Chronic Lung Allograft Dysfunction Patients Research Articles

Changes in HCMV immune cell frequency and phenotype are associated with chronic lung allograft dysfunction.

Human cytomegalovirus (HCMV) infection is common and often severe in lung transplant recipients (LTRs), and it is a risk factor associated with chronic lung allograft dysfunction (CLAD). The complex interplay between HCMV and allograft rejection is still unclear. Currently, no treatment is available to reverse CLAD after diagnosis, and the identification of reliable biomarkers that can predict the early development of CLAD is needed. This study investigated the HCMV immunity in LTRs who will develop CLAD. This study quantified and phenotyped conventional (HLA-A2pp65) and HLA-E-restricted (HLA-EUL40) anti-HCMV CD8+ T (CD8 T) cell responses induced by infection in LTRs developing CLAD or maintaining a stable allograft. The homeostasis of immune subsets (B, CD4T, CD8 T, NK, and γδT cells) post-primary infection associated with CLAD was also investigated. At M18 post-transplantation, HLA-EUL40 CD8 T responses were less frequently found in HCMV+ LTRs (21.7%) developing CLAD (CLAD) than in LTRs (55%) keeping a functional graft (STABLE). In contrast, HLA-A2pp65 CD8 T was equally detected in 45% of STABLE and 47.8% of CLAD LTRs. The frequency of HLA-EUL40 and HLA-A2pp65 CD8 T among blood CD8 T cells shows lower median values in CLAD LTRs. Immunophenotype reveals an altered expression profile for HLA-EUL40 CD8 T in CLAD patients with a decreased expression for CD56 and the acquisition of PD-1. In STABLE LTRs, HCMV primary infection causes a decrease in B cells and inflation of CD8 T, CD57+/NKG2C+ NK, and δ2-γδT cells. In CLAD LTRs, the regulation of B, total CD8 T, and δ2+γδT cells is maintained, but total NK, CD57+/NKG2C+ NK, and δ2-γδT subsets are markedly reduced, while CD57 is overexpressed across T lymphocytes. CLAD is associated with significant changes in anti-HCMV immune cell responses. Our findings propose that the presence of dysfunctional HCMV-specific HLA-E-restricted CD8 T cells together with post-infection changes in the immune cell distribution affecting NK and γδT cells defines an early immune signature for CLAD in HCMV+ LTRs. Such a signature may be of interest for the monitoring of LTRs and may allow an early stratification of LTRs at risk of CLAD.

Extracorporeal Photopheresis in CLAD: A 15-Year Single Centre Experience

<h3>Purpose</h3> Use of extracorporeal photopheresis (ECP) in chronic lung allograft dysfunction (CLAD) extends over 25 years. Various single centre reports suggest some benefit, but unequivocal data is lacking. Methodology and changes in CLAD definitions have hindered comparisons, and uncertainty remains about ECP efficacy in current CLAD phenotypes. This study summaries our experience of ECP in CLAD, assessing outcomes and determining treatment duration. <h3>Methods</h3> A retrospective review of all patients receiving ECP for CLAD was performed. Data from legacy patients was reanalysed according to current definitions. Patients were treated with 2-cycles fortnightly, or occasionally 1-cycle weekly for 6 months depending on resources. Primary outcomes were nature of ECP response: stabilized, partial response or non-response and graft survival. Secondary endpoints include time to ECP response and the effect of CLAD phenotype shift during ECP, particularly regarding timing and development of restrictive spirometry. <h3>Results</h3> In total 369 patients commenced ECP for CLAD. Median follow-up was 27 [IQR 8.6-70.6] months. Graft stabilization was achieved in 140/369 (38%) patients. A further 85/369 (23%) of patients exhibited slowing in FEV₁ decline but failed to stabilize during the study. The remaining 144/369 (39%) demonstrated no appreciable response to ECP. Graft survival in the latter group was poorest, with 3-year survival of 18%. Among transient responders 3-year survival was surprisingly good at almost 75%, with almost all stabilized patients surviving 3 years. Patients commenced on ECP for restrictive allograft syndrome (RAS) did poorly. Bronchiolitis obliterans patients who subsequently developed restrictive features, subsequently developed ECP responses similar to RAS. A reliable prediction of lack of ECP response was evident after 106 [71 - 181] days. <h3>Conclusion</h3> ECP appears to offer a survival benefit in some CLAD patients. A sizeable minority of patients stabilized and this conferred the best prognosis. Any demonstrable effect on dFEV1 within 6 months of initiation may support ECP continuation. Should no change in dFEV1 occur in this period, treatment withdrawal should be considered. Restrictive phenotypes, developing either prior to or during ECP treatment confer a very poor prognosis.

