INTRODUCTION:Prolonged invasive mechanical ventilation (IMV) after lung transplantation is an appealing early prognostic outcome as it can be reproducibly assessed both prospectively and retrospectively. Whether use of IMV at 72 h after lung transplantation is associated with post-transplant graft survival is unknown. METHODS:We performed a retrospective cohort study of 1511 participants in the multi-center Lung Transplant Outcomes Group cohort (2011-2018). Using Cox proportional hazards models and restricted mean survival time, we investigated whether IMV at 72 h was associated with post-transplant graft survival. We secondarily evaluated whether IMV at 72 h was concordant with severe primary graft dysfunction (PGD). RESULTS:Participants requiring IMV at 72 h after transplant were sicker at transplantation (higher lung allocation score [LAS], increased extracorporeal membrane oxygenation, or IMV bridge) and more likely to have severe PGD. Use of IMV at 72 h was associated with 55% (95% CI 26%-92%) increased hazards of death or re-transplantation after adjustment for age, ECMO, diagnosis, LAS, and intra-operative transfusion. The association between IMV and graft survival was modified by severe PGD (p-for interaction 0.002) but not by pre-transplant ECMO (p-for interaction 0.88) or pre-transplant IMV (p-for interaction 0.92). IMV was associated with increased risk of death or re-transplantation among those with PGD (HR 2.35, 95% CI 1.43-3.85) but not among those without PGD (HR 1.04, 95% CI 0.77-1.41). CONCLUSION:Requirement of IMV at 72 h is an important early post-transplant outcome associated with post-transplant survival. This appears driven by those with severe PGD.

Background:We aimed to identify data-driven FEV1 trajectory phenotypes post-chronic lung allograft dysfunction (CLAD), relate these phenotypes to patient factors and future graft loss, and develop a classification approach for prospective patients. Methods:We studied adult first lung recipients with probable CLAD from two prospective multicenter cohorts: CTOT-20 (n=206) and LTOG (n=1418). FEV1 trajectories over the first nine months post-CLAD were characterized using joint latent class mixed models, jointly modelling time-to-graft loss to account for informative censoring. Models were fit independently in both cohorts and also only among LTOG bilateral recipients. A classification and regression tree (CART) model was derived in LTOG bilateral recipients and applied to CTOT-20 bilateral recipients. Findings:Four distinct early FEV1 trajectory classes were identified in CTOT-20, with large differences in nine-month graft loss (72·3%, 31·1%, 2·2%, 0%). In LTOG, similar trajectory patterns were reproduced, with an additional class demonstrating early post-CLAD FEV1 improvement. Among bilateral recipients, trajectory classes showed a clear risk gradient, including a high-risk class with 100% graft loss and a low-risk class with no early graft loss. A CART model incorporating clinical and spirometric variables demonstrated good discrimination in LTOG bilateral recipients (multiclass AUC 0·85) and consistent class assignment and trajectory patterns when applied to CTOT-20. Interpretation:We identified reproducible, clinically meaningful early post-CLAD FEV1 trajectory phenotypes with differential graft loss risk. These phenotypes and a pragmatic classification tool may support risk stratification, trial enrichment, and improved prognostication for patients and clinicians. Funding:National Institutes of Health, Cystic Fibrosis Foundation.

Pulmonary ischemia-reperfusion injury (IRI) is a major cause of primary graft dysfunction in lung transplantation. Porcine models better simulate physiological conditions and are important for pre-clinical studies; however, comprehensive immune assessment of porcine lungs in IRI has not been performed. We aimed to evaluate immune cells and activation states in porcine IRI models and hypothesized that myeloid and lymphoid cells would infiltrate and activate following IRI. Two sets of porcine orthotopic lung transplants were performed: a 4 h reperfusion (n = 7) and a 72 h survival model (n = 6). Both were compared to a control group without lung injury (n = 6). Lung samples were processed into single cell suspensions and cryopreserved. Thawed samples were stained with anti-porcine antibodies and analyzed by flow cytometry. Absolute counts of neutrophils and CD14+ monocytes increased in the allograft at 4 h and remained stable over 72 h post-transplant. CD14-CD163+ monocytes and conventional dendritic cells continued to increase by 72 h post-transplant. Lymphoid cell numbers were unchanged overall, but T cells showed increased CD25 expression and a memory phenotype at 4 h. Our analysis revealed early myeloid cell infiltration post-IRI which developed into increased inflammatory and antigen-presenting cell populations by 72 h post-transplant. A transient rise in T cell activation markers was noted, consistent with rodent models. Our findings contribute to our understanding of immunological events in porcine pulmonary IRI, a model that better mimics the clinical setting. Our flow cytometry panels allow for improved immunologic analyses of porcine models in preclinical transplantation research.

