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Cumulative epithelial injuries after lung transplantation (LT) play a major role in CLAD pathogenesis. Epithelial club cells are specifically depleted in CLAD. We hypothesized that club cell apoptosis occurs in CLAD and at an earlier injury stage during B-grade airway-centered ACR. Lung biopsies were collected at the time of retransplantation from 20 consecutive patients undergoing a second LT for CLAD and from 13 control lungs (4 excess donor lung tissues, 9 lobectomies). Transbronchial biopsies (TBB) and bronchoalveolar lavage (BAL) were obtained from LT patients (post first bilateral LT 2010-2015) at the time of B-grade ACR =0 or ≥1 (categorized as stable or unstable based on a concurrent >10% drop in lung function), within 25 months post-LT. Biopsies were immunostained with antibodies for epithelial cells (Pan-cytokeratin), club cells (club cell secretory protein, CCSP) and a cell death detection kit for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptotic cells. M30 and M65, fragments of cytokeratin-18 released during epithelial cell apoptosis and total cell death, respectively, were measured in the BAL using ELISA. Compared to controls, CLAD lungs had a significantly higher frequency of apoptotic cells (p=0.02) and a higher frequency of apoptotic club cells (p=0.08) (figure 1A-C). Club cell frequency was lower in TBBs from unstable ≥B1 ACR patients compared to stable (p=0.10) (figure 1D). However, a drop in lung function occurring at the time of ≥B1 ACR showed no association with frequency of apoptotic epithelial cells, apoptotic club cells, or the concentration of M30 or M65 in the BAL. Increased epithelial cell apoptosis were observed in lungs affected by CLAD but were not identified during airway ACR, possibly due to the earlier phase of the inflammatory process or potential sampling error of the small biopsies. Further study is needed to understand the timing of epithelial cell injury and death in CLAD pathogenesis. [ABSTRACT FROM AUTHOR]

Acute fibrinous and organizing pneumonia (AFOP) is an unusual histopathologic pattern characterized by the formation of intra-alveolar plugs of fibrin deposition and associated organizing pneumonia. AFOP is considered to be a form of rejection and portends a dismal prognosis. Here, we present the case of a young male patient who initially underwent a double lung transplantation for cystic fibrosis. After 42 months of regular follow-up, he experienced rapidly progressive respiratory failure. Acute rejection and opportunistic lung infections were suspected. The clinical conditions rapidly deteriorated despite treatment with broad-spectrum antibiotics and high-dose steroids. Therefore, AFOP was suspected owing to: 1) acute clinical presentation; 2) pulmonary computerized tomographic data; 3) typical histopathologic findings on transbronchial biopsieseconds, and 4) lack of response to different treatments. The patient required an emergency bilateral lung retransplantation 44 months after the initial transplantation. The histopathologic analysis of the explanted lungs confirmed the diagnosis of AFOP. Two years after the 2nd transplant, the patient is alive and well. To the best of our knowledge, this is the 1st case of a patient experiencing AFOP following lung transplantation who was successfully rescued by a 2nd bilateral lung retransplantation. [ABSTRACT FROM AUTHOR]

Long-term survival after a lung transplantation (LTx) remains limited by Chronic Lung Allograft Dysfunction (CLAD). B-grade airway-centered acute rejection (AR) has been associated with subsequent CLAD. B-grade AR is considered a cause of airway epithelial damage, but the type of injury and specific epithelial cells at risk are poorly understood. We aimed to assess the detailed morphology of lung allograft epithelium during B-grade AR. We retrospectively identified all transbronchial biopsies (TBBx) with airway-centered AR (B-grade ≥B1), collected 2-25 months after LTx from patients with a first bilateral LTx performed 2010-2015: 38 TBBx from 36 patients. 15 A0B0 TBBx from separate patients were matched to the ≥B1 TBBx according to time post LTx. Blinded semi-quantitative (scores 0-4) grading was performed of each airway using Hematoxylin & Eosin-stained sections to assess features of airway fibrosis and epithelial changes previously associated with CLAD. Immunofluorescence staining was performed using antibodies against club cell secretory protein (CCSP) and Muc-5AC. B-grade AR with concurrent ≥10% decline in lung function was associated with an increased risk of CLAD compared to stable B-grade AR and stable B0 TBBx (p=0.01) (figure 1A). Obliterative airway fibrosis (p=0.001) and epithelial flattening (p=0.0004) scores were higher in patients with ≥B1 compared to B0 (figure 1B). We found that epithelial hyperplasia increased over time after transplant (p=0.003) (figure 1C). The percentage of cells positive for CCSP and Muc-5AC was not different between ≥B1 and B0 TBBx (figure 1D) , was not associated with time between LTx and TBBx, and did not correlate with our epithelial grading results. The combination of B-grade AR and declining lung function is a predictor of CLAD. B-grade AR is associated with histologic obliterative airway fibrosis and epithelial flattening. Further analyses of specific epithelial cell types and changes during B-grade AR are ongoing. [ABSTRACT FROM AUTHOR]

CLAD, the main limitation to long-term survival after a lung transplantation (LTx), has two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). There is great need for better characterization of cells within the lung allograft tissue. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a high dimensional tissue imaging system allowing a comprehensive and multiparametric in situ exploration of tissue microenvironments at a single cell level, using heavy metal-tagged antibodies. Explanted lung tissue was obtained at the time of retransplantation from 4 BOS patients, 4 RAS patients, and 4 control donor lungs. Paraffin-embedded lung tissue was sectioned and stained with 35 heavy-metal-tagged antibodies selected to identify structural and immune proteins of interest. For each sample, three 1 mm2 regions of interest, centered on an airway, were ablated and data was analyzed using MCD viewer and HistoCAT software. To identify specific cell subsets identified by combinations of markers, we used tSNE to create a 2D representation of cells organised in 50 immune and non-immune clusters according to the similarity of their antibody-binding in 35-dimensional space (Figure 1A). We present several key examples of our findings: CLAD lungs had significantly reduced club cells (Figure 1B-D, H) and a trend towards reduced basal cells (Figure 1B-D, I). A Ki67-high basal cell population was mostly present in RAS samples (Figure 1B-D, I-J) and in close proximity to memory T cells (neighborhood analysis not shown, Figure 1L). Memory CD8+ T cells were more frequent in CLAD lungs (Figure 1E-G, K) with prominent regulatory T cells in RAS (Figure 1E-G). IMC allowed us to identify distinct basal cells and their proximity to cytotoxic memory T cells in CLAD lungs. IMC is a powerful technology for detailed cellular analysis within intact organ structures that may shed further light on CLAD mechanisms. [ABSTRACT FROM AUTHOR]

