Lung-associated Self-antigens Research Articles

Lung Transplant Candidates With Pretransplant Gastroesophageal Reflux and Antibodies to Lung Self-antigens Have Shorter CLAD-free Survival After Transplant.

Background.Pre–lung transplant (LTx) gastroesophageal reflux (GER) and circulating antibodies against the lung self-antigens (SAbs) collagen V and K-alpha-1 tubulin may predispose recipients to chronic lung allograft dysfunction (CLAD). We aimed to study the association of pre-LTx GER or pre-LTx SAbs with CLAD.Methods.In this retrospective analysis of patients who underwent LTx between 2015 and 2019, pre-LTx GER and SAbs were dichotomously defined as present or absent. The study group comprised recipients with either GER‚ SAbs, or both, and the control group comprised recipients without GER or SAbs. Endpoints included CLAD and survival.Results.Ninety-five LTx recipients were divided into a study group (n = 71; 75%) and a control group (n = 24; 25%). Pretransplant GER was associated with pre-LTx SAbs (odds ratio [95% confidence intervals], 5.022 [1.419-17.770]; P = 0.012). In addition, the study group (either GER‚ SAbs, or both) had a higher risk of CLAD (hazard ratio [95% confidence intervals], 8.787 [1.694-45.567]; P = 0.010) and lower CLAD-free survival after LTx than the control group (P = 0.007); however, overall survival was similar between the 2 groups (P = 0.618).Conclusions.GER was associated with elevated SAbs in LTx candidates, and either GER, SAbs, or both were associated with CLAD in LTx recipients. This association suggests that GER may cause an immune response to normally sequestered lung-associated self-antigens that drives ongoing lung injury.

Global Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Lung Transplant Recipients.

Lung transplant recipients (LTxRs) with acute rejection (AR) and chronic rejection (bronchiolitis obliterans syndrome [BOS]) induce circulating exosomes known to contain donor human leukocyte antigens and lung-associated self-antigens. Here, we sought to identify proteomic signatures in circulating extracellular vesicles (EVs) that differentiate LTxRs in 4 groups: stable, AR, BOS, or respiratory viral infection (RVI). EVs were isolated from plasma from patients in each group via ultracentrifugation. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography–tandem mass spectrometry. We identified 2 unique proteins for AR, 4 for RVI, 24 for BOS, and 8 for stable LTxRs. Differential analysis of AR, BOS, RVI, and stable proteins identified significantly deregulated proteins (p < 0.05, log2(fold change) > ±1) in each condition (31, 2, and 2, respectively). EVs from LTxRs with AR contained proteins involved in immunoglobulin, complement regulation, coagulation, and innate and adaptive immune response pathways. EVs from LTxRs with BOS revealed enriched immunoglobulin receptors and a carboxypeptidase N catalytic chain. EVs from LTxRs with RVI had an enriched macrophage-stimulating factor. We found unique signatures in LTxRs with AR, BOS, and RVI, highlighting complex immune mechanisms underlying lung allograft rejection. Proteomic signatures in LTxRs’ circulating EVs provided insights into immunological mechanisms of graft rejection and RVI.

Respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection

BACKGROUNDRespiratory viral infections can increase the risk of chronic lung allograft dysfunction after lung transplantation, but the mechanisms are unknown. In this study, we determined whether symptomatic respiratory viral infections after lung transplantation induce circulating exosomes that contain lung-associated self-antigens and assessed whether these exosomes activate immune responses to self-antigens.METHODSSerum samples were collected from lung transplant recipients with symptomatic lower- and upper-tract respiratory viral infections and from non-symptomatic stable recipients. Exosomes were isolated via ultracentrifugation; purity was determined using sucrose cushion; and presence of lung self-antigens, 20S proteasome, and viral antigens for rhinovirus, coronavirus, and respiratory syncytial virus were determined using immunoblot. Mice were immunized with circulating exosomes from each group and resulting differential immune responses and lung histology were analyzed.RESULTSExosomes containing self-antigens, 20S proteasome, and viral antigens were detected at significantly higher levels (p < 0.05) in serum of recipients with symptomatic respiratory viral infections (n = 35) as compared with stable controls (n = 32). Mice immunized with exosomes from recipients with respiratory viral infections developed immune responses to self-antigens, fibrosis, small airway occlusion, and significant cellular infiltration; mice immunized with exosomes from controls did not (p < 0.05).CONCLUSIONSCirculating exosomes isolated from lung transplant recipients diagnosed with respiratory viral infections contained lung self-antigens, viral antigens, and 20S proteasome and elicited immune responses to lung self-antigens that resulted in development of chronic lung allograft dysfunction in immunized mice.

