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.     

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.     

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.     

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.     

Allelic and Epitopic Characterization of Intra–Kidney Allograft Anti‐HLA Antibodies at Allograft Nephrectomy

The reasons for the increased incidence of de novo anti–human leukocyte antibody (HLA) donor‐specific antibodies (DSAs) observed after kidney allograft nephrectomy are not fully understood. One advocated mechanism suggests that at graft loss, DSAs are not detected in the serum because they are fixed on the nonfunctional transplant; removal of the kidney allows DSAs to then appear in the blood circulation. The aim of our study was to compare anti‐HLA antibodies present in the serum and in the graft at the time of an allograft nephrectomy. Using solid‐phase assays, anti‐HLA antibodies were searched for in the sera of 17 kidney transplant patients undergoing allograft nephrectomy. No anti‐HLA antibodies were detected in the graft if they were not also detected in the serum. Eleven of the 12 patients who had DSAs detected in their sera also had DSAs detected in the grafts. Epitopic analysis revealed that most anti‐HLA antibodies detected in removed grafts were directed against the donor. In summary, our data show that all anti‐HLA antibodies that were detected in grafts were also detected in the sera. These intragraft anti‐HLA antibodies are mostly directed against the donor at an epitopic level but not always at an antigenic level.     

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.     

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.     

Objective Estimates Improve Risk Stratification for Primary Graft Dysfunction after Lung Transplantation

Primary graft dysfunction (PGD) is a major cause of early mortality after lung transplant. We aimed to define objective estimates of PGD risk based on readily available clinical variables, using a prospective study of 11 centers in the Lung Transplant Outcomes Group (LTOG). Derivation included 1255 subjects from 2002 to 2010; with separate validation in 382 subjects accrued from 2011 to 2012. We used logistic regression to identify predictors of grade 3 PGD at 48/72 h, and decision curve methods to assess impact on clinical decisions. 211/1255 subjects in the derivation and 56/382 subjects in the validation developed PGD. We developed three prediction models, where low‐risk recipients had a normal BMI (18.5–25 kg/m²), chronic obstructive pulmonary disease/cystic fibrosis, and absent or mild pulmonary hypertension (mPAP<40 mmHg). All others were considered higher‐risk. Low‐risk recipients had a predicted PGD risk of 4–7%, and high‐risk a predicted PGD risk of 15–18%. Adding a donor‐smoking lung to a higher‐risk recipient significantly increased PGD risk, although risk did not change in low‐risk recipients. Validation demonstrated that probability estimates were generally accurate and that models worked best at baseline PGD incidences between 5% and 25%. We conclude that valid estimates of PGD risk can be produced using readily available clinical variables.     

HLA Mismatching Favoring Host‐Versus‐Graft NK Cell Activity Via KIR3DL1 Is Associated With Improved Outcomes Following Lung Transplantation

Chronic lung allograft dysfunction (CLAD) is linked to rejection and limits survival following lung transplantation. HLA‐Bw4 recipients of HLA‐Bw6 grafts have enhanced host‐versus‐graft (HVG) natural killer (NK) cell activity mediated by killer cell immunoglobulin‐like receptor (KIR)3DL1 ligand. Because NK cells may promote tolerance by depleting antigen‐presenting cells, we hypothesized improved outcomes for HLA‐Bw4 recipients of HLA‐Bw6 grafts. We evaluated differences in acute cellular rejection and CLAD‐free survival across 252 KIR3DL1+ recipients from University of California, San Francisco (UCSF). For validation, we assessed survival and freedom from bronchiolitis obliterans syndrome (BOS), retransplantation, or death in 12 845 non‐KIR typed recipients from the United Network for Organ Sharing (UNOS) registry. Cox proportional hazards models were adjusted for age, gender, ethnicity, transplant type, and HLA mismatching. HVG‐capable subjects in the UCSF cohort had a decreased risk of CLAD or death (hazard ratio [HR] 0.57, 95% confidence interval [CI] 0.36–0.88) and decreased early lymphocytic bronchitis. The HVG effect was not significant in subjects with genotypes predicting low KIR3DL1 expression. In the UNOS cohort, HVG‐capable subjects had a decreased risk of BOS, retransplant, or death (HR 0.95, 95% CI 0.91–0.99). Survival improved with the higher‐affinity Bw4‐80I ligand and in Bw4 homozygotes. Improved outcomes in HVG‐capable recipients are consistent with a protective NK cell role. Augmentation of NK activity could supplement current immunosuppression techniques.     

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.     

Transfer of donor anti‐HLA antibody expression to multiple transplant recipients: A potential variant of the passenger lymphocyte syndrome?

Antibody‐mediated rejection, whereby transplant recipient B cells and/or plasma cells produce alloreactive anti‐human leukocyte antigen (HLA) antibodies, negatively influences transplant outcomes and is a major contributor to graft loss. An early humoral immune response is suggested by the production of anti‐HLA donor‐specific antibodies (DSA) that can be measured using solid phase assays. We report the early posttransplant coexistence of a shared anti‐HLA antibody profile in 5 solid organ transplant recipients who received organs from the same donor. Retrospective analysis of the donor's serum confirmed the presence of the same anti‐HLA profile, suggesting the transfer of donor‐derived anti‐HLA antibodies, or the cells that produce them, to multiple solid organ transplant recipients. The time frame and extent of transfer suggest a novel variant of the passenger lymphocyte syndrome. These findings have important implications for the consideration of all posttransplant antibody measurements, particularly the interpretation of non‐DSAs in the sera of 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.     

A Memory B Cell Crossmatch Assay for Quantification of Donor‐Specific Memory B Cells in the Peripheral Blood of HLA‐Immunized Individuals

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow–residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA‐specific memory B cells are scarce and insufficient in quantifying the complete donor‐specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor‐specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme‐linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II–specific memory B cells per million IgG‐producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor‐specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross‐match assay to quantify donor‐specific memory B cells in patients with a history of sensitizing events.     

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.     

Assessing the utilization of high‐resolution 2‐field HLA typing in solid organ transplantation

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA‐matching status between donor‐recipient pairs and assessing patients’ anti‐HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next‐generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high‐resolution 2‐field HLA typing (HR‐2F) in SOT by retrospectively evaluating NGS‐typed pre‐ and post‐SOT cases. HR‐2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre‐ and posttransplant scenarios were identified as being better served by HR‐2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR‐2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti‐HLA antibody issues.     

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.