Upper Tract Microbiome Modifications After Lung Transplantation and Its Impact in CLAD

<h3>Purpose</h3> Survival after lung transplantation is limited in large part due to the high incidence of chronic lung allograft dysfunction (CLAD). Infection is a recognized risk factor for the development of CLAD, and both acute infection and chronic lung allograft colonization with microorganisms increase the risk for CLAD. The aim of our study was to analyze nasopharyngeal (NP) microbiome composition and modifications following lung transplantation, with a focus on its relationship with CLAD. <h3>Methods</h3> This was a longitudinal study in which we analyzed bacterial microbiome in NP swab samples in 68 lung transplanted (LT) patients the day of the transplant, when patients were discharged, at 2-5 months and 12 months after lung transplantation. We also analysed NP swab samples from 10 healthy subjects. CLAD was assessed after 2 years of follow-up. DNA was extracted from the NP swabs. To create the amplicon library, the hyper-variable region (V4) of the 16S ribosomal RNA gene was amplified by standard PCR. Amplicons were purified and sequenced in barcoded pools using Illumina MiSeq technology. Raw sequence reads were demultiplexed by using idemp. The resulting single-end reads were processed using the Dada2 pipeline obtaining an amplicon sequence variant table to which taxonomy was assigned. Alpha diversity, including Shannon and Observed indices, and beta diversity metrics were calculated. We compared these metrics and particular taxa relative abundances between samples of LT patients at different time points and samples of healthy subjects. <h3>Results</h3> From the 68 studied patients, 13 developed CLAD during the two years of follow-up. We observed a significant decrease in the Observed index at the day of the discharge and 2-5 months after transplant when compared with healthy subjects. One year after transplant, CLAD patients had higher Shannon diversity than LT patients who did not develop the dysfunction. On the other hand, a lower beta diversity was observed between healthy subjects and patients with no CLAD. Besides, we detected an increased genus in NP swab samples associated with no CLAD also observed in NP samples from healthy subjects. <h3>Conclusion</h3> Nasopharyngeal overall microbiome composition, as well as relative abundances of particular taxa of non-CLAD patients, was closest to the one from healthy subjects suggesting a better outcome of the transplant if better microbiome normalization.

Characterization of B Cells in Lungs of Chronic Lung Allograft Dysfunction Patients by Single-Cell RNA Sequencing

