When tissue is the issue: A histological review of chronic lung allograft dysfunction

Although chronic lung allograft dysfunction (CLAD) remains the major life‐limiting factor following lung transplantation, much of its pathophysiology remains unknown. The discovery that CLAD can manifest both clinically and morphologically in vastly different ways led to the definition of distinct subtypes of CLAD. In this review, recent advances in our understanding of the pathophysiological mechanisms of the different phenotypes of CLAD will be discussed with a particular focus on tissue‐based and molecular studies. An overview of the current knowledge on the mechanisms of the airway‐centered bronchiolitis obliterans syndrome, as well as the airway and alveolar injuries in the restrictive allograft syndrome and also the vascular compartment in chronic antibody‐mediated rejection is provided. Specific attention is also given to morphological and molecular markers for early CLAD diagnosis or histological changes associated with subsequent CLAD development. Evidence for a possible overlap between different forms of CLAD is presented and discussed. In the end, “tissue remains the (main) issue,” as we are still limited in our knowledge about the actual triggers and specific mechanisms of all late forms of posttransplant graft failure, a shortcoming that needs to be addressed in order to further improve the outcome of lung transplant recipients.     

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.     

Blood CD9+ B cell,a biomarker of bronchiolitis obliterans syndrome after lung transplantation

Bronchiolitis obliterans syndrome is the main limitation for long‐term survival after lung transplantation. Some specific B cell populations are associated with long‐term graft acceptance. We aimed to monitor the B cell profile during early development of bronchiolitis obliterans syndrome after lung transplantation. The B cell longitudinal profile was analyzed in peripheral blood mononuclear cells from patients with bronchiolitis obliterans syndrome and patients who remained stable over 3 years of follow‐up. CD24^hiCD38^hi transitional B cells were increased in stable patients only, and reached a peak 24 months after transplantation, whereas they remained unchanged in patients who developed a bronchiolitis obliterans syndrome. These CD24^hiCD38^hi transitional B cells specifically secrete IL‐10 and express CD9. Thus, patients with a total CD9⁺ B cell frequency below 6.6% displayed significantly higher incidence of bronchiolitis obliterans syndrome (AUC = 0.836, PPV = 0.75, NPV = 1). These data are the first to associate IL‐10‐secreting CD24^hiCD38^hi transitional B cells expressing CD9 with better allograft outcome in lung transplant recipients. CD9‐expressing B cells appear as a contributor to a favorable environment essential for the maintenance of long‐term stable graft function and as a new predictive biomarker of bronchiolitis obliterans syndrome–free survival.     

T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase‐9 via a C‐C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction

Chronic lung allograft dysfunction (CLAD) is the major limitation of long‐term survival after lung transplantation. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Alloimmune reactions and epithelial‐to‐mesenchymal transition have been suggested in BOS. However, little is known regarding the role of allogenicity in epithelial cell differentiation. Primary human bronchial epithelial cells (BECs) were treated with activated T cells in the presence or absence of transforming growth factor (TGF)‐β. The expression of epithelial and mesenchymal markers was investigated. The secretion of inflammatory cytokines and matrix metalloproteinase (MMP)‐9 was measured in culture supernatants and in plasma from lung transplant recipients (LTRs): 49 stable, 29 with BOS, and 16 with RAS. We demonstrated that C‐C motif chemokine 2 secreted by T cells supports TGF‐β–induced MMP‐9 production by BECs after binding to C‐C chemokine receptor type 2. Longitudinal investigation in LTRs revealed a rise in plasma MMP‐9 before CLAD onset. Multivariate analysis showed that plasma MMP‐9 was independently associated with BOS (odds ratio [OR] = 6.19, p = 0.002) or RAS (OR = 3.9, p = 0.024) and predicted the occurrence of CLAD 12 months before the functional diagnosis. Thus, immune cells support airway remodeling through the production of MMP‐9. Plasma MMP‐9 is a potential predictive biomarker of CLAD.     

Vendor‐specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+Foxp3+ regulatory T cell levels

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4⁺Foxp3⁺ regulatory T cells (T_regs) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad‐spectrum antibiotics decreases T_regs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3‐driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of T_regs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through T_reg‐dependent mechanisms and challenges the practice of indiscriminate broad‐spectrum antibiotic use in the perioperative period.     