Abstract Background Chronic lung allograft dysfunction (CLAD) is the major cause of late mortality after lung transplantation and includes two principal phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). RAS and other phenotypes with RAS-like opacities (RLO) on chest imaging have a poorer prognosis. Despite clear clinical and pathological differences, molecular distinctions between phenotypes remain poorly defined. We aimed to explore gene transcriptional profiles across CLAD phenotypes and relevant controls. Methods We performed bulk RNA sequencing on explanted lung tissue from 45 lung transplant recipients with end-stage CLAD (20 with RLO and 25 without RLO). Samples from twenty-seven control donor and lobectomy lungs and sixteen idiopathic pulmonary fibrosis (IPF) lungs served as comparators. Non-negative matrix factorization (NMF) was used to identify latent transcriptomic signatures, which were correlated with clinical, radiologic, and histopathologic features. Results NMF identified seven distinct gene signatures that segregated CLAD phenotypes. RLO-CLAD lungs were enriched for extracellular matrix remodeling and B-cell/plasma cell–associated signatures, overlapping partly with IPF, whereas non-RLO-CLAD showed relative enrichment of epithelial injury and surfactant-response pathways. Signatures related to epithelial homeostasis and ciliary/microtubule function were progressively reduced from control lungs to non-RLO-CLAD and were most suppressed in RLO-CLAD. Conclusions RLO-CLAD and non-RLO-CLAD, aligning with RAS and BOS phenotypes, show distinct transcriptomic signatures. RLO-CLAD is characterized by profibrotic and humoral immune signatures with profound epithelial dysfunction, whereas non-RLO-CLAD shows relative enrichment of epithelial injury responses. These data provide molecular stratification of CLAD and support the development of phenotype-specific biomarkers and targeted therapies.

BACKGROUND:Invasive fungal infections (IFIs) are of major concern in lung transplant recipients. Although Aspergillus fumigatus is the predominant pathogen, non-A fumigatus mould infections (NAFMIs) also account for a substantial proportion of IFIs. We aimed to describe the epidemiology, clinical spectrum, risk factors, and outcomes of NAFMIs after lung transplantation. METHODS:We performed a retrospective, international, multicentre, case-control study across nine tertiary care centres in Canada and Europe. We included adult (aged ≥18 years) lung transplant recipients. Cases were defined as individuals with proven or probable IFIs caused by a mould other than A fumigatus (NAFMI group). Exclusion criteria were a concomitant infection with A fumigatus and a non-A-fumigatus mould and isolated positivity of galactomannan in serum or bronchoalveolar lavage. Two controls per case were included: one with IFI due to A fumigatus (A fumigatus control group) and one free of any IFI (IFI-free control group). 1:1:1 matching was done according to centre and time after transplantation (for IFI-free controls). We reviewed medical records to assess the clinical and radiological presentation and clinical outcomes (including 12-week therapeutic response, 12-month survival, development of chronic lung allograft dysfunction [CLAD] and post-transplant lymphoroliferative disorder) and used logistic regression models to assess risk factors for IFIs. The primary outcomes were the description of the microbial aetiologies of NAFMI, survival and CLAD 12-month post-IFI, and identification of factors associated with NAFMI. FINDINGS:Between Jan 1, 2010, and Jan 1, 2020, we included 120 individuals in the NAFMI group and 240 individuals in the control groups (n=120 in each control group). Median age at transplantation was 56 years (IQR 44-63); 170 (47%) of 360 participants were female and 190 (53%) were male. Only eight (7%) of 120 individuals in the NAFMI group had a polyfungal or mixed infection. Aspergillus flavus (26 [22%] of 120) and Aspergillus niger (24 [20%]) were the most common pathogens in the NAFMI group, followed by Scedosporium apiospermum (9[8%]). Respiratory symptoms predominated in individuals with IFI, and clinical presentations were similar in individuals with NAFMI and A fumigatus IFI. 12-month mortality was comparable across groups (NAFMI: 37/118 [31%], A fumigatus: 36/117 [31%], p=1; IFI-free controls: 25/120 [21%], p=0·11 compared with NAFMI). CLAD at 12-month post-IFI occurred at similar frequencies in cases and controls (NAFMI: 13/108 [12%], A fumigatus 17/111 [15%], p=0·69; IFI-free controls: 8/110 [7%], p=0·36 compared with NAFMI). Pre-transplant immunosuppression (adjusted odds ratio 3·82, 95% CI 1·55-9·38; p=0·0039) and pre-transplant intensive care unit stay (3·07, 1·01-9·34; p=0·048) were independently associated with NAFMI occurrence, when compared with IFI-free controls. INTERPRETATION:NAFMIs mostly involve non-fumigatus Aspergillus spp. Diagnostic features of NAFMIs are similar to those of A fumigatus infections; pre-transplant immunosuppression can increase the risk of NAFMI onset. These findings reinforce the importance of a thorough investigation when a mould is isolated from the airways after lung transplantation. FUNDING:None.