<h3>Purpose</h3> The primary factor limiting long-term survival in lung transplant recipients is chronic lung allograft dysfunction (CLAD), which manifests as fibrosis of the airways and/or lung parenchyma. The high incidence of CLAD despite the use of T cell-targeted immunosuppressive therapies suggests that other immune cells may contribute to its development. While multiple studies point towards a role of B cells in CLAD, knowledge regarding the specific populations of B cells prevalent in lungs of CLAD patients is lacking. We aimed to define the transcriptional signatures of B cell populations in explanted CLAD lungs by single-cell RNA sequencing (scRNAseq). <h3>Methods</h3> Single cell suspensions were made from 4 samples of CLAD lung explants. B cells were isolated by flow sorting and transcriptionally profiled using 10X genomics (10,000 cells, 50,000 reads/cell) within 2 hours. The transcriptional heterogeneity of B cells in CLAD lungs was analyzed using the R package Seurat. <h3>Results</h3> Based on flow cytometry, B cells (CD45+/CD3-/CD19+) were present in the lungs of all four CLAD patients, consistent with previously published reports of B cell-rich tertiary lymphoid organs in CLAD lungs. ScRNAseq analysis of expression levels of immunoglobulins, transcription factors, and surface markers of conventional B cells subsets revealed the presence of both naïve and antigen-experienced B cells in CLAD lungs. We identified populations of transitional B cells, naïve follicular B cells, germinal center cells, plasmablasts, and memory B cells. Notably, we identified a distinct population of class-switched B cells (IgM/IgD low, IgG/IgA high, and CD38+). In addition, our analysis revealed that many B cells expressed markers of antigen presentation including CD74, CIITA, and HLA-DMA, suggesting that B cells in CLAD lungs may function as antigen-presenting cells. <h3>Conclusion</h3> Our scRNAseq results show the prevalence of both naïve and antigen-experienced B cells in the lungs of CLAD patients. Further analyses will aim to identify specific B cell populations that may be associated with allograft dysfunction.

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

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

N-myc-interactor mediates microbiome induced epithelial to mesenchymal transition and is associated with chronic lung allograft dysfunction

Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking the microbiome to CLAD are poorly delineated. We investigated a possible mechanism involved in microbial modulation of mucosal response leading to CLAD with the hypothesis that a Proteobacteria dominant lung microbiome would inhibit N-myc-interactor (NMI) expression and induce epithelial to mesenchymal transition (EMT). Explant CLAD, non-CLAD, and healthy nontransplant lung tissue were collected, as well as bronchoalveolar lavage from 14 CLAD and matched non-CLAD subjects, which were followed by 16S rRNA amplicon sequencing and quantitative polymerase chain reaction (PCR) analysis. Pseudomonas aeruginosa (PsA) or PsA-lipopolysaccharide was cocultured with primary human bronchial epithelial cells (PBEC). Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate NMI expression and EMT in explants and in PsA-exposed PBECs. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of EMT regulator NMI. 16S rRNA amplicon analyses revealed that CLAD patients have a higher abundance of phyla Proteobacteria and reduced abundance of the phyla Bacteroidetes. At the genera level, CLAD subjects had an increased abundance of genera Pseudomonas and reduced Prevotella. Human CLAD airway cells showed a downregulation of the N-myc-interactor gene and presence of EMT. Furthermore, exposure of human primary bronchial epithelial cells to PsA resulted in downregulation of NMI and induction of an EMT phenotype while NMI upregulation resulted in attenuation of this PsA-induced EMT response. CLAD is associated with increased bacterial biomass and a Proteobacteria enriched airway microbiome and EMT. Proteobacteria such as PsA induces EMT in human bronchial epithelial cells via NMI, demonstrating a newly uncovered mechanism by which the microbiome induces cellular metaplasia.

Gene Expression Profiling and Pathway Enrichment Analysis in Long-Term Survivors after Lung Transplantation with Normal Allograft Function

