Bronchiolitis obliterans in lung transplantation: the good, the bad, and the future

FEATURED NEW INVESTIGATOR Bronchiolitis obliterans in lung transplantation: the good, the bad, and the future ERIC J. GROSSMAN and REBECCA A. SHILLING* CHICAGO, ILL

Lung transplantation remains the hope for many incurable pulmonary diseases, such as cystic fibrosis, pulmonary fibrosis, and chronic obstructive pulmonary disease. Remarkable progress has been made in improving outcomes, although the incidence of acute rejection remains more than 50% in the 1st year, and the 5-year graft survival is still less than 50% primarily because of the development of chronic rejection and graft dysfunction. Chronic rejection is characterized by the development of obliterative bronchiolitis in allografts and manifests as bronchiolitis obliterans syndrome in humans with no effective treatment. Previous studies support a role for alloreactive T cells in the development of bronchiolitis obliterans syndrome, but the specific mechanisms are unknown. One major stumbling block to research in the field of lung transplantation has been the lack of physiologic models to study the disease in the laboratory. We will review the current understanding of the immunology of the pathogenesis of obliterative bronchiolitis and will discuss exciting new advances from the laboratory as well as the implications for future research in lung transplantation. (Translational Research 2009;153:153–165) Abbreviations: APC ¼ antigen presenting cell; BOS ¼ bronchiolitis obliterans syndrome; DC ¼ dendritic cell; HLA ¼ human leukocyte antigen; HTT ¼ heterotopic tracheal transplant; I/R ¼ ischemia/reperfusion; ISHLT ¼ International Society of Heart and Lung Transplantation; LB ¼ lymphocytic bronchiolitis; LPS ¼ lipopolysaccharide; MHC ¼ major histocompatibility complex; NK ¼ natural killer; OB ¼ obliterative bronchiolitis; OTT ¼ orthotopic tracheal transplant; PGD ¼ primary graft dysfunction; TCR ¼ T-cell receptor; Th1 ¼ T helper 1; TLR ¼ toll-like receptor; Treg ¼ regulatory T cell; VEGF ¼ vascular endothelial growth factor

A

dvances in the field of solid organ transplantation have been intimately linked to advances in the field of immunology. Despite the 1905 proclamation of Carrel and Guthrie1 that the surgical

challenges of organ transplantation had been solved, it was not until the implementation of immunosuppression that solid organ transplantation became a viable treatment option for patients with end-stage disease.1,2 In

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Rebecca A. Shilling, MD, is Assistant Professor in the Department of Medicine, Section of Pulmonary and Critical at the University of Chicago. Her article is based on a presentation given at the Combined Annual Meeting of the Central Society for Clinical Research and Midwestern Section American Federation for Medical Research held in Chicago, Ill, April 2008. From the Department of Surgery and Section of Pulmonary and Critical Care Medicine, Department of Medicine and Committee on Immunology, The University of Chicago, Chicago, Ill.

Submitted for publication September 27, 2008; revision submitted January 19, 2009; accepted for publication January 20, 2009.

Supported by Grant K08 AI 059105 from the National Institute of Health and the Louis Block Fund.

doi:10.1016/j.trsl.2009.01.005

Reprint requests: Rebecca A. Shilling, Section of Pulmonary and Critical Care Medicine, The University of Chicago, MC6076, Room M658, 5841 S. Maryland Avenue, Chicago, Ill 60637.; e-mail: rshillin@ medicine.bsd.uchicago.edu. 1931-5244/$ – see front matter Ó 2009 Mosby, Inc. All rights reserved.

