to secrete Ig), CD19 Cre , CD19 Cre x TLR4fl/fl and CD19 Cre x MyD88fl/fl. Lung recipients were immunosuppressed with costimulatory blockade on post-operative day (POD) 0 (CD40L Ab) and POD 2 (CTLA4Ig) to induce allograft acceptance. On POD 21 recipients received intratracheal saline or 2 x 105 pfu/g body weight of live P. aeruginosa (clinical isolate M57-15) and then treated with ciprofloxacin from POD 22 to 25. On POD 42 peripheral blood was assessed for DSA and graft tissue evaluated for C4d deposition or quantitated for plasma cells (PC) by flow cytometric analysis (FACS). P. Aeruginosa infection stimulated high amounts of DSA generation and marked C4d deposition. In contrast, C4d deposition was nearly absent in uMT and AID/uS KO recipients. Infected recipients with TLR4 or MyD88 targeted-deficiency in B cells had sharply lower DSA when compared to WT controls. P. aeruginosa infection drove a 4-fold expansion of allograft resident PCs that was largely dependent on maintaining B cell TLR4-MyD88 signaling. In contrast, the spleen and bone marrow had only modest changes in PC abundance despite infection. Surprisingly, re-transplantation of P. aeruginosa-infected lung allografts into secondary uMT KO recipients revealed that lung allograft-resident PCs are capable of producing large amounts of DSA. Collectively, these data show that P. aeruginosa infection abrogates established lung transplant tolerance through engaging innate immune signaling pathways within the B cell compartment. Additionally, we make the novel observation that the lung is a suitable environment for PC development with implications for clinical AMR.

HDAC6-specific inhibitor suppresses Th17 cell function via the HIF-1α pathway in acute lung allograft rejection in mice.

Background: Previous animal experiments and clinical studies indicated the critical role of Th17 cells in lung transplant rejection. Therefore, the downregulation of Th17 cell function in lung transplant recipients is of great interest. Methods: We established an orthotopic mouse lung transplantation model to investigate the role of histone deacetylase 6-specific inhibitor (HDAC6i), Tubastatin A, in the suppression of Th17 cells and attenuation of pathologic lesions in lung allografts. Moreover, mechanism studies were conducted in vitro. Results: Tubastatin A downregulated Th17 cell function in acute lung allograft rejection, prolonged the survival of lung allografts, and attenuated acute rejection by suppressing Th17 cell accumulation. Consistently, exogenous IL-17A supplementation eliminated the protective effect of Tubastatin A. Also, hypoxia-inducible factor-1α (HIF-1α) was overexpressed in a lung transplantation mouse model. HIF-1α deficiency suppressed Th17 cell function and attenuated lung allograft rejection by downregulating retinoic acid-related orphan receptor γt (ROR γt) expression. We showed that HDAC6i downregulated HIF-1α transcriptional activity under Th17-skewing conditions in vitro and promoted HIF-1α protein degradation in lung allografts. HDAC6i did not affect the suppression of HIF-1α-/- naïve CD4+ T cell differentiation into Th17 cell and attenuation of acute lung allograft rejection in HIF-1α-deficient recipient mice. Conclusion: These findings suggest that Tubastatin A downregulates Th17 cell function and suppresses acute lung allograft rejection, at least partially, via the HIF-1α/ RORγt pathway.

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

552 Exploring the Role of Gastrointestinal Motility and Non-Acid Reflux in Lung Transplant Prognosis and Risk of Acute Rejection

INTRODUCTION: Lung transplant patients are at risk of developing acute rejection leading to bronchiolitis obliterans syndrome (BOS). Lung transplant patients are also known to have and/or develop gastroesophageal reflux. Acid gastroesophageal reflux has been correlated with risk of acute rejection and consequently BOS. There is however a paucity of data correlating non-acid reflux with risk of acute rejection. Our study investigated the role of non-acid reflux with lung transplant rejection. METHODS: 131 lung transplant recipients who had post-transplant evaluation between 2012 and 2019 were studied. A total of 64 lung transplant recipients had at least 3 months of post-transplant follow up consisting of 24 hour impedance pH testing, and high-resolution manometry (HREM). Patient characteristics were summarized with counts and percentages, means and SD, or medians and interquartile ranges (IQR) as appropriate to scale and distribution. The characteristics of those with and without rejection diagnoses was compared with Fisher's exact test for categorical variables and the Wilcoxon rank sum test for quantitative variables. Analyses were conducted in the open source R software environment. RESULTS: 60 of 64 patients (94%) were alive at one-year post- lung transplant. The majority of patients had bilateral transplants and were Caucasian males with average age of 59 years. At one year there were 12 deaths. Patients with a diagnosis of acute rejection were less likely to exhibit gastroparesis (1% vs. 9%) and abnormal pH (28% vs. 45%). Acute rejection patients were more likely to exhibit GERD (53% vs. 45%), more non-acid episodes (median 19 vs. 15.5), higher reported symptom association (51% vs. 41%), on impedance pH; abnormal UES (56% vs. 45%), and abnormal body (86% vs. 77%) on HREM (Table 1). However, no differences were statistically significant (all P &gt; .18), and conclusions cannot be generalized. CONCLUSION: Patients undergoing lung transplantation were found to have a high incidence of abnormal gastroesophageal motility studies, including both acid and non-acid reflux. Developing a protocolized pre- and post- transplant motility evaluation process for lung transplant patients may help identify those at greatest risk for acute rejection. Continued evaluation post-transplantation to establish therapeutic interventions in both acid and non-acid reflux patients to prevent lung transplant rejection appears warranted.

OR20 Nhlbi-sponsored prospective study to validate the use of donor-derived cell-free DNA to detect lung-transplant rejection - interim results

OR20 Nhlbi-sponsored prospective study to validate the use of donor-derived cell-free DNA to detect lung-transplant rejection - interim results

Acute cellular rejection in lung transplantation

Lung transplantation is an important therapy for end-stage respiratory failure in patients who have exhausted other therapeutic options. The lung is unique among solid-organ transplants in that it is exposed to the outside environment, and undergoes continuous stimulation from infectious and non-infectious agents, which may play a part in upregulating the immune response to the allograft. Despite induction immunosuppression and the use of aggressive maintenance regimens, acute allograft rejection is still a major problem, especially in the first year after transplant, with important diagnostic and therapeutic challenges. As well as being responsible for early graft failure and death, acute rejection also initiates alloimmune responses that predispose patients to chronic lung allograft dysfunction, in particular bronchiolitis obliterans syndrome. Cellular responses to human leukocyte antigens (HLAs) on the allograft have traditionally been considered the main mechanism of acute rejection, although the influence of humoral immunity is increasingly recognised. Here, we present two cases of acute cellular rejection (ACR) in the early post-transplant period and review the pathophysiology, diagnosis, clinical presentation and treatment of ACR.