BACKGROUND: Exposure to air pollution post lung transplant has been shown to decrease graft and patient survival. This study examines the impact of air pollution exposure in the first 3 months post-transplant on baseline (i.e. highest) forced expiratory volume in 1s (FEV1) achieved and development of chronic lung allograft dysfunction (CLAD).
METHODS: Double-lung transplant recipients (n=82) were prospectively enrolled for comprehensive indoor and personal environmental monitoring at 6- and 12-weeks post-transplant and followed for >4 years. Associations between clinical and exposure variables were investigated using an exposomics approach followed by analysis with a Cox Proportional Hazards model. Multivariable analyses were used to examine the impact of air pollution on baseline % predicted FEV1 (defined as the average of the 2 highest values achieved post-transplant) and risk of CLAD.
RESULTS: Multivariable analysis revealed a significant inverse relationship between personal black carbon (BC) levels and baseline % FEV1. The multivariable model indicated that patients with higher-than-median exposure to BC (>350 ng/m3) attained a baseline % FEV1 that was 8.8% lower than those with lower-than-median BC exposure (p = 0.019). Cox proportional hazards model analysis revealed that patients with high personal BC exposure had a 2.4 times higher hazard risk for CLAD than patients with low BC exposure (p = 0.045).
CONCLUSIONS: Higher personal BC levels during the first 3 months post-transplant decreases baseline FEV1 and doubles the risk of CLAD. Strategies to reduce BC exposure early following lung transplant may help improve lung function and long-term outcomes.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) limits survival following lung transplant, but substantial lung damage occurs before diagnosis by traditional methods. We hypothesized that small airway gene expression patterns could identify CLAD risk before spirometric diagnosis and predict subsequent graft failure.
METHODS: Candidate genes from 4 rejection-associated transcript sets were assessed for associations with CLAD or graft failure in a derivation cohort of 156 small airway brushes from 45 CLAD cases and 37 time-matched controls with >1-year stable lung function. Candidate genes not associated with CLAD and time to graft failure were excluded, yielding the Airway Inflammation 2 (AI2) gene set. Area under the receiver operating curve (AUC) for CLAD and competing risks of death or graft failure were assessed in an independent validation cohort of 37 CLAD cases and 37 controls.
RESULTS: Thirty-two candidate genes were associated with CLAD and graft failure, comprising the AI2 score, which clustered into 3 subcomponents. The AI2 score identified CLAD before its onset, in early and late post-CLAD brushes, as well as in the validation cohort (AUC 0.69-0.88). The AI2 score association with CLAD was independent of positive microbiology, CLAD stage, or CLAD subtype. However, transcripts most associated with CLAD evolved over time from CLAD onset. The AI2 score predicted time to graft failure and retransplant-free survival in both cohorts (p ≤ 0.03).
CONCLUSIONS: This airway inflammation gene score is associated with CLAD development, graft failure, and death. Future studies defining the molecular heterogeneity of airway inflammation could lead to endotype-targeted therapies.

BACKGROUND: Donor human leukocyte antigen (HLA)-specific antibodies (DSA) and non-HLA antibodies can cause allograft injury, possibly leading to chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx). It remains unclear whether these antibodies are produced locally in the graft or derived solely from circulating plasma cells. We hypothesized that DSA and non-HLA antibodies are produced in CLAD lungs.
METHODS: Lung tissue was prospectively collected from 15 CLAD patients undergoing re-LTx or autopsy. 0.3g of fresh lung tissue was cultured for 4 days without or with lipopolysaccharide or with CD40L: resulting lung culture supernatant (LCS) was sampled. Protein eluate was obtained from 0.3g of frozen lung tissue. Mean fluorescence intensity (MFI) of DSA and non-HLA antibodies was measured by Luminex and antigen microarray, respectively.
RESULTS: LCS from all 4 patients who had serum DSA at lung isolation were positive for DSA, with higher levels measured after lung tissue stimulation with CD40L (CD40L+LCS). Of these, only 2 had detectable DSA in the lung eluate. MFI of non-HLA antibodies from CD40L+LCS correlated with those from lung eluate, but not with those from sera. Flow cytometry showed higher frequencies of activated lung B cells for patients whose CD40L+LCS was positive for DSA (n=4) or high non-HLA antibodies (n=6) compared to those with low local antibodies (n=5). Immunofluorescence staining showed CLAD lung lymphoid aggregates with local antibodies contained larger numbers of IgG+ plasma cells and greater IL-21 expression.
CONCLUSIONS: We show that DSA and non-HLA antibodies can be produced within activated B cell-rich lung allografts.