Long-term survival after a lung transplantation (LTx) remains limited by Chronic Lung Allograft Dysfunction (CLAD), which includes two main phenotypes: bronchiolitis obliterans syndrome (BOS), characterized by airway fibrosis and obliteration, and restrictive allograft syndrome (RAS), presenting with parenchymal and sub-pleural fibrosis (2019 ISHLT consensus). There can be overlap between these phenotypes. We aimed to detail and quantify pathological features of human CLAD sub-types. Peripheral (from outer 1/3 of the lung tissue) and central (from middle 1/3) paraffin-embedded explanted lung samples were obtained from 20 consecutive patients undergoing a second LTx for CLAD (phenotyped clinically prior to retransplant), from 3 lobes: left lower lobe (LLL), left upper lobe (LUL) and right upper lobe (RUL). Thirteen lung samples, collected from lobectomies or pre-transplant donor lungs, were used as "normal" controls. Sections were stained with Hematoxylin & Eosin and Elastic Trichrome. Blinded semi-quantitative grading was performed: the percentage of evaluable lung tissue area affected by parenchymal fibrosis was estimated; peri-airway fibrosis and bronchiolitis obliterans (BO) were graded 0-4 for each airway. Pleural fibrosis, epithelial flattening and hyperplasia were graded on a scale 0-4 (fig 1A-C). CLAD lung samples had higher scores for all parameters compared to controls. There was a notable overlap in pathological scores between BOS (n=11) and RAS (RAS n=8, Mixed n=1), with a wide range of scores in both conditions. Parenchymal fibrosis score was significantly higher in RAS compared to BOS (p=0.0007), but other scores were not significantly different (figure 1D). BO was significantly heterogeneous between the lobes (p=0.008) (figure 1E). Our results confirm that BO and parenchymal fibrosis can coexist to different degrees of severity in both patients with RAS and BOS. This scoring strategy will be useful for pathologic correlations in future studies. [ABSTRACT FROM AUTHOR]

Long-term survival after lung transplantation remains limited, mostly because of Chronic Lung Allograft Dysfunction (CLAD), which has two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our tools for assessing lung allograft cells are limited by lung tissue autofluorescence. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a mass spectroscopic technology that allows high-resolution, multi-dimensional characterization of lung cell surface and intracellular proteins at a single cell level, using heavy metal-tagged antibodies. Explanted lung tissue was obtained at the time of retransplantation from one BOS and one RAS patient. One donor lung sample was used as control (figure 1A-C). Paraffin-embedded lung tissue was sectioned and stained with 24 heavy-metal-tagged antibodies selected to identify structural and immune proteins of interest. Three 1.5mm2 regions of interest in each sample were ablated and data was analyzed using MCD viewer and HistoCAT software. Visualization of individual antibody binding patterns showed differences between samples, such as greater E-cadherin staining (epithelial cells) in airways of normal lung compared to those of CLAD (figures 1D-F). Spatial distribution of B and T cells within a lymphoid aggregate in the RAS sample is shown (figure 1G-J). We also used an exploratory tSNE strategy to create a 2D representation of cells clustered according to the similarity of their antibody-binding in 24-dimensional space (figure 1K). Precise localization of specific cell populations such as B cell and memory T cell subsets can then be achieved by first identifying them on the tSNE plot and then highlighting them on the IMC image (figures 1L-N). Our results indicate that IMC is a powerful tool for precise localization of cells within lung tissue samples, allowing both hypothesis-driven comparisons as well as exploratory analyses. [ABSTRACT FROM AUTHOR]

Airway epithelial injury is thought to be the final common pathway leading to airway fibrosis in chronic lung allograft dysfunction (CLAD). While cumulative post-transplant inflammatory insults represent CLAD risk factors, we lack adequate methods to measure epithelial changes during such events. We hypothesize that early detection of allograft epithelial injury will reveal relevant pathways in CLAD pathogenesis and predict subsequent CLAD. We aimed to use transbronchial brushings (TBBr) to sample distal airway epithelial cells and assess their characteristics in the context of allograft dysfunction. In this study, TBBr were collected from patients undergoing routine surveillance bronchoscopies at 3, 6, 9, and 12 months post-transplant, as well as patients with acute lung allograft dysfunction (ALAD), defined as a >=10% drop in FEV 1 compared to previous baseline. Cytospins were assessed with H&E and immunofluorescence stains (Figure 1A-G). Multi-colour flow cytometry panels were developed to identify populations of epithelial and immune cells (Figure 1H). In this cross-sectional analysis of TBBr from 22 patients (10 stable, 12 ALAD), we were able to identify important epithelial and mucosal immune cell subsets. Some patients with ALAD exhibited an increase in club cells, which act as epithelial cell progenitors and regenerate in response to epithelial damage (Figure 1I, p=NS). Additionally, a higher percentage of epithelial cells expressed the activation marker CD54 (ICAM-1) (Figure 1J, p=NS). In contrast, T and B lymphocytes were similar between patient groups. This study shows that TBBr can successfully isolate airway epithelial and mucosal immune cells for relevant analyses of specific cell subsets and activation states. Trends were observed in club cells and epithelial cell populations at the time of ALAD as well as over time. Our multi-platform approach will generate new hypotheses on the role of epithelial cells in CLAD pathogenesis. [ABSTRACT FROM AUTHOR]