Chronic lung allograft dysfunction (CLAD) is the major barrier for long-term survival in lung transplant recipients (LTRs). CLAD remains a diagnosis of exclusion with poor responses to therapies. A molecular diagnostic for CLAD is needed to risk-stratify LTRs for prognosis and identify new targets to mitigate CLAD progression. We used weighted gene correlation network analysis on the airway brush-derived airway transcriptome to identify immune pathways and markers relevant to CLAD. Weighted gene correlation network analysis was performed on RNA sequencing from airway brushings of 37 LTRs with CLAD compared with 37 stable LTRs. We analyzed gene coexpression networks (modules) for their biological significance and association with CLAD. Three gene modules were positively correlated with CLAD, its severity, allograft dysfunction, and survival. These enriched components of the acute phase response, type 1 adaptive immunity, and innate immunity, respectively. A fourth module correlated with protection and was inversely correlated with the other modules. We validated our findings by identification of downstream protein and eicosanoid levels in the bronchoalveolar lavage, and an external validation cohort where module expression differentiated LTRs with CLAD and correlated with worse survival. The CLAD airway transcriptome enriches for coexpression networks associated with network modules that correlate with allograft dysfunction and survival.

Donor-derived cell-free DNA (dd-cfDNA) is a validated, highly sensitive, plasma molecular biomarker of allograft injury after solid organ transplantation. Robust experiences with dd-cfDNA testing after kidney and heart transplantation have generated interest in this biomarker within the lung transplantation (LTx) community. A growing body of evidence now provides increased insight into dd-cfDNA utility for molecular monitoring of lung allograft health after transplantation. The expanding understanding of lung allograft injury to appropriately frame the advancing role of dd-cfDNA in the evolution of the diagnostic approach after LTx is described. Performance characteristics of both laboratory-based shotgun-sequenced testing from the Genome Transplant Dynamics (GTD) and Genomic Research Alliance for Transplantation (GRAfT) consortia, as well as commercially available central lab-based algorithmic next-generation sequenced dd-cfDNA tests for lung transplant recipients (LTR) (AlloSure, CareDx and Prospera, Natera) are described. Kinetics of dd-cfDNA in LTRs over time, in multiple different clinical scenarios, from several investigator groups are aggregated. Phenotypes of lung allograft injury, such as acute lung allograft dysfunction, and associated dd-cfDNA patterns and performance are identified in alignment with established definitions and evolving molecular injury insights. Certain patterns of molecular injury that may predict long-term outcomes including chronic lung allograft dysfunction and mortality are examined. Lastly, clinical approaches to testing and interpretation of dd-cfDNA results in LTRs, a practical approach to using dd-cfDNA, and a rational framework for interpreting dd-cfDNA results in LTRs are presented.