Plasma-soluble (s)HLA-G and sHLA-E are immunoregulatory proteins that balance the activation of NKG2A+ immune cells. In lung-transplant recipients (LTRs), dysregulated NKG2A+ natural killer cell responses may result in high-level human cytomegalovirus (HCMV) replication as well as chronic lung allograft dysfunction (CLAD), and especially the development of rapidly deteriorating CLAD is associated with high mortality. We thus analyzed the kinetics and function of sHLA-G and sHLA-E in follow-up samples of N = 76 LTRs to evaluate whether these immunoregulatory proteins are associated with the risk for CLAD and high-level HCMV replication. Here, we demonstrate that rapidly deteriorating CLAD LTRs are hallmarked by continually low (<107 ng/ml) sHLA-G levels. In contrast, high sHLA-E levels were associated with the following development of high-level (>1,000 copies/ml) HCMV episodes. Thus, sHLA-G and sHLA-E may serve as novel biomarkers for the development of rapidly deteriorating CLAD and high-level HCMV replication in LTRs.

Ex vivo lung perfusion (EVLP) has demonstrated encouraging short- and medium-term outcomes with limited data available on its long-term outcomes. This study assesses (1) EVLP long-term outcomes and (2) EVLP era-based sub-analysis in addition to secondary outcomes of recipients with EVLP-treated donor lungs compared with recipients of conventionally preserved donor lungs in unmatched and propensity score-matched cohorts. Double lung transplants performed between 1st January 2012 and 31st December 2021 were included. A total of 57 recipients received EVLP-treated lungs compared to 202 unmatched and 57 matched recipients who were subjected to non-EVLP-treated lungs. The EVLP group had a significantly lower mean PaO2/FiO2 ratio and significantly higher mean BMI than the non-EVLP group in the unmatched and matched cohorts. The proportion of smoking history in the unmatched cohort was significantly higher in the EVLP group, while a similar smoking history was demonstrated in the matched cohorts. No difference was demonstrated in overall freedom from death and retransplantation between the groups in the unmatched and matched cohorts (unmatched: hazard ratio (HR) 1.28, 95% confidence interval (CI) 0.79-2.07, P = 0.32; matched: HR 1.06, 95% CI 0.59-1.89). P = 0.89). In the unmatched cohort, overall freedom from chronic allograft dysfunction (CLAD) was significantly different between the groups (HR 1.64, 95% CI 1.07-2.52, P = 0.02); however, the cumulative CLAD incidence was similar (HR 0.72, 95% CI 0.48-1.1, P = 0.13). In the matched cohort, the overall freedom from CLAD (HR 1.69, 95% CI 0.97-2.95, P = 0.06) and cumulative CLAD incidence (HR 0.91, 95% CI 0.37-2.215, P = 0.83) were similar between the groups. The EVLP era sub-analysis of the unmatched cohort in 2012-2014 had a significantly higher cumulative CLAD incidence in the EVLP group; however, this was not demonstrated in the matched cohort. All secondary outcomes were similar between the groups in the unmatched and matched cohorts. In conclusion, transplantation of marginal donor lungs after EVLP evaluation is non-detrimental compared to conventionally preserved donor lungs in terms of mortality, retransplantation, cumulative CLAD incidence, and secondary outcomes. Although the unmatched EVLP era of 2012-2014 had a significantly higher cumulative CLAD incidence, no such finding was demonstrated in the matched cohort of the same era.