The long-term survival after a lung transplantation remains limited by Chronic Lung Allograft Dysfunction (CLAD), which includes two main phenotypes: bronchiolitis obliterans syndrome (BOS), characterized by airway fibrosis and obliteration, and restrictive allograft syndrome (RAS), presenting with parenchymal fibrosis (PF) and restriction (2019 ISHLT consensus). It has been noted that there can be overlap between these phenotypes. We aimed to detail and quantify pathological features of human CLAD sub-types. Peripheral and central paraffin-embedded explanted left upper lobe lung samples were obtained from 20 consecutive patients undergoing second lung transplantation for CLAD (phenotyped clinically at retransplant). Sections were stained with Hematoxylin & Eosin and Elastic-Trichrome. Blinded semi-quantitative grading was performed: percentage of evaluable lung tissue area affected by PF was estimated; Peri-airway fibrosis (P-AF) and bronchiolitis obliterans (BO) were graded 0-4 for each airway. Pleural fibrosis (PLF), epithelial flattening (EF) and hyperplasia (EH) were graded on a scale 0-4 (fig 1A-C). There was notable overlap in pathological scores between BOS (n=11) and RAS/Mixed (RAS n=8, Mixed n=1), although there was a trend towards higher PF, P-AF and EH scores in RAS (fig 1D, E). Samples with BO grades >=2 had higher EH grades (p=0.003) and tended to have lower EF grades (p=0.11). Samples with P-AF >=2 had higher EH grades (p=0.002) and lower EF grades (p=0.01). Also, 9 patients had a respiratory infection at the time of retransplantation (1 BO, 8 RAS): PF scores were higher in RAS infected patients (p=0.02). Our results confirm that BO and PF can coexist to different degrees of severity in both patients with RAS and BOS. Divergent epithelial changes are seen and may be important in CLAD phenotype evolution. Sampling heterogeneity may be confounding these results and we plan to confirm these by grading multiple samples across 4 lobes. [ABSTRACT FROM AUTHOR]

Airway epithelial injury is thought to be the final common pathway leading to airway fibrosis in chronic lung allograft dysfunction (CLAD). While cumulative post-transplant inflammatory insults represent CLAD risk factors, we lack adequate methods to measure epithelial changes during such events. We hypothesize that early detection of allograft epithelial dysregulation and injury will reveal relevant pathways in CLAD pathogenesis and predict subsequent CLAD. We aim to use transbronchial brushings (TBBr) to sample distal airway epithelial cells and assess their characteristics in the context of allograft dysfunction. In this study, TBBr are being collected from patients undergoing routine surveillance bronchoscopies at 3, 6, 9, and 12 months post-transplant, as well as patients with acute lung allograft dysfunction (ALAD), defined as a >=10% drop in FEV 1 compared to previous baseline (Figure 1A). Multi-colour flow cytometry panels were developed to identify populations of epithelial and immune cells. Cell cytospins were assessed with immunofluorescence (Figure 1B-C). In this preliminary interim analysis of TBBr from 14 patients (6 stable and 8 with ALAD), we were able to identify important epithelial and mucosal immune cell subsets. In patients with ALAD, there was a trend towards decreased club cells, which act as epithelial cell progenitors and have anti-inflammatory properties (Figure 1D-E). Additionally, there was a trend towards increased epithelial cell expression of CD54 (ICAM), an activation marker (Figure 1E). In contrast, intraepithelial T and B lymphocytes were not found to be different in these patient groups. This preliminary study shows that TBBr can successfully isolate airway epithelial and mucosal immune cells for identification of specific cell populations. Follow-up studies from this project will include recruitment of additional subjects, longitudinal sample analyses, and association of cellular changes with later clinical outcomes. [ABSTRACT FROM AUTHOR]

The long-term survival after lung transplantation remains limited, mostly because of Chronic Lung Allograft Dysfunction (CLAD), which has two different phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our tools for assessing lung allograft cells are limited by the autofluorescence of the lung tissue. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a mass spectroscopic technology that allows high-resolution, multi-dimensional characterization of lung cell surface and intracellular proteins at a single cell level, using heavy metal-tagged antibodies. Explanted CLAD lung tissue was obtained at the time of retransplantation from two patients: one with BOS and one with RAS (figures 1A,1B). Paraffin-embedded lung tissue was sectioned, stained with 33 heavy-metal-tagged antibodies, selected to identify structural and immune proteins of interest. Three 9mm2 regions of interest in each sample were ablated and data was analyzed using MCD viewer and HistoCAT software. We identified and located multiple cell subsets. As examples, we show staining of E-cadherin (strongly present in bronchial and alveolar walls in BOS and absent in fibrous areas in RAS) (figures 1C, 1D), Vimentin (expressed by mesenchymal cells around bronchi) (figures 1E, 1F), and CD45+CD20+ B cells subpopulation (figures 1G, 1H). We also used an exploratory tSNE strategy to create a 2D representation of cells clustered according to their similarity in 33-dimensional space (figure 1I). Precise localization of specific or rare cell populations such as defined T cell subsets and epithelial basal cells (figures 1J-K) can be achieved by first identifying them on the tSNE plot and then highlighting them on the IMC image (figures 1J, 1K). Our results indicate that IMC is a powerful tool for precise localization of cells within lung tissue samples, allowing both hypothesis-driven comparisons as well as exploratory analyses. [ABSTRACT FROM AUTHOR]