Chronic lung allograft dysfunction (CLAD) is the main limiting factor for long-term survival after lung transplantation (LT). However, there is a small number of LT patients which are long-term survivors with good allograft function (LTS). The aim of the present study was to compare the gene expression profile of CLAD and LTS patients and performing an enrichment analysis by using Reactome annotation database. We analyzed whole-blood gene expression profiles of 60 double lung transplant recipients who were included in this multicenter cross-sectional study: 30 patients with CLAD and 30 patients with a stable allograft function 10 years after lung transplantation (LTS). Expression analysis were performed by microarray technology (ClariomTM D Arrays). We identified 458 differentially expressed genes (DEGs) between LTS and CLAD patients. Among them, 201 genes were up-regulated and 257 genes were down-regulated. Differentially expressed genes included MMP-8, LTF, TCN1 and OLFM4. Enrichment analysis showed involvement of neutrophil degranulation, class A/1 (Rhodopsin-like receptors), cell surface interaction at the vascular wall, antimicrobial peptides, interleukin-4 and 13 signaling, role of phospholipids in phagocytosis and activation of matrix metalloproteinases pathways. It seems that neutrophil degranulation, matrix metalloproteinases and antimicrobial peptides gene expression pathways within others could be involve in differences between CLAD and LTS patients. Study financed by Instituto de Salud Carlos III (PI13/01076); the European Regional Development Fund (FEDER), FUCAP, Astellas, Novartis and Chiesi.

Characterization Of Chronic Lung Allograft Dysfunction With Oscillometry

Chronic lung allograft dysfunction (CLAD) affects up to 70% of lung transplant (LTx) recipients and is the major impediment to long-term survival. CLAD is a retrospective diagnosis of exclusion when other causes for sustained drop of ≥20% of forced expiratory volume in 1s or forced vital capacity have been ruled out. No effective treatments exist, likely due to irreversible lung damage at diagnosis. Oscillometry (Osc) is a promising pulmonary function test that is particularly sensitive to changes in small airways, the site of CLAD onset. Our aim is to characterize Osc in patients with early CLAD. In a prospective study that began in December 2017, all new double LTx recipients are enrolled for Osc prior to routine spirometry. CLAD will be diagnosed according to ISHLT guidelines. By March 2020 we anticipate enrolment of 300 patients and 30 to have CLAD. We will compare Osc in CLAD patients with those who remain CLAD free after ≥6 mths follow-up. Univariate and mixed models will be used to examine spirometry vs Osc parameters of small airway disease: namely, X5 (reactance at 5Hz), Ax (area of reactance) and R5-19 (difference of resistance at 5 and 19 Hz). Receiver operating characteristic area under the curve (AUC) will be used to assess the predictive power of each. In interim analysis of patients enrolled by 31 July 2019, 13/197 had CLAD [all with bronchiolitis obliterans syndrome (BOS)], with median time of diagnosis at 24 wks post-LTx (range 10-59). Osc was abnormal and significantly different in CLAD vs. 87 CLAD-free recipients who had follow-up of ≥ 6 mths (median 40 wks). The mean Osc measurements at time of CLAD diagnosis vs last follow-up of CLAD-free patients are: X5 = -3.7 vs -2.1 cmH20.s/L, Ax = 31.6 vs 15.9 cmH20/L and R5-19 = 1.5 vs 0.8 cmH20.s/L (p < 0.05 for all). Using X5 to predict BOS, the AUC was 84%; a cut-off of X5 < -2.4 cmH20.s/L had sensitivity 73% and specificity 92%. Early analysis suggests Osc has excellent predictive capacity for BOS. Future work will test whether Osc can predict CLAD onset while spirometry is normal.

Forced Vital Capacity for Defining Restrictive Allograft Syndrome and Mixed Phenotype in Lung Transplant Recipients

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.

Circulating Follicular Helper T Cells are Decreased in Chronic Lung Allograft Dysfunction