Chronic Lung Allograft Dysfunction: Immune Responses Induced by Circulating Exosomes with Lung-Associated Self-Antigens.

Exosomes, nanovesicles shown to regulate physiological processes in vivo, have been implicated in pathological conditions including cancer, autoimmune disease, infectious disease, neurodegenerative disease, and allograft rejection. Studies of lung transplant recipients with primary graft dysfunction, respiratory viral infection, and (acute) rejection have demonstrated circulating exosomes containing donor-mismatched human leukocyte antigen and lung-associated self-antigens, K-alpha 1 tubulin and collagen V, indicating that exosomes are originating from the transplanted organ. These circulating exosomes likely play a role in activating immune responses that lead to increased risk of chronic lung allograft dysfunction, as exosomes efficiently present their antigens to the immune system by all known pathways of antigen recognition (i.e., direct, indirect, and semidirect pathways). Here, we discuss exosome biogenesis, describe their contents, and address the mechanism of exosome-mediated activation of immune responses that lead to allograft rejection, especially after lung transplantation.

Circulating Exosomes with Distinct Properties during Chronic Lung Allograft Rejection.

Circulating exosomes containing donor HLA and lung-associated self-antigens (SAg) are thought to play an important role in allograft rejection after human lung transplantation. We characterized exosomes isolated from serum of 10 lung transplant recipients (LTxR) diagnosed with bronchiolitis obliterans syndrome (BOS) and compared them with exosomes isolated from serum of 10 stable LTxR. Lung-associated SAg (K-α-1-tubulin [Kα1T] and collagen V [Col-V]), MHC class II molecules, costimulatory molecules CD40, CD80, and CD86, and transcription factors class II MHC trans-activator, NF-κB, hypoxia-inducible factor 1-α, IL-1R-associated kinase 1, MyD88, and 20S proteasome were detected in exosomes from BOS, but not stable LTxR. In contrast, adhesion molecules were present in both groups. C57BL/6 mice immunized with exosomes from BOS but not stable LTxR demonstrated Ab to SAg (Col-V, 33.5 ± 15.7 versus 10.4 ± 6.4, p = 0.021; Kα1T, 925 ± 403 versus 317 ± 285, p = 0.044) and HLA (mean fluorescence intensity: BOS, 8450; stable, 632; p < 0.05). Furthermore, splenic lymphocytes demonstrated increased frequency of lung SAg-specific IL-17 (Col-V, 128 ± 46 versus 31 ± 21, p = 0.013; Kα1T, 194 ± 47 versus 67 ± 43, p = 0.014) and IFN-γ (Col-V, 165 ± 79 versus 38 ± 40, p = 0.042; Kα1T, 232 ± 64 versus 118 ± 39, p = 0.012). Reduced levels of IL-10-producing cells were seen in BOS exosome immunized mice compared with mice immunized with stable exosomes (Col-V, 59 ± 23 versus 211 ± 85, p = 0.016; Kα1T, 78 ± 49 versus 295 ± 104, p = 0.017). Owing to the unique immune-stimulating properties of exosomes induced during rejection, we propose that they play an important role in eliciting both alloantigen- and SAg-specific immunity, leading to chronic rejection after lung transplantation.