Many scientific societies recommend SARS‐CoV‐2 vaccination for solid-organ transplant recipients. The immunogenicity of two or three vaccine doses in lung transplant (LTx) recipients is unclear. The aim of this study was to evaluate the humoral response to the vaccine in LTx and heart-lung transplant (HLTx) recipients. We conducted a prospective study of LTx and HLTx recipients at seven centers in France. Anti-spike-protein antibody titers after two or three SARS‐CoV‐2 vaccine injections were measured. We studied 2186 patients (1091 [51%] males) with a median age of 49 [45-55] years. Double LTx was performed in 1792 (82%) patients. The main reasons for LTx were chronic obstructive pulmonary disease (n=656, 30%), fibrosis (n=459, 21%), and cystic fibrosis (n=350, 16 %). Median time from LTx to vaccination was 59 [29-108] months and mean time from the last vaccine dose to serological testing was 3 months [1.5-3.8]. We used WHO definitions to classify antibody titers as negative (&lt. 30 BAU/mL), suboptimal (30-260 BAU/mL), or protective (&gt 260 BAU/mL). Of the first 1081 patients, 270 (25%) were partially vaccinated and 649 (60%) fully vaccinated (three doses or history of COVID-19 then two doses); Among these patients,133 (12%) were infected by covid. Of the 649 fully vaccinated patients, 461 (71%), 84 (13%), and 97 (15%) had negative, suboptimal, and protective antibody titers, respectively. The proportion of patients with protective titers was 8% vs. 18% in patients vaccinated within 5 years vs. 5 or more years after LTx, respectively. Among covid-infected patients, 48% developed a protective rate, whether fully or partially vaccinated. LTx recipients usually fail to develop protective antibody titers in response to SARS-CoV-2 vaccination. Once further data are collected, we will seek to identify risk factors for a poor antibody response. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the 2019 ISHLT CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed 2010-2015. Classification of CLAD phenotypes was based on the 2019 ISHLT consensus document. Survival after CLAD onset was assessed using Kaplan-Meier and Cox proportional hazards models. Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed and 19 (10.9%) undefined phenotype. Twenty-six patients (14.9%) did not match any of these four categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The remaining 45 (25.9%) patients were combined and re-categorized based on the presence or absence of characteristic RAS-like opacities on chest imaging: those with RAS-like opacities at CLAD onset had significantly worse allograft survival compared to patients with BOS (HR=2.14, 95% CI 1.17-3.93, P=0.014) and similar survival to RAS or mixed phenotype. The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for the risk-stratification of patients who do not match the major CLAD phenotypes. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation and results from repeated acute injuries to the graft epithelium. Epithelial club cells are known to have protective, anti-inflammatory, and regenerative roles and are depleted in CLAD. Mechanisms driving club cell loss in CLAD are unknown. Our single cell RNA sequencing (scRNAseq) data showed that TRAIL transcript was upregulated in CLAD club cells compared to control donor lung club cells. TRAIL induces apoptotic cell death by binding to its receptors (TRAIL-R1 and TRAIL-R2). To validate the scRNAseq data, we here further investigate the expression of TRAIL and its receptors at the protein level. We hypothesized that CLAD develops as a consequence of club cell death induced by TRAIL signaling during episodes of acute lung allograft dysfunction (ALAD). Lung transplant recipients' (LTR) airway brushings were obtained 3-12 months post-LT and processed for flow cytometry. TRAIL, TRAIL-R1, TRAIL-R2 expression was examined in club cells in LTRs with or without ALAD, defined as a >=10% drop in forced expiratory volume in 1 second from prior. After exclusion of non-epithelial cells, EpCAM+ epithelial cells and EpCAM+CCSP+ club cells were identified. Our findings show that the frequencies of both TRAIL+ club cells and TRAIL+ non-club epithelial cells were significantly higher in ALAD LTRs compared to non-ALAD LTRs (Fig. 1A-C). Further, TRAIL-R1 expression on club and non-club epithelial cells tended to be higher in ALAD compared to non-ALAD LTRs (Fig. 1A, D-E). TRAIL-R2 expression did not differ between ALAD and non-ALAD LTRs. Our preliminary data show an upregulation of TRAIL within epithelial cells found in ALAD airway brushings compared to control. The co-localization of TRAIL and TRAIL-R1 in club cell suggests that autocrine or paracrine cell death might be potential mechanisms for club cell loss in CLAD. [ABSTRACT FROM AUTHOR]

Lung transplantation is a therapeutic option for certain end-stage lung diseases. The phone call for lung transplantation is a major event in the life of these patients; as a result, it can generate significant stress. We herein present the case of a 58-year-old female patient with end-stage chronic obstructive pulmonary disease (COPD) who, while on the lung transplantation waiting list, received such a call. Complete transplant work-up, including cardiac tests undertaken shortly before, had revealed no contraindication to lung transplantation. She was admitted with severe acute respiratory failure, and her extensive work-up was compatible with pulmonary edema due to takotsubo cardiomyopathy. The lung transplantation was thus cancelled, owing to the patient's health condition and the poor quality of the graft as well. The patient stayed in the intensive care unit for several days, requiring noninvasive ventilation. The left ventricular function recovered completely within 10 days postdiagnosis, and the patient was discharged 13 days after her admission. The patient was transplanted 1 month thereafter, without any particular problems; she is currently, 8 months post-transplantation, in good condition. In the given case, the call for lung transplantation could have generated emotional stress severe enough to lead to takotsubo cardiomyopathy. [ABSTRACT FROM AUTHOR]

