Lung Allograft Rejection Research Articles

Bone marrow stem cells modified with human interleukin 10 attenuate acute rejection in rat lung allotransplantation.

The aim of this study was to investigate new therapeutic options to attenuate acute rejection in a rat lung allograft model. Cell-based gene therapies have recently been reported as a novel curative option in acute and chronic diseases for which conventional treatments are not available. We studied the effect of human interleukin 10 (hIL-10) on expressing bone marrow-derived mesenchymal stem cells (BMSCs) in combination with cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Lung allotransplantation was performed from male Brown Norway donor to male Fisher (F344) rats. Rat BMSCs were transfected with hIL-10 in vitro and introduced in the graft prior to implantation. Group A (n = 5) received CsA intraperitoneally (2.5 mg/kg body weight) for 5 days post-transplant; Group B (n = 5) received BMSC and CsA and Group C (n = 5) received hIL-10-BMSC before implantation and CsA. Graft function was assessed by blood gas levels only from the graft on day 5; tissue was sampled for histological grading of rejection and measurement of the wet-to-dry ratio. All Group A control animals showed severe signs of rejection. On Day 5, all grafts in Group C showed improved gas exchange (mean arterial partial pressure of oxygen 222.2 ± 40.38 mmHg vs 92.36 ± 20.92 mmHg in Group B and 42.72 ± 18.07 mmHg in Group A). Histological examination revealed moderate-to-severe rejection in all animals in Group A [International Society for Heart and Lung Transplantation Level III B (ISHLT)] in contrast to low-to-moderate rejection in Group B (II-IIIA) and much improved histological grade in Group C (I-IIA). Moreover, the wet-to-dry ratio was also reduced in Group C (4.8 ± 1.19 compared with 4.78 ± 0.62 in Group B and 9.36 ± 0.90 in Group A). The hIL-10 BMSC represent a promising novel method for localized cell-based gene therapy for acute rejection in a rat lung allograft model.

Zbtb7a induction in alveolar macrophages is implicated in anti-HLA–mediated lung allograft rejection

Chronic rejection significantly limits long-term success of solid organ transplantation. De novo donor-specific antibodies (DSAs) to mismatched donor human leukocyte antigen after human lung transplantation predispose lung grafts to chronic rejection. We sought to delineate mediators and mechanisms of DSA pathogenesis and to define early inflammatory events that trigger chronic rejection in lung transplant recipients and obliterative airway disease, a correlate of human chronic rejection, in mouse. Induction of transcription factor zinc finger and BTB domain containing protein 7a (Zbtb7a) was an early response critical in the DSA-induced chronic rejection. A cohort of human lung transplant recipients who developed DSA and chronic rejection demonstrated greater Zbtb7a expression long before clinical diagnosis of chronic rejection compared to nonrejecting lung transplant recipients with stable pulmonary function. Expression of DSA-induced Zbtb7a was restricted to alveolar macrophages (AMs), and selective disruption of Zbtb7a in AMs resulted in less bronchiolar occlusion, low immune responses to lung-restricted self-antigens, and high protection from chronic rejection in mice. Additionally, in an allogeneic cell transfer protocol, antigen presentation by AMs was Zbtb7a-dependent where AMs deficient in Zbtb7a failed to induce antibody and T cell responses. Collectively, we demonstrate that AMs play an essential role in antibody-induced pathogenesis of chronic rejection by regulating early inflammation and lung-restricted humoral and cellular autoimmunity.

Association of Local Intrapulmonary Production of Antibodies Specific to Donor Major Histocompatibility Complex Class I With the Progression of Chronic Rejection of Lung Allografts.

Antibody-mediated rejection may lead to chronic lung allograft dysfunction, but antibody-mediated rejection may develop in the absence of detectable donor-specific antibody (DSA) in recipient serum. This study investigated whether humoral immune responses develop not only systemically but locally within rejected lung allografts, resulting in local production of DSA. Lewis rats received orthotopic left lung transplantation from Lewis (syngeneic control) or Brown-Norway (major histocompatibility complex-mismatched allogeneic) donor rats. Rats that underwent allogeneic lung transplantation were subsequently administered cyclosporine until day 14 (short immunosuppression) or day 35 (long immunosuppression). The lung grafts and spleens of recipient animals were tissue cultured for 4 days, and the titer of antibody against donor major histocompatibility complex molecules was assayed by flow cytometry. Explanted lung grafts were also evaluated pathologically. By day 98, DSA titers in supernatants of lung graft (P = 0.0074) and spleen (P = 0.0167) cultures, but not serum, from the short immunosuppression group were significantly higher than titers in syngeneic controls. Cultures and sera from the long immunosuppression group showed no production of DSA. Microscopically, the lung grafts from the short immunosuppression group showed severe bronchiole obliteration and parenchymal fibrosis, along with lymphoid aggregates containing T and B cells, accompanying plasma cells. These findings suggestive of local humoral immune response were not observed by days 28 and 63. DSA can be locally produced in chronically rejected lung allografts, along with intragraft immunocompetent cells. Clinical testing of DSA in serum samples alone may underestimate lung allograft dysfunction.