(326) - Antibodies to Human Leukocyte Antigen Class I and Lung Associated Self-Antigens, K-alpha-1 Tubulin and Collagen V Induces Exosomes Containing Self-Antigens from Human Primary Airway Epithelial and Endothelial Cells

(326) - Antibodies to Human Leukocyte Antigen Class I and Lung Associated Self-Antigens, K-alpha-1 Tubulin and Collagen V Induces Exosomes Containing Self-Antigens from Human Primary Airway Epithelial and Endothelial Cells

(327) - Exosomes Isolated from Lung Transplant Recipients Diagnosed with Bronchiolitis Obliterans Syndrome, But Not Stable, Can Induce Immune Responses to Lung Associated Self-Antigens, K-alpha-1 Tubulin and Collagen V

(327) - Exosomes Isolated from Lung Transplant Recipients Diagnosed with Bronchiolitis Obliterans Syndrome, But Not Stable, Can Induce Immune Responses to Lung Associated Self-Antigens, K-alpha-1 Tubulin and Collagen V

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.

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 3.5years 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.

(255) - Donor Derived Exosomes Expressing Lung Associated Self-antigens (Collagen V and K alpha 1 Tubulin) in Acute and Chronic Rejection Following Human Lung Transplantation

Conclusion: Significant regional variation exists in the use of 1A status exceptions, suggesting differences in listing strategies between HTx centers. This practice has the potential to introduce bias into a system designed to be equitable. Waitlist survival in patients listed using a status exception is similar to those listed status 1B, indicating these patients are lower acuity and may not require 1A status. While pending changes to the allocation system may eliminate the need for some previously utilized 1A exceptions, developing a consensus on “acceptable” 1A exceptions may even their use across regions and improve equity. Careful review of pediatric heart allocation policies is needed to optimize patient outcomes and ensure a fair and unbiased allocation system.

De novo development of dsa and autoantibodies to lung associated self-antigens is associated with upregulation of transcription factor Zbtb7a: Role in chronic rejection following human lung transplantation

Aim Administration of anti-MHC class I (H-2Kb) leads to induction of transcription factor Zinc finger and BTB domain containing 7A (Zbtb7a) leading to inflammatory cascade and chronic rejection in a murine obliterative airway disease model. Development of DSA and Abs to lung-associated self-antigens (K-alpla-1 Tubulin (K α 1T) and Collagen V (Col-V) are significant risk factors in development of bronchiolitis obliterans syndrome (BOS) following human lung transplantation (LTx). The current study focused on the role of Zbtb7a in development of BOS following human LTx. Methods We analyzed 25 LTx patients that developed BOS years after LTx as well as 10 stable LTx performed at Barnes-Jewish Washington University. Kinetics of Zbtb7a expression was studied in bronchoalveolar lavage (BAL) cells. Quantitative PCR analysis was performed to evaluate mRNA levels and data was compared with the DSA, autoantibody and immunosuppression. Results Increases in the Zbtb7a expression was observed in BAL cells collected at visits immediately prior to clinical BOS diagnosis compared with earlier visits. Zbtb7a expression was significantly (p 0.01) increased (150%) in pre-BOS BAL cells. On the contrary, stable patients did not demonstrate any change in Zbtb7a levels. All of the BOS+ patients had developed DSA and Abs to K α 1T and Col-V. Zbtb7a expression in 13 patients who received Ab directed therapy following DSA demonstrated significant reduction along with decrease in DSA and Abs to lung self-antigens. Therefore, Zbtb7a activation is an important inducer of the DSA and autoantibody. Conclusions Up regulation of Zbtb7a was positively associated with de novo DSA and Abs to lung-associated self-antigens. Ab directed therapy significantly reduced Zbtb7a expression. We propose that Zbtb7a may be used as a prognostic marker in evaluating Ab-induced inflammation in the lung as well as efficacy of Ab directed therapy following development of DSA.