T cells are the primary driver of ACR-associated immune responses following lung transplantation (LTx) and may mediate the progression of ACR to Chronic Lung Allograft Dysfunction (CLAD). We previously reported that the CD8+, but not regulatory, T cell content of first grade A1 transbronchial biopsies (TBBs) from stable patients was related to future CLAD. Here, we used IMC, a technique that enables highly multiplexed tissue imaging and acquisition of single-cell data, to identify intragraft T cell phenotypes that may be associated with subsequent CLAD. Patients with a first episode of A1B0 ACR, who remained CLAD-free for at least 5 years (n=3) or who developed CLAD within 2 years (n=3) following LTx were selected. TBBs were sectioned and stained with 35 heavy metal-conjugated antibodies including both lineage-defining and functional markers. For each sample, four regions of interest (ROI) measuring 800 mm2 were laser ablated. Raw data was analyzed using QuPath and HistoCAT. Co-expression of markers was used to identify cell phenotypes. t-SNE was used to plot single-cell data in two dimensions. PhenoGraph was used to identify 37 cell clusters based on antibody binding similarity as represented on a heatmap (Figure 1A), highlighted on the t-SNE plot (Figure 1B). Antibody binding patterns were visualized and cell clusters were mapped back to future CLAD/CLAD-free ROIs and highlighted on constructed images (Figure 1C, D). The ratio of CD45RO+CD8+CD27+ T cells (Cluster 7) to total cells was significantly increased in the future CLAD group (p=0.006) (Figure 1E). In keeping with our prior observations, there was no significant difference in the proportion of CD45RO+CD4+FOXP3+ T cells (Cluster 33) (Figure 1F). These results suggest that in asymptomatic patients with a first episode of A1 rejection, greater memory CD8+ T cell content may be of prognostic importance, possibly indicating poorer long-term outlook. Validation in additional patients is underway. [ABSTRACT FROM AUTHOR]

Survival after lung transplantation (LTx) still remains limited by chronic lung allograft dysfunction (CLAD). Prior studies showed an association between graft cellular expression of the checkpoint HLA-G and acceptance, whereas conversely, soluble HLA-G (sHLA-G) in plasma/BAL was associated with acute rejection. We investigated whether plasma levels of extra-vesicular (EVs)-bound HLA-G could predict CLAD. We used data for 79 LTx recipients from COLT (Cohort For Lung Transplantation) cohortwith ≥1 available blood samples at 6-, or 12-months post-Tx, all with a stable function within the 1st year. Among them, 41 developed subsequent CLAD (Group CLAD=33 BOS and 8 RAS) and 38 remained STABLE at 3 years. 20 heathy individuals (HI) were negative controls. EVs from plasma were isolated by size exclusion chromatography and characterized by nanoparticule tracking analysis. Plasma levels of EVs-bound HLA-G (sHLA-G EV) and of total soluble-HLA-G (sHLA-G TOT =sHLA-G EV + free sHLA-G) were measured by ELISA. Uni- and multivariate were performed to assess association of EVs levels and CLAD/survival. In stable patients, mean plasma levels of sHLA-G EV at M6 and M12, were significantly increased as compared to those of HI (M6: 30.2 vs 15,1 ng/mL, p=0.008, and M12: 37.8 vs 15.1 ng/mL, p=0.0009, respectively). In BOS patients, a decreased plasma levels of sHLA-G EV were observed at M6 and M12 as compared to stable patients, with a significant difference at M12 (mean sHLA-G EV of 23.7 ng/mL vs 38.7 p=0.02). In those BOS patients, sHLA-G EV levels remained increased as compared to HI at M6 (p=0.01). In RAS patients, sHLA- GEV levels was similar to stable patients at M6 and M12, and increased as compared to HI (M12: 40,5 vs 15,6 ng/mL, p=0.004). Among the whole cohort, a higher freedom from CLAD was observed in patients with sHLA-G EV level > first IQR (=21.3 g/ml) at M12 post-LTx (p=0.01, Figure 1). Our data suggest that an early decrease of sHLA-G EV levels after LTx may be predictive of subsequent CLAD onset. [ABSTRACT FROM AUTHOR]

Phenotyping chronic lung allograft dysfunction (CLAD) in single lung transplant (SLTX) recipients is challenging, due to the native lung contribution to pulmonary function tests (PFT). We aimed to assess the ISHLT CLAD classification and its prognostic performance in SLTX. In this retrospective cohort of adult, first, SLTX from 2009 to 2017, patients with persistent drop in FEV1>20% were assessed by 2 independent reviewers to determine CLAD status and phenotype, specifically noting presence or absence of RAS-like opacities. Disagreements were resolved by a 3rd reviewer. Interobserver agreement (IOA) was calculated by Cohen's Kappa. Association of CLAD phenotypes with time to death/retransplant (ReTx), adjusted for age, sex, CMV mismatch and native lung condition, were assessed with Cox models. Out of 172 SLTX patients, 92 had a persistent drop in FEV1>20%, of whom 67 got a diagnosis of CLAD. We noted a moderate IOA for CLAD status (Kappa 0.69) and poor IOA for phenotype (Kappa 0.5). When applying the ISHLT criteria strictly (based on exact cut-offs for PFT, along with imaging), 34 patients had BOS (50.7%), 9 RAS/mixed (13.4%), 7 undefined (10.4%), and 17 unclassified (25.5%). We found no association of these strict phenotypes with death/ReTx (Fig A). When using adjudicated CLAD phenotypes, 31 patients had BOS (46.3%), 15 RAS/mixed (22.4%), 2 Undefined (3%), and 19 Unclassified (28.3%). Using these adjudicated phenotypes, RAS/mixed was significantly associated with higher risk of death/ReTx (HR 2.98, 95%CI [1.39-6.4]) (Fig B). Finally, the specific adjudication of RAS-like opacities had the best IOA (Kappa 0.73). Presence of RAS-like opacities was a strong predictor of death/ReTx (HR 2.3, 95%CI [1.2-4.5]) (Fig C). PFT interpretation is challenging in SLTX. Using a classification relying more on imaging analysis that harboured good IOA, we obtained better prognostic performances than with a strict application of published physiologic cut-offs. [ABSTRACT FROM AUTHOR]