The impact of first untreated subclinical minimal acute rejection on risk for chronic lung allograft dysfunction or death after lung transplantation

Acute cellular rejection (ACR) is a significant risk factor for chronic lung allograft dysfunction (CLAD). Although clinically manifest and higher grade (≥A2) ACR is generally treated with augmented immunosuppression, management of minimal (grade A1) ACR remains controversial. In our program, patients with subclinical and spirometrically stable A1 rejection (StA1R) are routinely not treated with augmented immunosuppression. We hypothesized that an untreated first StA1R does not increase the risk of CLAD or death compared to episodes of spirometrically stable no ACR (StNAR). The cohort was drawn from all consecutive adult, first, bilateral lung transplantations performed between 1999 and 2017. Biopsies obtained in the first‐year posttransplant were paired with (forced expiratory volume in 1 second FEV₁). The first occurrence of StA1R was compared to a time‐matched StNAR. The risk of CLAD or death was assessed using univariable and multivariable Cox proportional hazards models. The analyses demonstrated no significant difference in risk of CLAD or death in patients with a first StA1R compared to StNAR. This largest study to date shows that, in clinically stable patients, an untreated first A1 ACR in the first‐year posttransplant is not significantly associated with an increased risk for CLAD or death. Watchful‐waiting approach may be an acceptable tactic for stable A1 episodes in lung transplant recipients.     

Interleukin 6 trans-signaling is a critical driver of lung allograft fibrosis

Histopathologic examination of lungs afflicted by chronic lung allograft dysfunction (CLAD) consistently shows both mononuclear cell (MNC) inflammation and mesenchymal cell (MC) fibroproliferation. We hypothesize that interleukin 6 (IL-6) trans-signaling may be a critical mediator of MNC-MC crosstalk and necessary for the pathogenesis of CLAD. Bronchoalveolar lavage (BAL) fluid obtained after the diagnosis of CLAD has approximately twofold higher IL-6 and soluble IL-6 receptor (sIL-6R) levels compared to matched pre-CLAD samples. Human BAL-derived MCs do not respond to treatment with IL-6 alone but have rapid and prolonged JAK2-mediated STAT3 Tyr705 phosphorylation when exposed to the combination of IL-6 and sIL-6R. STAT3 phosphorylation within MCs upregulates numerous genes causing increased invasion and fibrotic differentiation. MNC, a key source of both IL-6 and sIL-6R, produce minimal amounts of these proteins at baseline but significantly upregulate production when cocultured with MCs. Finally, the use of an IL-6 deficient recipient in a murine orthotopic transplant model of CLAD reduces allograft fibrosis by over 50%. Taken together these results support a mechanism where infiltrating MNCs are stimulated by resident MCs to release large quantities of IL-6 and sIL-6R which then feedback onto the MCs to increase invasion and fibrotic differentiation.     

Detection of chronic lung allograft dysfunction using ventilation‐weighted Fourier decomposition MRI

Chronic lung allograft dysfunction (CLAD) remains the leading cause of morbidity and mortality after lung transplantation. Diagnosis requires spirometric change, which becomes increasingly difficult with advancing CLAD. Fourier decomposition magnetic resonance imaging (FD‐MRI) permits acquisition of ventilated‐weighted images during free‐breathing. This study evaluates FD‐MRI in detecting CLAD in selected patients after bilateral lung transplantation (DLTx). DLTx recipients demonstrating CLAD at various stages participated. Radiologists remained blinded to clinical status until completion of image analysis. Image acquisition used a 1.5‐T MR scanner using a spoiled gradient echo sequence. After FD processing and regional fractional ventilation (RFV) quantification, the volume defect percentage at 2 thresholds (VDP_1,2), median lung RFV and quartile coefficient of dispersion (QCD) were calculated. Sixty‐two patients participated. CLAD was present in 29/62 (47%) patients, of whom 17/62 (27%) had forced expiratory volume in 1 second ≤65% at image acquisition. VDP₁ was higher among these participants compared to other groups (P < .001). Increased VDP₁ was associated with subsequent graft loss, with values >2% showing reduced survival, independent of degree of graft dysfunction (P = .005). VDP₂ discriminated between presence or absence of CLAD (area under the curve = 0.71; P = .03). QCD increased significantly with advancing disease (P < .001). In conclusion, FD‐MRI‐derived parameters demonstrate potential in quantitative CLAD diagnosis and assessment after DLTx.     

A B cell–dependent pathway drives chronic lung allograft rejection after ischemia–reperfusion injury in mice

Chronic lung allograft dysfunction (CLAD) limits long‐term survival after lung transplant (LT). Ischemia–reperfusion injury (IRI) promotes chronic rejection (CR) and CLAD, but the underlying mechanisms are not well understood. To examine mechanisms linking IRI to CR, a mouse orthotopic LT model using a minor alloantigen strain mismatch (C57BL/10 [B10, H‐2^b] → C57BL/6 [B6, H‐2^b]) and isograft controls (B6→B6) was used with antecedent minimal or prolonged graft storage. The latter resulted in IRI with subsequent airway and parenchymal fibrosis in prolonged storage allografts but not isografts. This pattern of CR after IRI was associated with the formation of B cell–rich tertiary lymphoid organs within the grafts and circulating autoantibodies. These processes were attenuated by B cell depletion, despite preservation of allograft T cell content. Our observations suggest that IRI may promote B cell recruitment that drives CR after LT. These observations have implications for the mechanisms leading to CLAD after LT.     