Non-neuronal neuropeptide Y and its receptors during acute rejection of rat pulmonary allografts

This study tested the hypothesis that neuropeptide Y (NPY) and NPY receptors 1 (Y1) and 2 (Y2) participate in lung allograft rejection. Inflammation in grafts may include interaction between blood leukocytes and graft endothelial cells and marked accumulation of intravascular blood leukocytes. Fewer leukocytes accumulate in lung than in kidney allografts. Lung transplantion was performed in the Dark Agouti to Lewis rat strain combination. Intravascular and intraalveolar leukocytes were isolated from the grafts, and we evaluated the mRNA expression of NPY, Y1, and Y2 by real-time RT-PCR as well as the peptide expression of NPY by radioimmunoassay and immunohistochemistry. NPY and Y1 were expressed by pulmonary intravascular and intraalveolar leukocytes. Y1 was up-regulated by pulmonary intravascular and intraalveolar leukocytes during allograft rejection while Y2 could not be detected. Higher NPY expression levels in intravascular leukocytes were observed in lung compared to kidney allografts, which were investigated previously. Our findings suggest that an increased leukocytic expression of NPY in lung compared to kidney allografts results in a reduced accumulation of leukocytes in allograft vessels.

(407) - Repertoire Analysis of De Novo Non-HLA Antibodies Associated with Chronic Lung Allograft Rejection Following Human Lung Transplantation

(407) - Repertoire Analysis of De Novo Non-HLA Antibodies Associated with Chronic Lung Allograft Rejection Following Human Lung Transplantation

(328) - Increasing Frequency of CD127low Regulatory T Cells Early After Lung Transplantation Is Associated with Freedom from Chronic Lung Allograft Rejection and Survival in 503 Patients

(328) - Increasing Frequency of CD127low Regulatory T Cells Early After Lung Transplantation Is Associated with Freedom from Chronic Lung Allograft Rejection and Survival in 503 Patients

Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch

Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection. The method involves the initial development of a panel of probes in which each probe will specifically target a unique sequence of a human leukocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR (ddPCR) used as a quantitative assay to accurately track the trace amount of DcfDNA in an ample excess of recipient DNA background. The average false positive rate noted was about 1 per 800,000 molecules. Serially 2-fold diluted cfDNA, representing donor fractions of cfDNA, were spiked into a constant level of cfDNA representing the recipient cfDNA. The fraction of spiked cfDNA was measured and quantitative linearity was observed across seven serially diluted cfDNA samples. We were able to measure the minor portion of cfDNA as low as 0.2% of total cfDNA. We subsequently applied the method to a pilot set of 18 LTx recipients grouped into biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or stable groups. Serial plasma samples were used to identify the percentage of DcfDNA over total cfDNA. The level of DcfDNA was significantly elevated in patients diagnosed with acute rejection (10.30±2.80, n=18), compared to that from stable (1.71±0.50, n=24) or from BOS patients (2.52±0.62, n=20). In conclusion, we present results validating the application of digital PCR to quantify DcfDNA assay in primary clinical specimens, which demonstrate that DcfDNA can be used as an early non-invasive biomarker for acute lung allograft rejection.

Targeting the Canonical Nuclear Factor-κB Pathway with a High-Potency IKK2 Inhibitor Improves Outcomes in a Mouse Model of Idiopathic Pneumonia Syndrome