Immune Response to Tissue-Restricted Self-Antigens Induces Airway Inflammation and Fibrosis Following Murine Lung Transplantation

Immune responses against lung-associated self-antigens (self-Ags) are hypothesized to play a role in the development of chronic lung graft rejection. We determined whether immune responses to lung self-Ags, K-alpha-1-tubulin (Kα1T) and Collagen V (Col-V) in the absence of alloimmunity, could promote airway inflammation and fibrosis. Following syngeneic murine orthotopic lung transplantation (LTx) we administered antibodies (Abs) to either Kα1T or Col-V or in combination to both of these self-Ags. As compared to recipients of isotype control Abs, Kα1T Abs and/or Col-V Abs-treated recipients had marked lung graft cellular infiltration and bronchiolar fibrosis. This inflammation was also associated the accumulation of Kα1T and Col-V-specific interferon-γ+ and IL-17+ T cells. Notably, the administration of Abs to Kα1T led to cellular and humoral immune responses to Col-V prior to development of fibrosis, and vice versa, indicating that epitope spreading can occur rapidly in an alloantigen independent manner. Collectively, these data support a model of chronic LTx rejection where the progressive loss of self-tolerance through epitope spreading promotes airway fibrosis. Strategies that target autoreactive Abs may be useful to inhibit chronic rejection of lung grafts.

Efficacy of extracorporeal photopheresis in clearance of antibodies to donor-specific and lung-specific antigens in lung transplant recipients

Extracorporeal photopheresis (ECP) has been used to treat chronic rejection after lung transplantation (LTx). We investigated the effect of ECP on several immune parameters that have been associated with poor lung function, including donor-specific antibodies (DSA) to human leukocyte antigen (HLA), antibodies against the lung-associated self-antigens (SAg), Kα1-tubulin (Kα1T), collagen I and V, and circulating levels of pro-inflammatory and anti-inflammatory cytokines. Sera were collected from post-LTx patients diagnosed with bronchiolitis obliterans before and 6 months after initiation of ECP. DSA and cytokine levels were measured by Luminex (Invitrogen, Carlsbad, CA). Changes in lung function over the 6 months preceding and after the initiation of ECP were measured by retrospective analysis of spirometry performed at routine clinic visits. ECP was associated with a significant decline in DSA levels as well as antibodies to lung-associated SAg. ECP also reduced circulating levels of pro-inflammatory cytokines and increased levels of anti-inflammatory cytokines. These immunologic changes were associated with a significant 63% reduction in the rate of decline in forced expiratory volume in 1 second over a 1-year period. Though statistically insignificant, a higher rate of clearance of antibodies to lung-associated SAg was strongly associated with better response to ECP. ECP is associated with a reduction in the levels of circulating DSA, antibodies to lung-associated SAg (Kα1T, collagen I, and collagen V), and circulating levels of several pro-inflammatory cytokines. We propose that these changes contribute to the beneficial effect of ECP in reducing the decline in lung function.

Pre-transplant antibodies to Kα1 tubulin and collagen-V in lung transplantation: Clinical correlations

Immune responses to lung-associated self-antigens (SAgs) have been implicated in chronic lung allograft rejection. The goals of this study were to determine the prevalence of pre-existing antibodies (Abs) to the SAgs in pulmonary diseases and the association between pre-existing Abs to SAgs and the development of primary graft dysfunction (PGD), donor-specific antibodies (DSA), and chronic rejection. Pre- and post-transplant sera were analyzed from 317 lung transplant (LTx) recipients between 2000 and 2011 with diagnosis of chronic obstructive disease (n = 161), idiopathic pulmonary fibrosis (IPF; n = 50), cystic fibrosis (CF; n = 55), and others (n = 51). Samples were analyzed for Abs to SAgs by enzyme-linked immunosorbent assay, and DSA and cytokines by Luminex. The clinical diagnosis of PGD and bronchiolitis obliterans syndrome (BOS) was based on International Society for Heart and Lung Transplantation guidelines. The overall prevalence of Abs to SAgs was 22.71%, including 18% in chronic obstructive pulmonary disease (p = 0.033), 34% in IPF (p = 0.0006), 29% in CF (p = 0.0023), and 19.6% in other diagnoses (p = 0.044). The incidence of PGD (88% vs 54%, p < 0.05), DSA (70% vs 45%, p < 0.01), and BOS (90% vs 38% (p < 0.001) after LTx was significantly higher in patients with pre-LTx Abs to SAgs than without. Pro-inflammatory cytokines (interleukin-1β, interleukin-17, and interferon-γ) were elevated in patients who had pre-LTx Abs to SAgs, along with a reduction in anti-inflammatory interleukin-10. Patients with IPF and CF have the highest prevalence of Abs to SAgs. Patients with pre-existing Abs to SAgs are at increased risk for development of PGD, DSA, and BOS. Strategies to remove pre-existing Abs to SAgs should be considered to improve lung allograft outcome.