Humoral immunity is a fundamental element of chronic lung allograft dysfunction (CLAD) as shown by the presence of B cells and lymphoid follicles in the lung allograft. The formation of lymphoid follicles is under control of follicular helper T (TFH) cells, which also help B cells to differentiate into high-affinity alloantibody-producing plasma cells and memory B cells. The presence and role of circulating TFH cells in CLAD has not yet been investigated and is therefore the objective of our cross-sectional study. Whole blood samples were collected from healthy controls (HC) (n=7) and lung transplant recipients (n=31: stable n=12, CLAD n=19). After peripheral blood mononuclear cell isolation, quantification of CD3+ T cells, CD4+ T helper (TH) cells and CXCR5+PD1+ TFH cells was performed using flow cytometry (LSR Fortessa, BD Bioscience). One-way ANOVA with multiple comparisons was used for statistical analysis. No differences were found in CD3+ T lymphocytes between groups, but percentages of CD4+ TH cells within the T cell population were decreased in CLAD versus HC (p=0.005) and the same trend was seen in stable patients versus HC (p=0.07). The number of TFH cells within the TH cells was lower in HC (p=0.044) and in CLAD patients (p<0.0001) versus stable transplant patients. Circulating TFH cells are measurable in blood of lung transplant recipients by flow cytometry and are significantly decreased in patients with CLAD compared to stable lung transplant patients. TFH may play a role in immune activation after lung transplantation, however, further investigation is needed.

Identification and characterization of chronic lung allograft dysfunction patients with mixed phenotype: A single‐center study

Patients can change chronic lung allograft dysfunction (CLAD) phenotype, especially from BOS to mixed phenotype. Our aim was to further characterize these patients. Mixed CLAD was defined as a restrictive physiology with persistent CT opacities, after initial bronchiolitis obliterans syndrome (BOS) diagnosis. The incidence, prognosis, pulmonary function, radiology, pathology, and airway inflammation were compared between patients with restrictive allograft syndrome (RAS) and mixed CLAD. A total of 268 (44%) patients developed CLAD of which 47 (18%) were diagnosed with RAS "ab initio," 215 (80%) with BOS, and 6 (2%) an undefined phenotype. Twenty-five patients developed a mixed CLAD phenotype (24 BOS to mixed and 1 RAS to mixed). Survival after mixed phenotype diagnosis was comparable (P=.39) to RAS. More emphysema patients developed a mixed phenotype (P=.020) compared to RAS ab initio, while mixed CLAD patients had a lower FEV1 (P<.0001) and FEV1 /FVC (P=.0002) at diagnosis compared to RAS ab initio. CT scans in patients with the mixed phenotype demonstrated apical predominance of the opacities (P=.0034) with pleuroparenchymal fibroelastosis on histopathology. We further characterized patients with a mixed phenotype of CLAD. Although the survival after diagnosis was comparable to RAS ab initio patients, there was a difference in demography, pulmonary function, radiology, and pathology.

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

Chronic lung allograft dysfunction (CLAD) is a fatal condition that limits survival after lung transplantation (LTx). The pathological hallmark of CLAD is obliterative bronchiolitis (OB). A subset of patients present with a more aggressive CLAD phenotype, called restrictive allograft syndrome (RAS), characterized by lung parenchymal fibrosis (PF). The mouse orthotopic single LTx model has proven relevant to the mechanistic study of allograft injury. The minor-alloantigen-mismatched strain combination using C57BL/10(B10) donors and C57BL/6(B6) recipients reportedly leads to OB. Recognizing that OB severity is a spectrum that may coexist with other pathologies, including PF, we aimed to characterize and quantify pathologic features of CLAD in this model. Left LTx was performed in the following combinations: B10→B6, B6→B10, B6→B6. Four weeks posttransplant, blinded pathologic semi-quantitative assessment showed that OB was present in 66% of B10→B6 and 30% of B6→B10 grafts. Most mice with OB also had PF with a pattern of pleuroparenchymal fibroelastosis, reminiscent of human RAS-related pathology. Grading of pathologic changes demonstrated variable severity of airway fibrosis, PF, acute rejection, vascular fibrosis, and epithelial changes, similar to those seen in human CLAD. These assessments can make the murine LTx model a more useful tool for further mechanistic studies of CLAD pathogenesis.