Idiopathic pneumonia syndrome (IPS) is a noninfectious inflammatory disorder of the lungs that occurs most often after fully myeloablative allogeneic hematopoietic stem cell transplantation (HSCT). IPS can be severe and is associated with high 1-year mortality rates despite existing therapies. The canonical nuclear factor-(NF) κB signaling pathway has previously been linked to several inflammatory disorders of the lung, including asthma and lung allograft rejection. It has never been specifically targeted as a novel IPS treatment approach, however. Here, we report that the IκB kinase 2 (IKK2) antagonist BAY 65-5811 or “compound A,” a highly potent and specific inhibitor of the NF-κB pathway, was able to improve median survival times and recipient oxygenation in a well-described mouse model of IPS. Compound A impaired the production of the proinflammatory chemokines CCL2 and CCL5 within the host lung after transplantation. This resulted in significantly lower numbers of donor lung infiltrating CD4+ and CD8+ T cells and reduced pulmonary inflammatory cytokine production after allograft. Compound A's beneficial effects appeared to be specific for limiting pulmonary injury, as the drug was unable to improve outcomes in a B6 into B6D2 haplotype-matched murine HSCT model in which recipient mice succumb to lethal acute graft-versus-host disease of the gastrointestinal tract. Collectively, our data suggest that the targeting of the canonical NF-κB pathway with a small molecule IKK2 antagonist may represent an effective and novel therapy for the specific management of acute lung injury that can occur after allogeneic HSCT.

Donor-derived exosomes: key in lung allograft rejection?

In a recent publication, Gunasekaran and colleagues conducted a case-control observational study in patients who underwent bilateral lung transplantation (LTx), determining whether exosomes are generated during allograft rejection and define their origin and antigenic phenotype (1). LTx is the sole effective treatment to save the life for patients with end-stage lung disorders (2). One of the major drawbacks for the clinical outcome of LTx is allograft rejection by acute rejection (AR) or bronchiolitis obliterans syndrome (BOS) (2). A link between autoimmunity and immune responses to tissue restricted self-antigens (SAgs) such as K-alpha-1-tubulin (K-α1T) and Collagen V (Col-V) with acute or chronic lung allograft rejection has been proposed (3,4). However, the mechanisms underlying the evolution of acute or chronic lung rejection are poorly understood.

Mechanisms of graft rejection after lung transplantation.

To date, outcomes after lung transplantation are far worse than after transplantation of other solid organs. New insights into mechanisms that contribute to graft rejection and tolerance after lung transplantation remain of great interest. This review examines the recent literature on the role of innate and adaptive immunity in shaping the fate of lung grafts. Innate and adaptive immune cells orchestrate allograft rejection after transplantation. Innate immune cells such as neutrophils are recruited to the lung graft early after reperfusion and subsequently promote allograft rejection. Although it is widely recognized that CD4 T lymphocytes in concert with CD8 T cells promote graft rejection, regulatory Foxp3 CD4 T, central memory CD8 T cells, and natural killer cells can facilitate tolerance. This review highlights interactions between innate and adaptive immune pathways and how they contribute to lung allograft rejection. These findings lay a foundation for the design of new therapeutic strategies that target both innate and adaptive immune responses.

Lung Injury and Loss of Regulatory T Cells Primes for Lung-Restricted Autoimmunity.

Lung transplantation is a life-saving therapy for several end-stage lung diseases. However, lung allografts suffer from the lowest survival rate predominantly due to rejection. The pathogenesis of alloimmunity and its role in allograft rejection has been extensively studied and multiple approaches have been described to induce tolerance. However, in the context of lung transplantation, dysregulation of mechanisms, which maintain tolerance against self-antigens, can lead to lung-restricted autoimmunity, which has been recently identified to drive the immunopathogenesis of allograft rejection. Indeed, both preexisting as well as de novo lung-restricted autoimmunity can play a major role in the development of lung allograft rejection. The three most widely studied lung-restricted self-antigens include collagen type I, collagen type V, and k-alpha 1 tubulin. In this review, we discuss the role of lung-restricted autoimmunity in the development of both early as well as late lung allograft rejection and recent literature providing insight into the development of lung-restricted autoimmunity through the dysfunction of immune mechanisms which maintain peripheral tolerance.

OR16 Antibodies to donor human leukocyte antigen induce exosomes containing tissue-restricted self-antigens in acute and chronic rejection