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the United States, nearly 30,000 solid organ transplants are performed yearly. Lung transplant recipients face the worst posttransplant survival statistics.3 The development of chronic rejection signified by the pathologic diagnosis of obliterative bronchiolitis (OB) is the leading cause of death in lung allograft recipients. Bronchiolitis obliterans syndrome (BOS), which is the clinical correlate of OB, is found in 27% of recipients at 2.5 years and 51% at 5.6 years posttransplant, according to the latest available statistics from the International Society of Heart and Lung Transplantation (ISHLT) registry.4 OB/BOS can occur a few months to several years after transplant and is characterized by fibrous obliteration of the small airways.5 After transplantation, recipient T cells that recognize foreign major histocompatibility complex antigens from the donor infiltrate the graft and can induce episodes of acute rejection.6 Over time, the infiltration of recipient macrophages and myofibroblasts into the graft may result in a fibroproliferative phase and the development of OB/BOS.5 Studies in humans and animal models have implicated both allodependent and alloindependent factors in the development of BOS.5-7 Some of the previously identified risk factors have been acute rejection episodes, human leukocyte antigen (HLA) mismatches and HLA antibodies, lymphocytic bronchiolitis, ischemia-reperfusion (I/R) injury, and infection.5 Episodes of acute rejection are associated with damage to the airway epithelium and endothelium, which may provide a source of allo-antigens necessary for the persistence of alloimmune T cells in the lungs.5 Lymphocytic bronchiolitis (LB), which is thought to be a precursor to OB, may represent areas of continued proliferation and activation of alloreactive T cells. Previous work has found an association between LB and the development of BOS,8 and a recent article has correlated the severity of LB with the development of BOS.9 Whether the lymphocytic infiltration is organized into lymphoid aggregates that support T- and B-cell immunity is currently not known. Infection and I/R injury may promote BOS by inducing inflammation that leads to the initiation and perpetuation of the alloimmune response. I/R injury may be related to the lack of bronchial artery anastomosis after lung transplantation. Some have proposed that BOS may be decreased by bronchial artery revascularization based on data from small studies showing a possible delay in BOS in centers where this is performed.10,11 However, no large studies to date have confirmed these findings, and currently, bronchial artery anastomosis is not routinely performed in most centers. More recently, primary graft dysfunction (PGD)12,13 and autoimmunity14,15 have been associated with BOS. PGD, which is an early complication post transplantation, is characterized by acute lung injury and has an

unclear pathogenesis. The ISHLT has recently standardized the definition and severity grading of PGD using the chest X-ray and ratio of partial pressure of arterial oxygen to the fraction of inspired oxygen.16 Previous work had demonstrated that PGD was associated with worse outcomes,17,18 but in 2007, Daud et al12 found an association of PGD with increased risk of BOS independent of other risk factors for BOS.12 In a follow-up study, the authors found that the severity of PGD correlated with increasing risk for BOS.13 The mechanisms by which PGD may promote the development of OB/ BOS are only beginning to be elucidated and may be related to exposure of previously sequestered antigens in the lung. Haque et al19 as well as Yoshida et al20 had previously reported in a rat model of lung transplantation the role of autoimmunity to collagen V in lung allograft rejection. Collagen V had been found to be expressed in the lung during acute rejection; I/R injury and T-cell responses to collagen V were found during rejection in rat allografts.19,21 In 2007, Burlingham et al14 reported that the presence of CD41 T-cell responses to collagen V were associated with an increased incidence of OB/ BOS in humans after lung transplantation. Surprisingly, collagen V cellular immunity was associated more strongly with increased risk of BOS than acute rejection episodes, HLA mismatches, or induction of HLA antibodies.14 Goers et al15 have also reported the association of autoreactive epithelial-specific antibodies with BOS providing additional support for the hypothesis that autoimmunity mediates chronic lung allograft rejection. Interestingly, subsequent studies have demonstrated that anticollagen V cellular and humoral immunity are associated with PGD.22,23 Thus, autoimmunity may contribute to acute lung injury after lung transplantation and may play a role in perpetuating the chronic fibroproliferative process of BOS. CURRENT MODELS

An understanding of the current knowledge in lung transplant research requires a brief introduction to the models used to study OB. In the early 1980s, Marck et al24-26 demonstrated that lung transplantation in rats was technically feasible and used imaging modalities to assess allograft function. Despite its early successes, the rat model has subsequently yielded mixed results. Yasufuku et al27 have successfully used the rat model to study the pathogenesis of OB and have recently translated their findings to humans after lung transplantation.14 However, one major issue with the rat orthotopic model is that it does not develop OB consistently.28 Furthermore, rat models lack the breadth of reagents and the genetic manipulations available to study both innate and adaptive immunity in mice. An

