HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction

Donor selection in lung transplantation (LTx) is historically based upon clinical urgency, ABO compatibility, and donor size. HLA matching is not routinely considered; however, the presence or later development of anti‐HLA antibodies is associated with poorer outcomes, particularly chronic lung allograft dysfunction (CLAD). Using eplet mismatches, we aimed to determine whether donor/recipient HLA incompatibility was a significant predictor of CLAD. One hundred seventy‐five LTx undertaken at the Alfred Hospital between 2008 and 2012 met criteria. Post‐LTx monitoring was continued for at least 12 months, or until patient death. HLA typing was performed by sequence‐based typing and Luminex sequence‐specific oligonucleotide. Using HLAMatchmaker, eplet mismatches between each donor/recipient pairing were analyzed and correlated against incidences of CLAD. HLA‐DRB1/3/4/5+DQA/B eplet mismatch was a significant predictor of CLAD (hazard ratio [HR] 3.77, 95% confidence interval [CI]: 1.71–8.29 p < 0.001). When bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) were analyzed independently, HLA‐DRB1/3/4/5 + DQA/B eplet mismatch was shown to significantly predict RAS (HR 8.3, 95% CI: 2.46–27.97 p < 0.001) but not BOS (HR 1.92, 95% CI: 0.64–5.72, p = 0.237). HLA‐A/B eplet mismatch was shown not to be a significant predictor when analyzed independently but did provide additional stratification of results. This study illustrates the importance of epitope immunogenicity in defining donor–recipient immune compatibility in LTx.     

Impact of Allograft Injury Time of Onset on the Development of Chronic Lung Allograft Dysfunction After Lung Transplantation

The impact of allograft injury time of onset on the risk of chronic lung allograft dysfunction (CLAD) remains unknown. We hypothesized that episodes of late‐onset (≥6 months) allograft injury would produce an augmented CXCR3/ligand immune response, leading to increased CLAD. In a retrospective single‐center study, 1894 transbronchial biopsy samples from 441 lung transplant recipients were reviewed for the presence of acute rejection (AR), lymphocytic bronchiolitis (LB), diffuse alveolar damage (DAD), and organizing pneumonia (OP). The association between the time of onset of each injury pattern and CLAD was assessed by using multivariable Cox models with time‐dependent covariates. Bronchoalveolar lavage (BAL) CXCR3 ligand concentrations were compared between early‐ and late‐onset injury patterns using linear mixed‐effects models. Late‐onset DAD and OP were strongly associated with CLAD: adjusted hazard ratio 2.8 (95% confidence interval 1.5–5.3) and 2.0 (1.1–3.4), respectively. The early‐onset form of these injury patterns did not increase CLAD risk. Late‐onset LB and acute rejection (AR) predicted CLAD in univariable models but lost significance after multivariable adjustment for late DAD and OP. AR was the only early‐onset injury pattern associated with CLAD development. Elevated BAL CXCR3 ligand concentrations during late‐onset allograft injury parallel the increase in CLAD risk and support our hypothesis that late allograft injuries result in a more profound CXCR3/ligand immune response.     

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.     

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.     

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.     

Phenotyping Chronic Lung Allograft Dysfunction Using Body Plethysmography and Computed Tomography

Restrictive subtype of chronic lung allograft dysfunction (CLAD) was recently described after lung transplantation. This study compares different definitions of a restrictive phenotype in CLAD patients and impact on survival. Eighty‐nine CLAD patients out of 1191 screened patients (September 1987 to July 2012) were included as complete longitudinal lung volume measurements and chest computed tomography (CT) after CLAD onset was available. CT findings and lung volumes were quantified and survival was calculated for distinctive groups and predictive factors for worse survival were investigated. Graft survival in patients with total lung capacity (TLC) between 90% and 81% of baseline (BL) (n = 13, 15%) in CLAD course was similar to those with TLC >90% BL (n = 64, 56%; log‐rank test p = 0.9). Twelve patients (13%) developed a TLC ≤80% BL and 10 (11%) had significant parenchymal changes on CT, of whom 6 (46%) also had TLC ≤80% BL. CT changes correlated with TLC ≤80% BL (Φ‐coefficient = 0.48, p = 0.001). Patients with either TLC ≤80% or significant CT changes (n = 16, 18%) had a significantly reduced survival (log‐rank p < 0.001). Forced vital capacity loss at CLAD onset was associated with poorer survival but did not correlate with the TLC or CT changes. A restrictive subtype of CLAD may be defined by either TLC ≤80% BL or severe parenchymal changes on chest CT.     