BACKGROUND:Lung transplant recipients with lower club cell secretory protein (CCSP) levels in bronchoalveolar lavage fluid (BALF) early post-transplantation are at increased risk for chronic lung allograft dysfunction (CLAD). For CLAD risk stratification, we previously identified a potential risk threshold for reduced CCSP (protein-normalised CCSP <8.63 ng·µg-1). Here, we aim to validate this association in an independent patient set from a prospective observational cohort. METHODS:Total protein and CCSP were quantified in 1481 BALF samples collected over the first post-transplant year from 353 patients (validation cohort). A Cox model tested the association between time to first CCSP <8.63 ng·µg-1 and CLAD. If this threshold did not validate, we prespecified combining the discovery and validation cohorts to rederive a reduced CCSP risk threshold considering a larger number of CLAD events. In a subset, gene expression analyses were performed on allograft biopsies to examine molecular alterations at the time of reduced CCSP. RESULTS:BALF CCSP <8.63 ng·µg-1 in the first post-transplant year was not significantly associated with CLAD in the validation cohort (hazard ratio (HR) 1.41; p=0.208). However, in the combined cohort, a dense grid search, including the previously identified threshold of 8.63 ng·µg-1, revealed that the threshold of 8.63 ng·µg-1 had the largest HR for CLAD. Iterative resampling demonstrated robust reproducibility of the association between BALF CCSP <8.63 ng·µg-1 and CLAD risk across the combined cohort. Biopsies corresponding to CCSP <8.63 ng·µg-1 had a pro-inflammatory profile. CONCLUSIONS:Early post-transplant reductions in BALF CCSP identify lung recipients at increased CLAD risk and may associate with heightened allograft inflammation.

Rationale: Chronic lung allograft dysfunction (CLAD) hinders lung transplant success. A 2019 consensus refined CLAD diagnosis, introducing probable or definite CLAD based on persistence of lung function decline. Outcomes and risks for probable CLAD remain uncertain. Objectives: We sought to determine the prognosis and clinical risks for probable CLAD in a prospective multicenter cohort. Methods: Clinical Trials in Organ Transplantation-20 included 745 CLAD-eligible adult lung recipients at five centers and applied rigorous methods to prospectively adjudicate probable CLAD. The impact of probable CLAD on graft loss was determined using a Cox model that considered CLAD as a time-dependent covariate. Regularized Cox modeling with least absolute shrinkage and selection operator (LASSO) penalty was used to evaluate donor or recipient characteristics and the occurrence and timing of posttransplant events as probable CLAD risks. Similar analyses were performed for definite CLAD. Measurements and Main Results: Probable CLAD occurred in 29.7% of patients at 3 years posttransplant and conferred a marked increase in risk for graft loss (unadjusted hazard ratio = 4.38, P < 0.001). Most patients (80%) with probable CLAD progressed to definite CLAD. Cytomegalovirus infection and, specifically, late presence (>90 d posttransplant) of donor-specific alloantibodies, acute rejection, acute lung injury, or organizing pneumonia contributed the greatest independent information about probable CLAD risk. Definite CLAD risks were similar. Conclusions: Probable CLAD identifies patients at high risk for graft loss, supporting prospective identification of this condition for early initiation of CLAD-directed interventions. More effective strategies to prevent posttransplant cytomegalovirus, inhibit allospecific immunity, and reduce tissue injury are needed to reduce probable CLAD and improve lung recipient survival. Clinical trial registered with www.clinicaltrials.gov (NCT02631720).