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orthotopic lung transplant model in swine has been found to develop pathology consistent with OB.29 However, the work has not been replicated by others, and studies in swine are even more costly and limited than rats, which makes it difficult for many laboratories to contribute. Because of the limitations with orthotopic lung transplants in animals, investigators have turned to models using tracheal transplants as surrogates for lung transplantation. The first model, which was described by Hertz and colleagues in 1993,30 was a heterotopic tracheal transplant (HTT) model in the mouse. The model consists of transplanting a fully mismatched trachea into the subcutaneous tissue of the back of a recipient mouse. This model has been reproducible and extensively used for the study of OB.7 However, the model has been widely criticized for being nonphysiologic. An alternative model has been the orthotopic tracheal transplant (OTT) model, where the donor trachea is surgically attached to the recipient trachea.31 This model, although aerated and therefore more analogous to the natural physiology, also has its limitations. OTT grafts develop tissue edema and epithelial damage within the graft but fail to develop fibrous obliteration of the airway lumen.31 Investigators have recently improved on the tracheal model using the rat to allow transplant of an allograft trachea directly into the pulmonary parenchyma of the recipient rat lung.32,33 Similar to both the HTT and OTT models, intrapulmonary allografts developed fibrous obliteration of the lumen, as well as the typical histologic changes of OB. The model has the advantage of exposing the allograft to the intrapulmonary environment, which enables the study of resident lung antigen-presenting cells (APCs) and intrapulmonary targeted therapies. Nonetheless, the model is limited by the paucity of reagents for the rat, as well as by the fact that OB occurs in small airways, not the trachea. Recently, one group has been able to establish successfully a vascularized and aerated orthotopic murine lung transplant model.34 Isografts were found to be physiologically functional with normal levels of oxygenation, in contrast to allografts, which underwent rejection and were edematous after 7 days. This technically demanding mouse model of orthotopic vascularized aerated lung transplantation is the most physiologic model to date. Although evidence of an interstitial fibrotic process was observed, no obliteration of the airways consistent with OB could be demonstrated.34 The data from the current models overwhelmingly support the hypothesis that the alloimmune response underlies the pathogenesis of OB, but the limitations of these models have made it difficult for progress to be made in lung transplantation.

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ALLOIMMUNITY

The primary basis for rejection of solid organs is host recognition of non-self-donor antigens or the alloimmune response. The immune response to alloantigens is primarily initiated by a T-cell response, which may then promote a B-cell response that leads to alloantibody production. After transplantation, the T-cell receptor (TCR) on host T cells recognizes its cognate peptide major histocompatibility complex (MHC) present on donor cells. Why humans have evolved to have alloreactive T and B cells is unknown, but it may be a result of the inherent affinity of the TCR for MHC molecules.35 Humans all have allogeneic lymphocytes that circulate regardless of whether they have previously been exposed to alloantigens. These mostly naı¨ve lymphocytes can be activated after transplantation when T cells are presented with their cognate antigen in the right context. Prior to 1980, the direct pathway, in which recipient T cells recognize intact donor MHC molecules displayed on the surface of donor cells, either traditional hematopoietic APCs or other nonhematopoietic graft cells, was generally accepted as the only mechanism of allorecognition.36,37 However, in 1982, Lechler and Batchelor38 proposed an alternative mechanism called the indirect pathway, which is defined by recipient APCs that take up and process damaged donor cells and present donor-derived MHC peptides to recipient T cells via self-MHC:donor peptide complexes. This finding led to the understanding that T cells mediate allograft rejection via two distinct pathways: the direct and the indirect pathways (Fig 1). The direct pathway is characterized by alloreactive T cells with a high precursor frequency and a wide range of receptor specificities that can recognize numerous allo-MHC molecules.36,37 In contrast, T cells involved in the indirect pathway are aimed at a single or a few principal donor MHC peptides displayed on the surface of recipient MHC molecules.36,37 The direct and indirect pathways also seem to be differentiated chronologically. The direct pathway dominates the early posttransplant period, when numerous donor APCs can migrate to the draining lymph nodes and activate alloreactive recipient T cells.37 In contrast, the indirect pathway is likely to remain active throughout the life of the allograft either because of the infiltration of recipient APC in the allograft or the persistence of donor antigens in the lymphoid tissue.39 The indirect pathway may therefore be responsible for allorecognition later in the posttransplant period and may represent the basis of chronic allograft rejection.6 In lung transplant recipients, Stanford et al40 proposed that chronic lung rejection and OB was the result of not only the expected ongoing indirect alloreactivity but