Association of Higher CD4+CD25highCD127low,FoxP3+, and IL‐2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung‐transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4⁺CD25^high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL‐2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4⁺CD25^highCD127^low, CD4⁺CD25^highFoxP3⁺ and CD4⁺CD25^highIL‐2⁺ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.     

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL‐17. We have used this orthotopic mouse model to investigate the source of IL‐17A and the requirement for T cells producing IL‐17A. The major sources of IL‐17A were CD4⁺ T cells and γδ T cells. Depletion of CD4⁺ T cells led to a significantly decreased frequency and number of IL‐17A⁺ lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3‐deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL‐17A⁺ cells was not decreased in mice with STAT3‐deficient T cells due mainly to the presence of IL‐17A⁺ γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4⁺ T cells are required for OB development and expansion of IL‐17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.     

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.     

Adipose Gene Expression Profile Changes With Lung Allograft Reperfusion

Obesity is a risk factor for primary graft dysfunction (PGD), a form of lung injury resulting from ischemia–reperfusion after lung transplantation, but the impact of ischemia–reperfusion on adipose tissue is unknown. We evaluated differential gene expression in thoracic visceral adipose tissue (VAT) before and after lung reperfusion. Total RNA was isolated from thoracic VAT sampled from six subjects enrolled in the Lung Transplant Body Composition study before and after allograft reperfusion and quantified using the Human Gene 2.0 ST array. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed enrichment for genes involved in complement and coagulation cascades and Jak‐STAT signaling pathways. Overall, 72 genes were upregulated and 56 genes were downregulated in the postreperfusion time compared with baseline. Long pentraxin‐3, a gene and plasma protein previously associated with PGD, was the most upregulated gene (19.5‐fold increase, p = 0.04). Fibronectin leucine‐rich transmembrane protein‐3, a gene associated with cell adhesion and receptor signaling, was the most downregulated gene (4.3‐fold decrease, p = 0.04). Ischemia–reperfusion has a demonstrable impact on gene expression in visceral adipose tissue in our pilot study of nonobese, non‐PGD lung transplant recipients. Future evaluation will focus on differential adipose tissue gene expression and the development of PGD after transplant.     

Prophylactic Azithromycin Therapy After Lung Transplantation: Post hoc Analysis of a Randomized Controlled Trial

Prophylactic azithromycin treatment has been demonstrated to improve freedom from bronchiolitis obliterans syndrome (BOS) 2 years after lung transplantation (LTx). In the current study, we re‐evaluated the long‐term effects of this prophylactic approach in view of the updated classification system for chronic lung allograft dysfunction (CLAD). A retrospective, intention‐to‐treat analysis of a randomized controlled trial comparing prophylactic treatment with placebo (n = 43) versus azithromycin (n = 40) after LTx was performed. Graft dysfunction (CLAD), graft loss (retransplantation, mortality), evolution of pulmonary function and functional exercise capacity were analyzed 7 years after inclusion of the last study subject. Following LTx, 22/43 (51%) patients of the placebo group and 11/40 (28%) patients of the azithromycin group ever developed CLAD (p = 0.043). CLAD‐free survival was significantly longer in the azithromycin group (p = 0.024). No difference was present in proportion of obstructive versus restrictive CLAD between both groups. Graft loss was similar in both groups: 23/43 (53%) versus 16/40 (40%) patients (p = 0.27). Long‐term pulmonary function and functional exercise capacity were significantly better in the azithromycin group (p < 0.05). Prophylactic azithromycin therapy reduces long‐term CLAD prevalence and improves CLAD‐free survival, pulmonary function, and functional exercise capacity after LTx.     