BACKGROUND: T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. International Society for Heart and Lung Transplantation grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T-cell composition of A1 ACR lesions may have prognostic value; therefore, protein-level and epigenetic techniques were applied to transbronchial biopsy tissue to determine whether differential T-cell infiltration in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD.
METHODS: Sixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n = 13) and those remaining CLAD-free for 5 or more years (n = 49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n = 16; 8 early-CLAD and 8 no early-CLAD). Immunofluorescence was used to quantify CD4+, CD8+, and FOXP3+ cells. Separately, CD3+ cells were fluorescently labeled, micro-dissected, and the degree of Treg-specific demethylated region methylation was determined.
RESULTS: PhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of lung transplantation showed greater CD8+ T cell infiltration compared to those who remained CLAD-free for 5 or more years.
CONCLUSIONS: In asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of worse long-term outlook.

BACKGROUND: Due to development of chronic lung allograft dysfunction (CLAD), prognosis for patients undergoing lung transplantation (LTx) is still worse compared to other solid organ transplant recipients. Treatment options for slowing down CLAD progression are scarce with extracorporeal photopheresis (ECP) as an established rescue therapy. The aim of the study was to identify characteristics of responders and non-responders to ECP treatment, assess their survival, lung function development and by that define the subset of patients who should receive early ECP treatment.
METHODS: We performed a retrospective study of all LTx patients receiving ECP treatment at the University Hospital Zurich between January 2010 and March 2020. Patients were followed-up for a maximum period of 5 years. Mortality and lung function development were assessed by CLAD stage and by CLAD subtype before initiation of ECP treatment.
RESULTS: Overall, 105 patients received at least one ECP following LTx. A total of 57 patients (61.3%) died within the study period with a median survival of 15 months. Mortality was 57% for patients who started ECP at CLAD1, 39% for CLAD2, 93% for CLAD3, and 90% for CLAD4 (p < 0.001). Survival and lung function development was best in young patients at early CLAD stages 1 and 2. Response to ECP treatment was worst in patients with CLAD-RAS/mixed subtype (14.3%) and patients with ECP initiation in CLAD stages 3 (7.1%) and 4 (11.1%). Survival was significantly better in a subset of patients with recurrent acute allograft dysfunction and earlier start of ECP treatment (105 vs 15 months).
CONCLUSIONS: In this retrospective analysis of a large group of CLAD patients treated with ECP after LTx, early initiation of ECP was associated with better long-term survival. Besides a subset of patients suffering of recurrent allograft dysfunction, especially a subset of patients defined as responders showed an improved response rate and survival, suggesting that ECP should be initiated in early CLAD stages and young patients. ECP might therefore prevent long-term disease progression even in patients with CLAD refractory to other treatment options and thus prevent or delay re-transplantation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the leading long-term cause of poor outcomes after transplant and manifests by fibrotic remodeling of small airways and/or pleuroparenchymal fibroelastosis. This study evaluated the effect of pirfenidone on quantitative radiographic and pulmonary function assessment in patients with CLAD.
METHODS: We performed a single-center, 6-month, randomized, placebo-controlled trial of pirfenidone in patients with CLAD. Randomization was stratified by CLAD phenotype. The primary outcome for this study was change in radiographic assessment of small airways disease, quantified as percentage of lung volume using parametric response mapping analysis of computed tomography scans (PRMfSAD); secondary outcomes included change in forced expiratory volume in 1 second (FEV1), change in forced vital capacity (FVC), and change in radiographic quantification of parenchymal disease (PRMPD). Linear mixed models were used to evaluate the treatment effect on outcome measures.
RESULTS: The goal enrollment of 60 patients was not met due to the coronavirus disease of 2019 pandemic, with 23 patients included in the analysis. There was no significant difference over the study period between the pirfenidone vs placebo groups with regards to the observed change in PRMfSAD (+4.2% vs -0.4%; p = 0.22), FEV1 (-3.5% vs -3.6%; p = 0.97), FVC (-1.9% vs -4.6%; p = 0.41), or PRMPD (-0.6% vs -2.5%; p = 0.30). The study treatment tolerance and adverse events were generally similar between the pirfenidone and placebo groups.
CONCLUSIONS: Pirfenidone had no apparent impact on radiographic evidence of allograft dysfunction or pulmonary function decline in a single-center randomized trial of CLAD patients that did not meet enrollment goals but had an acceptable tolerance and side-effect profile.