Aim Immune responses to tissue-restricted self-antigens (SAgs) are known contributors to acute and chronic rejection in solid organ transplantation (Tx). The goal of this study was to determine whether a donor-specific antibody (DSA) induces exosomes during lung and kidney allograft rejection and to define its antigenic compositions. Methods Exosomes were isolated from sera and bronchoalveolar lavage (BAL) of lung Tx recipients with acute or chronic rejection who developed both DSA and antibodies to SAgs, and from sera of kidney Tx recipients with transplant glomerulopathy (TG) who developed both DSA and antibodies to kidney-associated SAgs. The exosomes were isolated by ultracentrifugation, and then purified by the sucrose cushion method. Expression of lung-associated SAgs Collagen-V and K α 1-tubulin was examined by immunogold labelling and visualized by transmission electron microscopy. The presence of lung-associated SAgs and kidney-associated SAgs (i.e., Collagen-IV, fibronectin), and exosome-specific marker annexin-V was detected by Western immunoblot. Results Exosomes isolated from patients with acute rejection, chronic rejection, and TG showed the presence of tissue-restricted SAgs. Lung-associated SAgs Collagen-V and K α 1-tubulin were confirmed on the surface of exosomes. Exosomes with Collagen-V were detectable in exosomes isolated from sera just 1 month after lung Tx in all patients with acute rejection, much earlier than it could be diagnosed clinically (i.e., 3 months). Similarly, exosomes from sera in patients with bronchiolitis obliterans syndrome contained Collagen-V 6 months after lung Tx, before clinical diagnosis was possible. Exosomes from patients with TG had significantly higher expression of kidney-associated SAgs compared with the stable group. Conclusions The exosomes isolated from lung Tx recipients with acute and chronic rejection who developed DSA and antibodies to lung-associated SAgs and from kidney Tx recipients with TG confirm the presence of tissue-restricted SAgs in these patients, while the respective stable control groups do not. We therefore conclude that antibodies to human leukocyte antigen after lung or kidney Tx induce exosomes containing SAgs, which may result in the spread of immune response to tissue-restricted SAgs. T. Mohanakumar: Grant/Research Support; Company/Organization ; NIH R21AI123034 .

Donor-Reactive Regulatory T Cell Frequency Increases During Acute Cellular Rejection of Lung Allografts.

Acute cellular rejection is a major cause of morbidity after lung transplantation. Because regulatory T (Treg) cells limit rejection of solid organs, we hypothesized that donor-reactive Treg increase after transplantation with development of partial tolerance and decrease relative to conventional CD4 (Tconv) and CD8 T cells during acute cellular rejection. To test these hypotheses, we prospectively collected 177 peripheral blood mononuclear cell specimens from 39 lung transplant recipients at the time of transplantation and during bronchoscopic assessments for acute cellular rejection. We quantified the proportion of Treg, CD4 Tconv, and CD8 T cells proliferating in response to donor-derived, stimulated B cells. We used generalized estimating equation-adjusted regression to compare donor-reactive T cell frequencies with acute cellular rejection pathology. An average of 16.5 ± 9.0% of pretransplantation peripheral blood mononuclear cell Treg cell were donor-reactive, compared with 3.8% ± 2.9% of CD4 Tconv and 3.4 ± 2.6% of CD8 T cells. These values were largely unchanged after transplantation. Donor-reactive CD4 Tconv and CD8 T cell frequencies both increased 1.5-fold (95% confidence interval [95% CI], 1.3-1.6; P < 0.001 and 95% CI, 1.2-1.6; P = 0.007, respectively) during grade A2 rejection compared with no rejection. Surprisingly, donor-reactive Treg frequencies increased by 1.7-fold (95% CI, 1.4-1.8; P < 0.001). Contrary to prediction, overall proportions of donor-reactive Treg cells are similar before and after transplantation and increase during grade A2 rejection. This suggests how A2 rejection can be self-limiting. The observed increases over high baseline proportions in donor-reactive Treg were insufficient to prevent acute lung allograft rejection.

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.

Diagnostic value of plasma and bronchoalveolar lavage samples in acute lung allograft rejection: differential cytology.

Diagnosis of acute lung allograft rejection is currently based on transbronchial lung biopsies. Additional methods to detect acute allograft dysfunction derived from plasma and bronchoalveolar lavage samples might facilitate diagnosis and ultimately improve allograft survival. This review article gives an overview of the cell profiles of bronchoalveolar lavage and plasma samples during acute lung allograft rejection. The value of these cells and changes within the pattern of differential cytology to support the diagnosis of acute lung allograft rejection is discussed. Current findings on the topic are highlighted and trends for future research are identified.

Lung Transplant Rejection and Surveillance in 2016: Newer Options

Lung allograft rejection is a major risk factor for the development of chronic lung allograft dysfunction. It is a significant cause of morbidity and mortality, and limits survival post lung transplantation, which is lower than any other solid organ transplant. The invasive nature of current methods of diagnosis which consists of histological diagnosis via transbronchial biopsy and the lack of sensitivity of clinical surveillance warrants the search for novel less invasive and more accurate methods of diagnosis. This review aims to highlight recent changes to current methods of surveillance and diagnosis as well as present some of the novel methods that are becoming available.