Long-term survival in lung transplantation is limited by the eventual development of chronic lung allograft dysfunction (CLAD), resulting in the loss of the transplanted lung. We hypothesized that analysis of the pulmonary T cell transcriptome would reveal novel T cell populations that drive CLAD pathogenesis. Human lung tissue samples (9 CLAD, 5 donor as healthy control) were obtained and processed into single cell suspensions. T cells were enriched through magnetic selection of CD45+ leukocytes followed by fluorescence-activated cell sorting. Sorted cells then underwent 5’ single cell RNA sequencing (scRNAseq) using 10x Genomics. scRNAseq data was analyzed using Seurat. T cell clusters of interest were validated at the protein level using spectral flow cytometry Comparison between lung samples revealed a cluster that was only present in CLAD samples (6/9 CLAD lungs vs. 0/5 donor lungs, Fig A), characterized by RNA expression of CD8, CD27 and HAVCR2 (TIM-3, Fig B). Flow cytometry analysis of T cells from CLAD lungs (n = 3) and donor lungs (n = 4) similarly showed that CD8+ CD27+TIM-3+ T cells were uniquely present in CLAD lungs (Fig C). Utilizing pseudotime trajectroy analysis to examine cellular changes, this cluster appears to be terminally differentiated (Fig D). We conclude that a CD8+ T cell population expressing CD27 and TIM-3 is enriched in CLAD lung T cells. This phenotype is suggestive of a memory/exhaustion phenotype that could play an important role in CLAD. New Frontiers in Research Fund - Transformation Grant Transplantation Immunology (TRAN)

BACKGROUND:Baseline lung allograft dysfunction (BLAD), defined as failure to achieve ≥ 80% predicted spirometry after lung transplant, is associated with reduced survival. This study aimed to determine the prevalence and character of computed tomography (CT) abnormalities in BLAD. METHODS:In this retrospective cohort study, we analyzed adult first-time double-lung transplant recipients (12/2017-10/2021) who had 12-month CT chest and concurrent BLAD/non-BLAD status assigned. Three radiologists used a semi-quantitative ordinal score to evaluate ground glass opacities (GGO), reticulation, consolidation, pleural effusion, bronchiectasis, and air-trapping. Machine learning-trained lung texture analysis provided CT radiomic data: CT-measured total lung capacity (CTTLC), pulmonary vessel volume (PVV), GGO, reticulation, and hyperlucency. Parametric response mapping measured functional small airways disease. Receiver operating characteristic analysis and logistic regression identified radiologic features of BLAD. RESULTS:BLAD patients (n = 59, 46%) had longer intubation duration, longer index hospitalization post-transplant, and lower donor-to-recipient total lung capacity (TLC) ratio than non-BLAD patients (n = 69, 54%). Radiologist-assessed scoring identified more pleural effusions in BLAD with no significant differences in GGO or air trapping. Computer-aided CT demonstrated more reticulation, GGO, and parenchymal density in BLAD, with no difference in functional small airways disease. CTTLC indexed for height was lower in BLAD, while PVV was higher. PVV was significantly associated with BLAD in univariable analysis (OR 2.30, 95% CI 1.38-3.83, p < 0.001), and remained strong after adjusting for age, sex, and native disease (OR = 2.65,95% CI 1.45-4.84, p = 0.002). CONCLUSIONS:Computer-aided CT elicited structural changes in BLAD not captured by limited-slice radiologist review. CTTLC and PVV were the strongest radiologic predictors of BLAD, likely reflecting restrictive physiology and vascular remodeling.

Clinical trials in lung transplantation have been hindered by a lack of clarity on the formulation and significance of endpoints for evaluating therapeutic efficacy. To address this challenge, a multidisciplinary working group from the International Society for Heart and Lung Transplantation developed consensus recommendations on endpoints beyond mortality. These endpoints include primary graft dysfunction (PGD), chronic lung allograft dysfunction (CLAD), acute cellular rejection (ACR), antibody-mediated rejection (AMR), immunosuppression-related complications, patient-reported outcomes (PROs), and pediatric-specific considerations. For each endpoint, a subgroup reviewed measurement best practices, assessed links to clinical benefit, and evaluated the evidence supporting their utility in clinical trial settings. Consensus was established through a Delphi process involving three rounds of voting. This document provides practical guidance for operationalizing these endpoints and outlines their optimal use in clinical trials. By standardizing trial design, these recommendations aim to accelerate the development of urgently needed therapies to improve lung transplantation outcomes.