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Fig 1. Mechanisms of initiating an alloimmune response. After lung transplantation, allorecognition may occur via direct, indirect, or semidirect antigen presentation to T cells. A, Direct allorecognition occurs when donor dendritic cells (D—blue), which display intact donor MHC:peptide complexes, migrate from lung allograft to lymph nodes. B, Indirect alloantigen occurs when recipient dendritic cells (R—pink) in the draining lymph nodes activate T cells with complexes of self-MHC and processed donor MHC peptides. C, The semidirect pathway may occur when intact donor MHC molecules are transferred from donor to recipient dendritic cells and subsequently are presented by recipient dendritic cells to T cells. D, Activated CD41 and CD81 T cells then return to the lung and may reject the allograft. (Color version of figure is available online.)

potentially an overactive and persistent direct response. The authors tested this hypothesis with 19 lung transplant recipients, 8 of whom had OB, where donor spleens had been frozen at the time of transplantation. Using purified recipient CD41 T cells from peripheral blood, the authors measured the frequency of either a direct response to intact donor splenocytes or an indirect response to fragments of donor spleen cells presented by recipient APCs. The authors found that patients with OB had significantly higher frequencies of indirect recognition of donor antigens when compared with patients without OB. However, low frequencies of T-cell reactivity to direct allorecognition were present in both patients with and without OB. These data, as well as findings from several other groups, reinforced the concept that chronic rejection was associated with the indirect pathway.41,42 The data also suggested that lower levels of direct alloantigen recognition do not provide protection against OB and that persistent direct allorecognition does not induce OB. It is possible that current immunosuppression strategies abrogate the direct immune response but fail to suppress an indirect T-cell response to the lung allograft. Although indirect allorecognition may dominate chronic lung rejection, evidence suggests that direct recognition by T cells can contribute to chronic rejection after donor APC have been depleted.43,44 CD81 T cells recognize antigens presented by class I MHC, which is present on all cells, unlike class II MHC (CD41 T cells), which is expressed primarily by specialized hematopoi-

etic cells. In mouse models, evidence indicates that CD81 T cells with direct class I MHC alloreactivity to the graft persist and contribute to the chronic destruction and subsequent obliterative airway disease of transplanted tracheal allografts.45 Furthermore, after transplantation in a rat model, class II MHC was found to be upregulated on the epithelium and endothelium of lung allografts.46 Increased class II MHC on lung allografts has also been reported in lung transplant patients with OB.47 The expression of class II on nonhematopoietic cells in an allograft may provide a means of direct allorecognition for CD41 T cells, although proof of this in vivo is lacking. The persistence of direct allorecognition may also be explained by extrapolating concepts newly proposed as the semidirect pathway of alloantigen presentation (Fig 1).48 Semidirect allorecognition describes the process in which recipient APCs may acquire intact donor MHC:peptide complexes through either cell–cell contact or exosomes.39 This additional pathway may enable recipient APCs to interact with both CD41 and CD81 T cells simultaneously, thus extending the role of direct allorecognition. It is possible in human lung transplantation that episodes of acute rejection may injure lung epithelium and endothelium leading to cellular fragments of donor MHC that can be taken up by recipient APC and presented to alloreactive T cells. However, no direct proof of the semidirect pathway occurring in lung transplantation has been reported. The direct and indirect pathways are not mutually exclusive, and

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although they are distinct, they may be coupled via the semi-direct pathway. Allorecognition is mediated not by a specific isolated and independent pathway, but through a continuum of MHC peptide presentation mediating both CD41 and CD81 T-cell activation. A better understanding of how these pathways contribute to the persistent immune response after lung transplantation and how they may escape immunosuppression will be necessary to move lung transplantation forward. INNATE IMMUNITY: TOLL-LIKE RECEPTOR (TLR) LIGANDS