Lung transplantation (TxP) is now a validated treatment of end-stage pulmonary diseases, but long-term survival are still hampered by the development of chronic allograft dysfunction (CLAD) affecting> 50% of patients. Bronchiolitis obliterans syndrome/obliterative bronchiolitis (BOS/OB) is the most common manifestation of CLAD. Survival after onset of BOS is poor (<50% at 3 years). OB is thought to arise from repeated injury to graft epithelial cells, leading to fibrous scarring and obliteration of the small airway lumen. The crucial role of a dysregulated fibrotic repair demonstrated in BOS strongly suggests the potential role of tyrosine kinase inhibitors (TKI) that target these growth factors involved in OB. The TKI Nintedanib, demonstrated as effective in the treatment of idiopathic pulmonary fibrosis (IPF) appears as a candidate molecule. Hence, we started a Nintedanib trial in LTx recipients with BOS because of : (i) High unmet medical need in BOS; (ii) fibrotic pathway involved in BOS; (iii) efficacy of nintedanib in IPF. A phase III clinical randomized trial to assess the efficacy of Nintedanib in LTx recipients with BOS. Inclusion criteria: n=80 LTx recipients with BOS 0p-1-2, with a decline of FEV1> 200ml within the previous year. Primary Objective : to assess Nintedanib efficacy in the reduction of the rate of decline of FEV1 (forced expiratory volume in 1 sec) at a dose of 150 mg twice daily (bid) compared to placebo over 6 months. Secondary Objectives : to assess Nintedanib efficacy and tolerance in the treatment of BOS 0p-1-2, based on the change over 6 months of : 6WT, QOL, BOS grade/graft failure, O2 saturation, KL-6, SPD, VEGF, PDGF parameters, and tolerance. The trial was started in December 2019 among 8 french LTx centers (Hôpital Foch, Bichat, HEGP/Cochin, Marie-Lannelongue, Marseille, Bordeaux, Strasbourg, and Grenoble), for a total duration of inclusion anticipated at 36 months. On October 15th 2020, 14 patients were already included. Infinitix-BOS is the first therapeutic randomized trial testing the efficacy of nintedanib in LTx recipients with BOS. In case of demonstrated effectiveness of Nintedanib, the benefit for LTx patients with BOS seems high in terms of stabilization of lung function and enhancement of survival. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) remains the main barrier to long-term survival after lung transplant. The main phenotypes of CLAD are bronchiolitis obliterans syndrome (BOS) with small airway fibrosis, and restrictive allograft syndrome (RAS) with extensive lung parenchymal fibrosis. Mechanisms of CLAD and the pathways active in BOS/RAS remain poorly understood. We hypothesized that bulk RNA sequencing (seq) of human CLAD lungs would help uncover pro-fibrotic pathways and phenotype-specific mechanisms. At time of retransplantation, we obtained CLAD lung tissue from 27 BOS and 18 RAS. Negative controls included donor lungs (18) and lobectomy samples done for suspected cancers (14); positive controls came from patients with idiopathic pulmonary fibrosis (IPF) (19). RNA was extracted and submitted for bulk RNAseq at a depth of 50 million paired-end 100-base-pair reads. Differentially expressed genes were detected using DESeq2 and NOIseq. Top differentially expressed genes in CLAD vs. negative controls included genes involved in cell-matrix interactions (COL10A1, ADAM9) and cell proliferation (CST, MDK). Gene Ontology analysis showed higher expression of genes associated with fibrosis and motility, whereas genes involved in defense and immunity were decreased due to immunosuppression in CLAD vs. negative controls. Analyses to compare RAS to BOS are ongoing. As we anticipated an imperfect correlation between clinical phenotypes and transcriptomic endotypes, we performed unsupervised clustering of all samples. A subset of RAS/mixed samples clustered together and in close proximity to IPF, while other distinct clusters appeared independent of clinical phenotypes: These transcriptomic endotypes will merit further exploration. This large cohort of lung samples allows a detailed analysis of the lung allograft transcriptome in different phenotypes of CLAD, enabling identification of the top differentially expressed genes and pathways in this poorly understood condition. [ABSTRACT FROM AUTHOR]

Lung transplant recipients have the lowest survival rate compared to all other solid organ transplants, the primary cause being chronic lung allograft dysfunction (CLAD) which manifests as two main clinical phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Pulmonary macrophages (MΦ) are a significant population of immune cells which serve homeostatic and immunoregulatory roles but may also perform proinflammatory and profibrotic functions when activated. MΦ contribution to CLAD is not clear. We used single-cell RNA sequencing (scRNAseq) to profile MΦ in BOS and RAS explanted lungs and to determine their roles in CLAD. 6 fresh lung samples (4 BOS, 2 RAS) from re-transplant cases were collected, processed, subjected to 10X Genomics chemistry for scRNAseq, and compared to 5 donor lung samples. R and Seurat package were used for quality control, dimensionality reduction, and annotation. Slingshot package was utilised for pseudotime trajectory analysis. Lung tissue showed high MΦ heterogeneity, with at least 20 unique clusters (Fig 1A). CLAD lungs had a higher presence of FCN1+ MΦ with high expression of AIF1 activation marker: these were annotated as activated proinflammatory AIF1+ MΦ (Fig 1B). In RAS samples, there was a reduction of alveolar FABP4+ MΦ (Fig 1C) and an enrichment of MΦ expressing fibrosis-associated genes (SPP1, MERTK, MMP9) that were annotated as pro-fibrotic SPP1+ MΦ (Fig 1D). Pseudotime trajectory analysis shows both AIF1+ and SPP1+ Φ were at an earlier stage of development compared to other clusters. Using scRNAseq, we have identified novel clusters of MΦ in CLAD lungs. Proinflammatory AIF1+ MΦ were found in all CLAD lungs, while a reduction in alveolar FABP4+ MΦ and enriched pro-fibrotic SPP1+ MΦ may contribute to the parenchymal lung fibrosis found in RAS. In vitro assays on sorted MΦ are under way to further characterise functions of these MΦ subsets and to explore their role in CLAD. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation. Different immune and non-immune insults contribute to a chronic inflammatory and fibrotic process that ultimately reduces the graft's function and survival. The renin-angiotensin-aldosterone system (RAA) has been shown to play a role in fibrogenesis in the kidney, heart and lung. Notably, 6 RAA signature proteins (RHOB, BST1, LYPA1, GLNA, TSP1, LAMB1) have been identified in urine and kidney tissue of patients with Interstitial Fibrosis and Tubular Atrophy. We hypothesize that RAA is involved in CLAD pathogenesis and that these RAA signature proteins could be used as biomarkers of CLAD in Bronchoalveolar Lavage (BAL). We performed immunofluorescence staining of angiotensin receptors (AGTR1 and AGTR2) and immunohistochemical staining of TSP1 in 10 explanted CLAD lungs (obtained at time of re-transplantation), compared to 5 control lungs (obtained from donors or lobectomies for lung nodule resections). Using mass spectrometry, we quantified two peptides for each RAA signature protein in the BAL of 40 lung transplant recipients undergoing bronchoscopy. We observed significantly more AGTR1+ cells in CLAD versus control lungs (p=0.04) (Fig1 a), while the percentage of AGTR2+ cells was not different. We also found a trend towards higher expression of TSP1, particularly in macrophages and fibrotic zones, in CLAD versus controls (Fig1b). Mass spectrometry-based assays were developed for BAL quantification of the RAA proteins (Fig1c). There was a trend towards higher concentration of peptides of RHOB, BST1 and LYPA1 peptides in the BAL of CLAD patients with concomitant acute >10% decline in lung allograft function in comparison to patients with stable lung function (Fig1d). Our pilot data support the hypothesis that RAA is involved in CLAD and that RAA-regulated proteins may help identify patients with active CLAD. We are currently validating these findings using a larger cohort and additional endpoints. [ABSTRACT FROM AUTHOR]