The histomorphological spectrum of restrictive chronic lung allograft dysfunction and implications for prognosis

AbstractChronic lung allograft dysfunction continues to be the main contributor to poor long-term allograft survival after lung transplantation. The restrictive phenotype of chronic lung allograft dysfunction carries a particularly poor prognosis. Little is known about the pathogenetic mechanisms involved in restrictive chronic lung allograft dysfunction. In this study, we performed histomorphological and immunohistochemical analysis of restrictive chronic lung allograft dysfunction lungs. Explant lung tissue from 21 restrictive chronic lung allograft dysfunction patients was collected and histopathologic patterns of rejection, fibrosis and vascular changes were scored after routine histochemical stains and additional immunohistochemistry for endothelial markers and C4d. In all, 75% of cases showed evidence of acute cellular rejection; lymphocytic bronchiolitis was absent in most lungs, whereas in 55% there was obliterative bronchiolitis. Almost half of the cases showed a pattern consistent with pleuroparenchymal fibro-elastosis (n=10), and a subset showed nonspecific interstitial pneumonia (n=5) or irregular emphysema (n=5). Fibrinous alveolar exudates were frequently seen in association with fibrosis (n=6), but no diffuse alveolar damage was found. Evidence of microvascular damage was present in most cases. An emphysematous pattern of fibrosis was associated with a better survival (P=0.0030), whereas fibrinous exudates were associated with a worse survival (P=0.0007). In addition to the previously described nonspecific interstitial pneumonia and pleuroparenchymal fibro-elastosis patterns in restrictive chronic lung allograft dysfunction, we are the first to describe a pattern of fibrosis-induced subpleural/paraseptal emphysema. This pattern confers a better survival, whereas fibrinous exudates are associated with a worse survival. We believe that our findings offer a pathogenetic theory for pleuroparenchymal fibro-elastosis in restrictive chronic lung allograft dysfunction, and show that restrictive chronic lung allograft dysfunction is an increasingly heterogeneous disease with presumably different mechanisms of subpattern formation.

The Lymphatic Phenotype of Lung Allografts in Patients With Bronchiolitis Obliterans Syndrome and Restrictive Allograft Syndrome.

Chronic lung allograft dysfunction (CLAD), presenting as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) is the major limiting factor of long-term survival in lung transplantation. Its pathogenesis is still obscure. In BOS, persistent alloimmune injury and chronic airway inflammation are suggested. One of the main tasks of the lymphatic vessel (LV) system is the promotion of immune cell trafficking. The formation of new LVs has been shown to trigger chronic allograft rejection in kidney transplants. We therefore sought to address the role of lymphangiogenesis in CLAD. Formalin-fixed paraffin-embedded tissue samples of 22 patients receiving a lung retransplantation due to BOS or RAS were collected. Lymphatic vessel density (LVD) was determined by immunohistochemical staining for podoplanin. Lung tissue obtained from 13 non-CLAD patients served as control. The impact of LVD on graft survival was assessed. Lymphatic vessel density in CLAD patients did not differ from those in control subjects (median number of LVs per bronchiole: 4.75 (BOS), 6.47 (RAS), 4.25 (control), P = 0.97). Moreover, the number of LVs was not associated with regions of cellular infiltrates (median number of LVs per bronchiole: with infiltrates, 5.00 (BOS), 9.00 (RAS), 4.00 (control), P = 0.62; without infiltrates, 4.5 (BOS), 0.00 (RAS), 4.56 (control), P = 0.74). Lymphatic vessel density did not impact the time to development of BOS or RAS in lung transplantation (low vs high LVD: 38.5 vs 86.0 months, P = 0.15 [BOS]; 60.5 vs 69.5 months, P = 0.80 [RAS]). Unlike chronic organ failure in kidney transplantation, lymphangiogenesis is not altered in CLAD patients. Our findings highlight unique immunological processes leading to BOS and RAS.