A challenge for improving lung transplantation outcomes is to dissect the unique mechanisms by which the lung, unlike other solid organs, may respond to stressors, such as I/R injury, infection, and environmental insults. The normal lung has dendritic cells, macrophages, epithelium, and endothelium, which all contribute to its defense against the environment and mediate the innate immune response. How these cells respond to stimuli impacts the adaptive immune response of T and B cells. The molecular basis for signaling between the innate immune response and environmental stimuli became much clearer with the discovery in 1997 of a human analog for the microbial recognition receptor identified in drosophila called ‘‘toll.’’49 Since that time, enormous excitement and interest has surrounded the study of the TLR subfamily.50 TLRs are a family of receptors that recognize pathogen-associated molecular patterns, which are expressed on infectious microorganisms. The ligation of TLRs on dendritic cells, which are the major antigen presenting cell, leads to dendritic cell maturation and upregulation of cytokines and costimulatory molecules; these signals are required for the adaptive immune response to be initiated. Interestingly, all TLR ligands are not exogenous, and a role for endogenous ligands in activation of the innate immune response has also been observed.51 The lung, with chronic exposure to microbes in the environment, significant I/R injury during transplantation, and risk of infection may be particularly vulnerable to activation of TLRs by endogenous and exogenous ligands after transplantation. Some have speculated that the unique exposure to the environment may underlie the poor outcomes found in organ transplantation of lung, skin, and intestine compared with other solid organs.52 One group has shown that the TLR4 agonist lipopolysaccharide (LPS) promotes an OB phenotype in a mouse model of allogeneic bone marrow transplantation, which suggests that the innate immune response can potentiate alloimmune-mediated fibrosis in the lung.53 TLR4 has also been implicated in mediating I/ R injury, and the deficiency of TLR4 in a mouse model of I/R injury reduced cytokine and chemokine produc-

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tion and significantly reduced vascular injury.54 TLR4 polymorphisms have also been linked to BOS in human lung transplant patients, and a decreased sensitivity to LPS was associated with less BOS.55,56 Taken together, these studies provide compelling evidence that the innate immune response to environmental stimuli affects lung transplantation. One possible mechanism by which TLR ligands may promote BOS is by augmenting the alloimmune response. In support of this, Kuo et al57,58 found that a respiratory viral infection promoted OB in a mouse model. Interestingly, the respiratory viral infection initially suppressed the alloimmune response and airway fibrosis at 30 days, but by 60 days the alloimmune response was increased and correlated with worse fibrosis compared with uninfected controls.57 The authors also found that preexisting immunity to the virus was not associated with any increase in airway fibrosis at 60 days. The data suggest that viral infections may promote BOS in humans by augmenting alloreactivity during the acute infection. However, previous vaccination or prior exposure to the virus may attenuate the infection and be protective. These results may explain why some studies have found a role for infection in promoting BOS and others have not.59 The potential for TLR ligands to affect the outcome of thoracic and solid-organ transplantation is the subject of several recent reviews and is beyond the scope of this review.60,61 A greater understanding of the innate immune response in the lung and its impact on promoting alloreactive T-cell responses may provide novel targets for therapeutics. INNATE IMMUNITY: DENDRITIC CELLS

The major APC type that links the innate immune response in the lung to activation of the alloimmune response is the dendritic cell (DC).62,63 DCs after transplantation migrate from the donor to the recipient’s lymph nodes and directly induce the alloimmune response (Fig 1). Studies have found that depletion of DCs significantly abrogates acute allograft rejection in both animal models and human kidney transplant patients.63 Because the mucosal surface of the lung is estimated to have a network of 500 to 750 dendritic cells per square millimeter, which is comparable with the network of Langerhans cells found in the skin, depletion of DC would be difficult prior to transplant of the lung.64 Furthermore, the lung also has several different types of DC characterized by anatomic location, cell surface receptors, and morphology, and their biology is unique to the lung, which makes studies in other organs difficult to extrapolate.62 DCs capture antigens from the allograft, the environment, or both; as they migrate to the draining lymph nodes, their phenotype matures, and they