Anti-major histocompatibility complex–induced obliterative airway disease: Selective role for CD4 and CD8 T cells in inducing immune responses to self-antigens

The goal of this study was to define the role of T-cell sub-sets in the pathogenesis of autoimmunity-induced obliterative airway disease by passive transfer of CD8+ or CD4+ T cells. Antibodies to major histocompatibility complex (MHC) class I were administered intrabronchially into C57BL/6 animals. Lungs were analyzed by histopathology and immunohistochemistry. The CD8+ and CD4+ T-cell sub-sets were purified from the lung-infiltrating cells and intrabronchially transferred. Frequency of cells secreting interleukin-17, interferon-γ, or interleukin-10 to self-antigens was enumerated by enzyme-linked immunospot assay. Myeloperoxidase and antibodies to self-antigens were determined by enzyme-linked immunosorbent assay. Cytokine and growth factor expression was determined by quantitative reverse-transcription polymerase chain reaction. Passive transfer of lung-infiltrating CD8 T cells isolated after anti-MHC class I administration, along with sub-optimal dose, induced significantly higher cellular infiltration (89.3% ± 7.9% vs 62.8% ± 10.1%, p < 0.05) vs the CD4 transfer group. Further, passive transfer of CD8 cells resulted in infiltration of neutrophils and macrophages, suggesting early injury response. In contrast, passive transfer of CD4+ T cells induced a significantly higher degree of luminal occlusion (29.3% ± 5.6% vs 8.6 ± 2.5%, p < 0.05) and fibrosis (54.4% ± 9.3% vs 10.2% ± 2.4%, p < 0.05) vs the CD8 group and B-cell infiltration, leading to immune responses to lung-associated self-antigens and fibrosis. Ligation of MHC molecules by its specific antibodies induced early injury with neutrophils, macrophages, and CD8 T cells, which leads to exposure of cryptic self-antigens and their presentation by the infiltrating CD4+ T cells and B cells, leading to the development of immune responses to self-antigens and culminating in obliterative airway disease.

T Regulatory Cells Play a Significant Role in Modulating MHC Class I Antibody-Induced Obliterative Airway Disease

The molecular mechanisms leading to the development of chronic lung allograft dysfunction following de novo development of antibodies to mismatched donor MHC remain undefined. We demonstrated that intrabronchial administration of antibodies to MHC class I resulted in induction of both innate and adaptive cellular immune responses characterized by a predominance of Th17 specific to lung associated self-antigens Kα1-tubulin and Collagen-V leading to the development of obliterative airway lesions (OAD), correlate of chronic rejection following human lung transplantation. To determine the role of regulatory T cells (Treg) in the pathogenesis of OAD, we administered anti-MHC class I to mice, in which Treg were depleted by conditional ablation of FoxP3+cells. Under this condition, we observed a threefold increase in pulmonary cellular infiltration, luminal occlusion and fibrous deposition when compared anti-MHC class I Ab administered mice maintaining FoxP3. OAD lesions were accompanied with enhanced accumulation of neutrophils along with self-antigen-specific Th17 and humoral responses. However, IL-17-blockade or adoptive transfer of Treg abrogated OAD. We conclude that Treg exerts a suppressive effect on anti-MHC induced IL-8-mediated neutrophil infiltration and innate immune responses that leads to inhibition of Th17 immune responses to lung associated self-antigens which is critical for development of OAD.