Humoral responses after lung transplantation.

In recent years, there has been increasing awareness and appreciation for the role of humoral immune responses in lung allograft rejection. This review summarizes our current understanding of this role and the associated challenges. Recent studies have described a syndrome of acute antibody-mediated rejection with a generally poor response to therapy and a high mortality. In addition, there is significant evidence implicating donor-specific human leukocyte antigen antibodies in the development of chronic lung allograft dysfunction. However, the optimal intervention to mitigate the risk of chronic lung allograft dysfunction after donor-specific human leukocyte antigen antibodies development remains unclear. There is mounting evidence that humoral immune responses play an important role in lung allograft rejection. However, therapeutic implications of this increased awareness have been limited. Indeed, there is insufficient evidence to adequately guide therapy, and the optimal treatment is unknown.

Role of interleukin-17A in early graft rejection after orthotopic lung transplantation in mice.

The cellular and molecular mechanisms underlying lung allograft rejection remain poorly understood. We investigated the potential role of interleukin (IL)-17A in lung transplant rejection in a mouse model, because previous studies in clinical and rodent models have implicated IL-17A in both acute and chronic rejection. To generate an orthotopic lung transplantation model, lungs from C57BL/6 or BALB/c mice were transplanted into C57BL/6 mice (isograft and allograft models, respectively). The effects of anti-IL-17A treatment in allograft recipients were investigated. The histological features and rejection status of isografts and allografts were assessed at 3, 7, and 28 days after transplantation, and differences in graft infiltrating cells and mRNA expression of relevant cytokines were quantified at 3 and 7 days after transplantation. As expected, isografts showed no obvious signs of rejection, whereas allografts exhibited minimal-to-mild rejection (grade A1-A2) by day 3 and moderate-to-severe rejection (grade A3-A4) by day 7, without evidence of obliterative bronchiolitis (OB). However, by 28 days, evidence of OB was observed in 67% (2/3) of allografts and severe rejection (grade A4) was observed in all. IL-17 mRNA expression in allografts was increased with rejection, and interferon (IFN)-γ and IL-6 mRNA expression levels followed a similar pattern. In contrast, IL-22 expression in allografts was only slightly increased. Antibody (Ab) neutralization of IL-17A diminished the signs of acute rejection at 7 days after transplantation in allografts, and this early protection was accompanied by a decrease in cellular stress according to histological evaluation, suggesting the involvement of IL-17A in the development of early post-transplantation lesions. Our data indicate that IL-17A is important in the pathophysiology of allograft rejection, and neutralization of IL-17A is a potential therapeutic strategy to preventing lung transplant rejection.

Differences of airway dimensions between patients with and without bronchiolitis obliterans syndrome after lung transplantation—Computer-assisted quantification of computed tomography

BackgroundThe aim of our retrospective study was to determine whether a dedicated software for assessment of airway morphology can detect differences in airway dimensions between patients with and without bronchiolitis obliterans syndrome (BOS), regarded as the clinical correlate of chronic lung allograft rejection. Methods12 patients with and 14 patients without diagnosis of BOS were enrolled in the study. Evaluation of bronchial wall area percentage (WA%) and bronchial wall thickness (WT) in all follow-up CT scans was performed using a semiautomatic airway assessment tool. We assessed temporal changes (ΔWA%, ΔWT) and compared these morphological parameters with forced expiratory volume in one second (ΔFEV1). ResultsIn patients with and without BOS, the temporal changes over the entire follow-up were 26.6% versus 16.2% for ΔFEV1 (p=0.034), 14.2% versus 5.4% for ΔWA% (p=0.003) and 0.212mm versus 0.064mm for ΔWT (p=0.011). ConclusionsWe detected significant differences of the temporal changes of airway dimensions (ΔWA%, ΔWT) between lung transplant recipients with and without BOS. We conclude that computer-assisted bronchial wall measurements in CT scans might complement the information from pulmonary function tests and establish as a non-invasive method to confirm BOS in lung transplant recipients in the future.

(271) - Intrapulmonary Local Production of Antibodies Specific to Donor Major Histocompatibility Complex Class I in Association with the Progression of Chronic Rejection in Lung Allografts

(271) - Intrapulmonary Local Production of Antibodies Specific to Donor Major Histocompatibility Complex Class I in Association with the Progression of Chronic Rejection in Lung Allografts