Our study aims to evaluate the effect of everolimus treatment on lung function in lung transplant (LT) patients with established chronic lung allograft dysfunction (CLAD).
METHODS: This retrospective study included LT patients in two reference LT units who started everolimus therapy to treat CLAD from October 2008 to October 2016. We assessed the variation in the maximum forced expiratory volume in the first second (FEV1) before and after the treatment.
RESULTS: Fifty-seven patients were included in this study. The variation in the FEV1 was -102.7 (149.6) mL/month before starting everolimus compared to -44.7 (109.6) mL/month within the first three months, +1.4 (63.5) mL/month until the sixth month, and -7.4 (46.2) mL/month until the twelfth month (p < 0.05). Glomerular filtrate remained unchanged after everolimus treatment [59.1 (17.5) mL/min per 1.73 m2 at baseline and 60.9 (19.6) mL/min per 1.73 m2, 57.7 (20.5) mL/min per 1.73 m2, and 57.3 (17.8) mL/min per 1.73 m2, at 1, 3, and 6 months, respectively] (p > 0.05). Everolimus was withdrawn in 22 (38.6%) patients. The median time to withdrawal was 14.1 (5.5-25.1) months.
CONCLUSIONS: This study showed an improvement in FEV1 decline in patients with CLAD treated with everolimus. However, the drug was withdrawn in a high proportion of patients.

BACKGROUND: Lung transplant recipients are traditionally monitored with pulmonary function testing (PFT) and lung biopsy to detect post-transplant complications and guide treatment. Plasma donor-derived cell free DNA (dd-cfDNA) is a novel molecular approach of assessing allograft injury, including subclinical allograft dysfunction. The aim of this study was to determine if episodes of extreme molecular injury (EMI) in lung transplant recipients increases the risk of chronic lung allograft dysfunction (CLAD) or death.
METHODS: This multicenter prospective cohort study included 238 lung transplant recipients. Serial plasma samples were collected for dd-cfDNA measurement by shotgun sequencing. EMI was defined as a dd-cfDNA above the third quartile of levels observed for acute rejection (dd-cfDNA level of ≥5% occurring after 45 days post-transplant). EMI was categorized as Secondary if associated with co-existing acute rejection, infection or PFT decline; or Primary if not associated with these conditions.
RESULTS: EMI developed in 16% of patients at a median 343.5 (IQR: 177.3-535.5) days post-transplant. Over 50% of EMI episodes were classified as Primary. EMI was associated with an increased risk of severe CLAD or death (HR: 2.78, 95% CI: 1.26-6.22, p = 0.012). The risk remained consistent for the Primary EMI subgroup (HR: 2.34, 95% CI 1.18-4.85, p = 0.015). Time to first EMI episode was a significant predictor of the likelihood of developing CLAD or death (AUC=0.856, 95% CI=0.805-0.908, p < 0.001).
CONCLUSIONS: Episodes of EMI in lung transplant recipients are often isolated and may not be detectable with traditional clinical monitoring approaches. EMI is associated with an increased risk of severe CLAD or death, independent of concomitant transplant complications.