Donor‐Derived Exosomes With Lung Self‐Antigens in Human Lung Allograft Rejection

The immunological role of exosomes in allograft rejection remains unknown. We sought to determine whether exosomes are induced during lung allograft rejection and to define the antigenic compositions of HLA, lung‐associated self‐antigens (SAgs) and microRNAs (miRNAs). Exosomes were isolated from sera and bronchoalveolar lavage fluid from 30 lung transplant recipients (LTxRs) who were stable or who had acute rejection (AR) or bronchiolitis obliterans syndrome (BOS). Exosomes were defined by flow cytometry for CD63 and western blotting for annexin V SAgs, collagen V (Col‐V) and Kα1 tubulin were examined by electron microscopy; miRNAs were profiled by a miRNA array. Donor HLA and SAgs were detected on exosomes from LTxRs with AR and BOS but not from stable LTxRs. Exosomes expressing Col‐V were isolated from sera from LTxRs 3 mo before AR and 6 mo before BOS diagnosis, suggesting that exosomes with SAgs may be a noninvasive rejection biomarker. Exosomes isolated from LTxRs with AR or BOS also contained immunoregulatory miRNAs. We concluded that exosomes expressing donor HLA, SAgs and immunoregulatory miRNAs are present in the circulation and local site after human lung transplantation and play an important role in the immune pathogenesis of acute allograft rejection and BOS.     

Montelukast for bronchiolitis obliterans syndrome after lung transplantation: a pilot study

Bronchiolitis obliterans syndrome (BOS) remains the major hurdle to improve long‐term survival after lung transplantation, as its treatment remains troublesome. In this pilot study, we investigated the effect of montelukast (a leukotriene receptor antagonist) on the FEV₁ decline after diagnosis of BOS and compared this with a control group. In both groups, 11 patients were included with BOS stage <3 and bronchoalveolar lavage (BAL) neutrophilia <15%, already being treated or concurrently being started on azithromycin. Control patients were selected retrospectively. After adding montelukast (10 mg/day) to the immunosuppressive regimen, the FEV₁ decline significantly decreased from 112 ± 26 ml/month before BOS diagnosis to 13 ± 13 ml/month after 6 months of montelukast therapy (P = 0.001). In the control group, there was no significant change in the rate of FEV₁ decline: 103 ± 20 ml/month before BOS diagnosis to 114 ± 27 ml/month (P = 0.55). Adding montelukast may be a promising treatment option in patients with low neutrophilic (<15%) BOS after lung transplantation, already or concurrently being treated with azithromycin.     

Current views on chronic rejection after lung transplantation

Chronic lung allograft dysfunction (CLAD) was recently introduced as an overarching term mainly to classify patients with chronic rejection after lung transplantation, although other conditions may also qualify for CLAD. Initially, only the development of a persistent and obstructive pulmonary function defect, clinically identified as bronchiolitis obliterans syndrome (BOS), was considered as chronic rejection, if no other cause could be identified. It became clear in recent years that some patients do not qualify for this definition, although they developed a chronic and persistent decrease in FEV₁, without another identifiable cause. As the pulmonary function decline in these patients was rather restrictive, this was called restrictive allograft syndrome (RAS). In the present review, we will further elaborate on these two CLAD phenotypes, with specific attention to the diagnostic criteria, the role of pathology and imaging, the risk factors, outcome, and the possible treatment options.     

The Prognostic Importance of Bronchoalveolar Lavage Fluid CXCL9 During Minimal Acute Rejection on the Risk of Chronic Lung Allograft Dysfunction

The clinical significance and treatment strategies for minimal acute rejection (grade A1), the most common form of acute rejection (AR), remain controversial. In this retrospective single‐center cohort study of 441 lung transplant recipients, we formally evaluate the association between minimal AR and chronic lung allograft dysfunction (CLAD) and test a novel hypothesis using bronchoalveolar lavage (BAL) CXCL9 concentration during minimal AR as a biomarker of subsequent CLAD development. In univariable and multivariable models adjusted for all histopathologic injury patterns, minimal AR was not associated with CLAD development. However, minimal AR with elevated BAL CXCL9 concentrations markedly increased CLAD risk in a dose–response manner. Minimal AR with CXCL9 concentrations greater than the 25th, 50th, and 75th percentile had adjusted hazard ratios (HRs) for CLAD of 1.1 (95% confidence interval [CI] 0.8–1.6), 1.6 (95% CI 1.1–2.3), and 2.2 (95% CI 1.4–3.4), respectively. Thus we demonstrate the utility of BAL CXCL9 measurement as a prognostic biomarker that allows discrimination of recipients at increased risk of CLAD development after minimal AR. BAL CXCL9 measurement during transbronchial biopsies may provide clinically useful prognostic data and guide treatment decisions for this common form of AR, as a possible strategy to minimize CLAD development.