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upregulate costimulatory markers necessary for efficient T-cell activation.62 The specific stimuli received by DC via TLRs and other receptors determine the particular type of T-cell response. T-cell responses differ by the secretion of cytokines, which define different subsets, such as T helper 1 (Th1) (interferon g, lymphotoxin), Th2 (interleukin-4 [IL-4], IL-5, and IL-13), or the newly recognized Th17 (IL-17).65 Alternatively, DCs may have a phenotype that is more tolerogenic and activates regulatory T cells (Treg) or anergizes T cells and makes them unresponsive. A Treg response is the likely outcome of inhalation of inert particles, such as allergens, which do not cause disease in most humans.66 Interestingly, data in small studies of patients with allograft tolerance show that DCs may be involved in promoting tolerance.63 These data have led some investigators to propose that DCs may be used as tolerogenic vaccines.63 The factors that affect migration of DC to the lymph nodes and their development into mature activating or tolerizing DC after lung transplantation are not known. The relevant costimulatory molecules expressed by DC during and after lung transplant are also not clear. Studies in animals have implicated the CD28-B7 and the CD40CD40L pathways as important mediators of OB, but the particular DC subsets involved have not been well defined.67-69 A substantial increase in a population of cells consistent with a DC phenotype that expressed high levels of MHC class II, CD40, and the CD28 ligand CD80 was found in allografts in the murine orthotopic lung transplantation model compared with isografts.70 These data suggest that DCs play a role in the initial antigen presentation after engraftment. Murine models have the advantage of the myriad of antibodies and reagents available for research, and additionally, the phenotype and functions of DC are well characterized in mice. More studies in this model may provide insight into which DC populations are relevant for lung rejection. Although the gold standard will be to understand DC biology in human lung transplantation, this standard is not easily reached. However, studies with other solid organ transplants have shown great promise for manipulating DCs to prevent allograft rejection and to promote allograft tolerance.63 Perhaps lung transplantation will eventually benefit from therapies targeted at DC. INNATE IMMUNITY: MACROPHAGES AND OTHER INNATE CELLS

Although DCs are the likely APCs responsible for initiating the alloimmune response, other innate cell types in the lung clearly modify the adaptive immune response. Macrophages, neutrophils, mast cells, and natural killer (NK) cells have been implicated in transplantation. Macrophages play a role in lung homeostasis and

pathogen defense and have been shown to be a source of growth factors thought to mediate the fibroproliferation characteristic of OB in humans.71 Depletion of macrophages in a rat model of heterotopic tracheal transplant prevented the development of OB, which suggests that macrophages play a causative role in OB lesions.72 Neutrophils have been found in the broncheoalveolar lavage of patients with BOS but were not found to affect OB in a mouse model, which suggests they may be a marker of disease and not a disease mediator.7 NK cells were found to affect chronic graft vasculopathy in a cardiac model of transplantation and have recently been implicated in human lung transplant patients.73-75 NK cells may be a future target for therapies to prevent OB.76 Most interesting is that mast cells recruited by Tregs have recently been implicated in mediating tolerance in a skin transplantation model.77 Mast cells have long been known to promote allergic responses in both atopic dermatitis and asthma, but recent studies have revealed a role for mast cells in negatively regulating immune responses.78 Their newly discovered role in tolerance is very intriguing for lung transplantation. Given the predominance of mast cells in lung tissue, it is interesting to speculate that they may play a role in lung allograft rejection. Although the various innate cell types found in the lung are likely to significantly modulate the immune response, the limitations of the models used for research in lung transplantation often make studying their contributions extremely difficult. Newer techniques will be needed to evaluate fully the contributions of these cell types in lung transplantation. ADAPTIVE IMMUNE RESPONSE

Studies in humans with BOS have consistently implicated persistent alloimmunity in the pathogenesis of OB. Although the association of MHC class II expression with the development of OB has long been recognized,47 more recently, direct evidence of persistent alloimmune humoral and cellular responses has been found. Humans with antidonor HLA antibodies have been found to be more likely to develop BOS79,80; as discussed above, anti–donor-specific indirect T-cell responses have also been associated with BOS in several studies.40,42 Data from animal models support a role for alloantibodies in promoting the pathogenesis of OB but have also found that they are not necessary to induce OB.81,82 An oligoclonal expansion of CD41 T cells in the peripheral blood was also found to be associated with the development of BOS, which suggests that specific CD41 T cells may expand and contribute to the pathogenesis of OB.83 Interestingly, DeBruyne et al84 previously showed that the response to acute lung allograft rejection was oligoclonal compared with the polyclonal T-cell response