Viral gastroenteritis causing diarrhea is a common complication observed in lung transplant recipients. Differently from the mild and typically self-limited disease seen in immunocompetent subjects, immunocompromised patients frequently have a more severe course. Norovirus and rotavirus are among the leading causes of severe gastroenteritis in transplant recipients. Specific treatment is unavailable, although good supportive treatment can significantly reduce morbidity. Previous studies have suggested that oral immunoglobulins may be used for the treatment of acute viral gastroenteritis after solid-organ transplantation. Herein, we conducted a retrospective chart review of 12 lung transplant recipients with norovirus-induced gastroenteritis who were treated with oral immunoglobulins for 2 days. Eleven patients were successfully treated, whereas 1 subject was only mildly improved. Four patients had at least 1 recurrence. No significant adverse effects were observed. We conclude that oral immunoglobulins may be clinically useful for lung transplant recipients with norovirus-induced gastroenteritis. [ABSTRACT FROM AUTHOR]

Airway epithelial injury is thought to be a key event in the pathogenesis of Chronic lung allograft dysfunction (CLAD). We investigated whether markers of epithelial activity and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS)-related phenotypes (including RAS, mixed phenotype, and all other patients with RAS-like opacities). CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free subjects were used as controls. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing apoptosis and overall death, respectively), were measured in BAL obtained within 6-months from CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Kruskal-Wallis and Mann-Whitney-U-test. Association between protein levels and graft survival was assessed using Cox proportional hazards models, adjusted for CMV serology mismatch status and phenotype. Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 26 CLAD-free controls were included. Median BAL levels were significantly higher in patients with CLAD compared to CLAD-free controls for M30 (124.5 vs. 88.9), MUC1 (6.8 vs. 3.28) and MUC16 (121.0 vs. 31.1). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes compared to BOS (160.9 vs. 114.6). In multivariable models, levels of M30 and MUC5B were associated with allograft survival after CLAD onset independent of phenotype (P<0.05 for all). Airway epithelial mucin and cell death markers are enhanced in the BAL fluid of patients with CLAD and can assist in differentiating between CLAD phenotypes. Abnormal airway mucin expression and epithelial cell death may be involved in the airway inflammatory response of CLAD and thus aid in future selection of targeted therapies. [ABSTRACT FROM AUTHOR]

Chronic Lung Allograft Dysfunction (CLAD) is a heterogeneous disease. Three major phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed phenotype, are defined based on combinations of lung physiology and chest imaging. Total lung capacity (TLC)≤90% of baseline is used to define RAS/Mixed; however, many centers do not routinely obtain TLC and use instead forced vital capacity (FVC)≤80%. The objective of the current study was to assess the diagnostic accuracy of FVC≤80% for determining RAS/Mixed in a large center that routinely monitors both TLC and FVC. This was a retrospective cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with CLAD were included. Spirometry, lung volumes, and chest imaging were available. RAS/mixed were determined based on 2019 ISHLT definitions using TLC≤90% and the presence of RAS-like opacities. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic accuracy of FVC≤80% combined with RAS-like opacities for diagnosis of RAS/Mixed were computed. A total of 174 patients developed CLAD: 25(14.4%) met criteria for RAS/mixed by TLC. The sensitivity, specificity, PLR, NLR and diagnostic accuracy for FVC to determine RAS/Mixed was 84.0% (95%CI 63.9-95.5), 95.3% (95%CI 90.6-98.1), 17.9 (95%CI 8.5-37.6), 0.17 (95%CI 0.07-0.41) and 93.7% (95%CI 89.0-96.8). Low FVC has an overall good diagnostic value for estimating RAS/mixed phenotype. Given the high specificity, it is possible to rule-in RAS/mixed phenotype with spirometry. However, it cannot be ruled-out confidently based on spirometry alone. In CLAD patients with a clinical suspicion of RAS/mixed phenotype and a preserved FVC, measurement of TLC is important for a more accurate diagnosis. [ABSTRACT FROM AUTHOR]

Bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), and mixed are major phenotypes of CLAD. In previous, older studies, CMV viremia has been associated with reduced survival. We hypothesized that, in a more modern era of CMV prophylaxis, CMV viremia would remain associated with death, but also represent a risk factor for CLAD and specifically RAS/mixed phenotype. This is a retrospective cohort study of all consecutive adult, first, bilateral-/single-lung transplants performed 2010-2016 who had ≥4 pulmonary function tests and ≥1 CMV-PCR measurement. CMV-PCR levels were divided into 3 categories: undetectable, <1000 IU/mL, and ≥1000 IU/mL. Risks for CLAD, RAS/mixed, or death/re-transplantation were assessed by Cox proportional hazards models using CMV-PCR as a time-dependent categorical variable, adjusting for variables listed in table. Of 645 patients, 257 developed CLAD (143 BOS, 44 RAS/mixed, 66 undefined/unclassified) and 388 were CLAD-free at last follow-up. In univariate analysis, CMV-PCR≥1000 IU/mL was a significant risk factor for CLAD (HR 1.33, p=0.05). In multivariate analysis, CMV-PCR was not a significant risk factor for CLAD, whereas CMV serostatus mismatch was (Table). CMV-PCR was not a significant risk factor for RAS/mixed (CMV-PCR<1000 IU/mL: HR 1.46, p=0.32. CMV-PCR≥1000 IU/mL: HR 1.43, p=0.33) in univariate analysis. Regarding time to death/re-transplantation, CMV-PCR was a significant risk factor in multivariate analysis, whereas CMV serostatus was not (Table). CMV viremia was significantly associated with death/re-transplantation. Conversely, CMV viremia was not strongly associated with CLAD or RAS/mixed, while CMV serostatus mismatch was. CMV serostatus mismatch may have an impact on CLAD through mechanisms independent of viremia. In light of these paradoxical results, we are further assessing the relationship between immunosuppression, immune function, and CMV. [ABSTRACT FROM AUTHOR]

Lung transplant (LT) recipients experience a low survival rate compared with other solid organ transplant recipients mainly due to chronic lung allograft dysfunction (CLAD). Acute lung allograft dysfunction (ALAD) episodes is a risk factor of subsequent CLAD. The contribution of lung macrophages (Macs) to ALAD and CLAD is not clear. We applied single-cell RNA sequencing (scRNAseq) to profile cells during quiescence, ALAD, and CLAD, in order to determine the role of Macs in dysfunctional lung allografts. Fresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (SLF) and 3 undergoing an episode of ALAD, as well as cells from 3 explanted CLAD lungs were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. Our data revealed that Macs are highly heterogeneous (∼12 transcriptionally distinct subsets from all 3 SLF). We identified four Mac subsets more prominent in ALAD BAL compared to stable (Fig 1A). Of the four, three populations were uniquely present in the CLAD lung samples compared to a control donor lung sample (Fig 1B). Based on pathway analysis and the top differentially expressed genes in BAL (Fig 1C) and CLAD lung (Fig 1D), we annotated them as proinflammatory (CXCL10+), Ig-regulated (FcγRIIb +), and metallothioneins-mediated inflammatory (MT) Macs. Pseudotime analysis suggested that CXCL10+ and FcγRIIb+ Macs represent an earlier stage of differentiation than other Macs (Fig 1E). Deconvolution demonstrated that donor Macs are lost with time post-transplant (Fig 1F) and absent from CLAD lungs (Fig 1G). Using scRNAseq, we observed Mac heterogeneity and identified specific subsets of Macs that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of Macs in allograft injury and dysfunction. [ABSTRACT FROM AUTHOR]

Airway epithelial injury is thought to be a key event in the pathogenesis of CLAD. We investigated whether markers of epithelial activation and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and the major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS) / mixed phenotype. This was a retrospective, cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. CLAD status and phenotypes were retrospectively determined based on 2019 ISHLT definitions. All patients with RAS/mixed phenotype were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free controls were randomly selected. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing epithelial cell apoptosis and overall death, respectively) were measured in BAL obtained within 6-months from CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Kruskal-Wallis and Mann-Whitney-U-test. Forty-five CLAD (27 BOS, 11 RAS, 7 mixed) and 26 CLAD-free controls were included. BAL levels of M30, MUC1 and MUC16 were significantly higher in patients with CLAD compared to CLAD-free controls. When comparing CLAD phenotypes, only M30 demonstrated a difference between RAS/mixed and BOS with a significantly higher M30 level in RAS/mixed patients. Airway epithelial mucin and cell death markers are enhanced in the BAL fluid of patients with CLAD and can assist in differentiating between CLAD phenotypes. Abnormal airway mucin expression and epithelial cell death may be involved in the airway inflammatory response of CLAD and thus aid in future selection of targeted therapies. [ABSTRACT FROM AUTHOR]

Chronic Lung Allograft Dysfunction (CLAD) is a heterogeneous disease. Three major phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed phenotype, are defined based on combinations of lung physiology and chest imaging. Total lung capacity (TLC)≤90% of baseline is used to define RAS/Mixed; however, many centers do not routinely obtain TLC and use instead forced vital capacity (FVC)≤80%. The objective of the current study was to assess the diagnostic accuracy of FVC≤80% for determining RAS/Mixed in a large center that routinely monitors both TLC and FVC. This was a retrospective cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with CLAD were included. Spirometry, lung volumes, and chest imaging were available. RAS/mixed were determined based on 2019 ISHLT definitions using TLC≤90% and the presence of RAS-like opacities. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic accuracy of FVC≤80% combined with RAS-like opacities for diagnosis of RAS/Mixed were computed. A total of 174 patients developed CLAD: 25(14.4%) met criteria for RAS/mixed by TLC. The sensitivity, specificity, PLR, NLR and diagnostic accuracy for FVC to determine RAS/Mixed was 84.0% (95%CI 63.9-95.5), 95.3% (95%CI 90.6-98.1), 17.9 (95%CI 8.5-37.6), 0.17 (95%CI 0.07-0.41) and 93.7% (95%CI 89.0-96.8). Low FVC has an overall good diagnostic value for estimating RAS/mixed phenotype. Given the high specificity, it is possible to rule in RAS/mixed phenotype with spirometry. However, it cannot be ruled out confidently based on spirometry alone. In CLAD patients with a clinical suspicion of RAS/mixed phenotype and a preserved FVC, measurement of TLC is important for a more accurate diagnosis. [ABSTRACT FROM AUTHOR]