Proteomic Characterization Reveals That MMP‐3 Correlates With Bronchiolitis Obliterans Syndrome Following Allogeneic Hematopoietic Cell and Lung Transplantation

Improved diagnostic methods are needed for bronchiolitis obliterans syndrome (BOS), a serious complication after allogeneic hematopoietic cell transplantation (HCT) and lung transplantation. For protein candidate discovery, we compared plasma pools from HCT transplantation recipients with BOS at onset (n = 12), pulmonary infection (n = 16), chronic graft‐versus‐host disease without pulmonary involvement (n = 15) and no chronic complications after HCT (n = 15). Pools were labeled with different tags (isobaric tags for relative and absolute quantification), and two software tools identified differentially expressed proteins (≥1.5‐fold change). Candidate proteins were further selected using a six‐step computational biology approach. The diagnostic value of the lead candidate, matrix metalloproteinase 3 (MMP3), was evaluated by enzyme‐linked immunosorbent assay in plasma of a verification cohort (n = 112) with and without BOS following HCT (n = 76) or lung transplantation (n = 36). MMP3 plasma concentrations differed significantly between patients with and without BOS (area under the receiver operating characteristic curve 0.77). Consequently, MMP3 represents a potential noninvasive blood test for diagnosis of BOS.     

Assessment of Tocilizumab (Anti–Interleukin‐6 Receptor Monoclonal) as a Potential Treatment for Chronic Antibody‐Mediated Rejection and Transplant Glomerulopathy in HLA‐Sensitized Renal Allograft Recipients

Extending the functional integrity of renal allografts is the primary goal of transplant medicine. The development of donor‐specific antibodies (DSAs) posttransplantation leads to chronic active antibody‐mediated rejection (cAMR) and transplant glomerulopathy (TG), resulting in the majority of graft losses that occur in the United States. This reduces the quality and length of life for patients and increases cost. There are no approved treatments for cAMR. Evidence suggests the proinflammatory cytokine interleukin 6 (IL‐6) may play an important role in DSA generation and cAMR. We identified 36 renal transplant patients with cAMR plus DSAs and TG who failed standard of care treatment with IVIg plus rituximab with or without plasma exchange. Patients were offered rescue therapy with the anti–IL‐6 receptor monoclonal tocilizumab with monthly infusions and monitored for DSAs and long‐term outcomes. Tocilizumab‐treated patients demonstrated graft survival and patient survival rates of 80% and 91% at 6 years, respectively. Significant reductions in DSAs and stabilization of renal function were seen at 2 years. No significant adverse events or severe adverse events were seen. Tocilizumab provides good long‐term outcomes for patients with cAMR and TG, especially compared with historical published treatments. Inhibition of the IL‐6–IL‐6 receptor pathway may represent a novel approach to stabilize allograft function and extend patient lives.     

Long‐Term Persistence of Donor Alveolar Macrophages in Human Lung Transplant Recipients That Influences Donor‐Specific Immune Responses

Steady‐state alveolar macrophages (AMs) are long‐lived lung‐resident macrophages with sentinel function. Evidence suggests that AM precursors originate during embryogenesis and populate lungs without replenishment by circulating leukocytes. However, their presence and persistence are unclear following human lung transplantation (LTx). Our goal was to examine donor AM longevity and evaluate whether AMs of recipient origin seed the transplanted lungs. Origin of AMs was accessed using donor–recipient HLA mismatches. We demonstrate that 94–100% of AMs present in bronchoalveolar lavage (BAL) were donor derived and, importantly, AMs of recipient origin were not detected. Further, analysis of BAL cells up to 3.5 years post‐LTx revealed that the majority of AMs (>87%) was donor derived. Elicitation of de novo donor‐specific antibody (DSA) is a major post‐LTx complication and a risk factor for development of chronic rejection. The donor AMs responded to anti‐HLA framework antibody (Ab) with secretion of inflammatory cytokines. Further, in an experimental murine model, we demonstrate that adoptive transfer of allogeneic AMs stimulated humoral and cellular immune responses to alloantigen and lung‐associated self‐antigens and led to bronchiolar obstruction. Therefore, donor‐derived AMs play an essential role in the DSA‐induced inflammatory cascade leading to obliterative airway disease of the transplanted lungs.