Epithelial and Erythrocyte Microvesicles From Bronchoalveolar Lavage Fluid Are Elevated and Associated With Outcome in Chronic Lung Allograft Dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major outcome limitation for lung transplant recipients (LTR) after the first year, and therapies targeting immunological pathways show only limited success. Because microvesicles (MV) are biomarkers of endothelial dysfunction and coagulation but are also involved in immunological responses, we hypothesized that MV, found in bronchoalveolar lavage (BAL) fluids (BALF) of LTR at CLAD diagnosis, are elevated and potential prognostic biomarkers. The BALF was collected from 37 LTR at time point of CLAD diagnosis and 37 LTR without any complication at routinely performed BAL. The MV concentration and origin were determined by flow cytometry by detection of different antigens. Patient- and transplant-related risk factors were included in a retrospective statistical analysis. The BALF-MV levels of epithelial and red blood cell (RBC) origin were significantly higher in CLAD patients (mean: 1533/μL and 158/μL) compared to controls (436/μL, 57/μL). The LTR with high levels of epithelial MV >580/μL showed a significantly shorter overall survival at 4 years after BAL (39.5%) compared to patients with low MV (66.4%) and this proofed to be an independent prognostic factor in multivariate Cox analysis (hazards ratio = 3.05). Furthermore, LTR with high levels of RBC MV ≥225/μL were also associated with worse disease-specific survival, with probabilities at 4 years after BAL of 85.8% vs. 36.0%. Epithelial and RBC BALF-MV are elevated at CLAD diagnosis, have a potential as biomarkers, and support the hypothesis of a pathway, including activation of coagulation and complement, endothelial barrier dysfunction, and microangiopathy.

Chronic lung allograft dysfunction: evolving practice.

Chronic lung allograft dysfunction (CLAD) was recently introduced as an overarching term covering different phenotypes of chronic allograft dysfunction, including obstructive CLAD (bronchiolitis obliterans syndrome), restrictive CLAD (restrictive allograft syndrome) and graft dysfunction due to causes not related to chronic rejection. In the present review, we will highlight the latest insights and current controversies regarding the new CLAD terminology, underlying pathophysiologic mechanisms, diagnostic approach and possible treatment options. Different pathophysiological mechanisms are clearly involved in clinically distinct phenotypes of chronic rejection, as is reflected by differences in histology, allograft function and imaging. Therefore, not all CLAD patients may equally benefit from specific therapies. The recent introduction of CLAD importantly changed the clinical practice in lung transplant recipients. Given the relative low accuracy of the current diagnostic tools, future research should focus on specific biomarkers, more sensitive pulmonary function parameters and imaging techniques for timely CLAD diagnosis and phenotyping. Personalized or targeted therapeutic options for adequate prevention and treatment of CLAD are required.

S100 Family Proteins in Human Chronic Lung Allograft Dysfunction: Potential Markers for Biologic Subtyping

Purpose Chronic lung allograft dysfunction (CLAD) remains a major cause of mortality and morbidity after transplantation. We have noted restrictive allograft syndrome (RAS) as a novel subtype of CLAD. We aimed to further characterize human CLAD subtypes by profiling damage-associated molecular pattern molecules in bronchoalveolar lavage fluid (BALF). Methods and Materials 17 consecutively identified specimens from patients with CLAD (5 RAS, 12 bronchiolitis obliterans syndrome (BOS) ) and 12 controls from patients with no CLAD were included. All the BALF specimens from CLAD cases were taken after their clinical onset. CLAD and its subtypes were defined based on pulmonary function test results. S100A8, S100A9, S100A12, S100P and high mobility group box-1 (HMGB-1) expression were measured in BALF by enzyme-linked immunosorbent assay. Results All of S100A8, S100A9, S100A12 and S100P were upregulated in RAS compared to controls (p figure 1 ] Conclusions In CLAD patients, we have noted a distinct difference in the expression of S100 family proteins in BALF. It appears that S100A9 is associated with RAS and not BOS. Hopefully, further characterization of molecular pathways involved in the development of CLAD subtypes will help to develop more accurate diagnostics and specifically directed therapies.