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found during infection in human lung transplant subjects. Taken together, these studies suggest that the alloimmune response during both acute and chronic rejection involves a limited subset of T cells, as well as B cells and alloantibodies, which may be exploited and targeted by future therapies. The mechanisms by which alloimmune adaptive responses promote the development of OB are only beginning to be understood. Most of the work in understanding the mechanisms of alloimmune injury to the lung has been performed in either rat models or in mouse models of tracheal transplant.85 From these studies, both CD41 and CD81 T cells have been established to play a role in the development of OB.82,85,86 Data from the murine orthotopic lung transplant model discussed above have expanded on these results.34,70,87 In these studies, both CD81 and CD41 T cells were present in the lung during acute rejection, although CD41 T cells were not necessary for acute rejection.34,70 Interferon-g and not IL-17 was the major cytokine produced by the T cells that infiltrated the lung. Interestingly, a portion of the T cells that infiltrated the lung had a Treg phenotype, which was determined by the expression of the transcription factor Foxp3 in CD41 T cells.70 These data suggest that the initial alloimmune response in the lung is predominantly a Th1 type response, and that Treg type T cells may be recruited, but they are not effective in suppressing inflammation. The data from this model regarding an early Th1 type response are consistent with data from humans showing that at early time points, Th1 cytokines and chemokines predominate.88 The increased early Th1-type response in humans was also associated with greater alloimmunity and a higher risk of BOS, which suggests that preventing Th1 responses may be beneficial.88 To date, the mouse orthotopic lung model has not developed OB even when acute rejection is prevented by immunosuppression, which makes testing this hypothesis in this model difficult.87 Another interesting finding was that alloreactive Treg cells may be present during acute rejection. Whether these Treg cells may be expanded to suppress the Th1 response and prevent allograft rejection is not known. One of the most exciting recent discoveries in the laboratory has been that an autoimmune response to the lung may develop in addition to the alloimmune response. Sumpter and Wilkes89 have previously reported that during lung transplantation associated injury, collagen V may be exposed to the immune system, which results in the generation of an autoimmune response. This discovery has recently been translated successfully to similar findings in humans. Subjects with BOS were more likely to have immunoreactivity to collagen V than lung transplant subjects without BOS.14 The cellular immune response to collagen V in

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these subjects was found to be dependent on the cytokine IL-17, as well as tumor necrosis factor-a and IL-1b. IL-17 is produced by a newly defined subset of T helper cells, Th17, that are important mediators of the inflammatory response to pathogens and have been linked to autoimmune disease.90 Evidence suggests that Th17 cells may not be suppressed by current treatments and that the macrolide antibiotic azithromycin, which has been used to treat BOS, may suppress Th17 responses.14 However, azithromycin does not treat all BOS successfully, which indicates that other mechanisms may be involved.91 The data on collagen V argue that an autoimmune response to previously unobserved self-antigens may establish a unique immunologic response that is not suppressed by current therapies. More evidence of autoimmunity playing a role in OB/ BOS comes from a recent study that demonstrates an association of epithelial-specific autoantibodies with BOS in humans.15 Unfortunately, diseases of autoimmunity in the lung are often difficult to treat, such as lupus pneumonitis, rheumatoid arthritis-related lung disease, and scleroderma. Alternative therapies, such as cytokine blockade and other biologics that target B cells like rituximab, have been promising and may prove useful in lung transplantation.92 Interestingly, Wilkes and colleagues have found that oral tolerance to collagen V can be induced in rats and can decrease allograft rejection by a Treg dependent mechanism.21 Oral tolerance may be a future therapy to prevent autoimmunity and mitigate OB. STRUCTURAL CELLS

Epithelium. Although the alloimmune response clearly plays a role in the pathogenesis leading to OB, the epithelium has long been thought to contribute to the fibrotic response in the lung. Numerous studies have implicated epithelial cells as both a major target of the immune response and as a causative agent in the creation of OB.93-95 It is hypothesized that severe damage to bronchiolar epithelium may lead to excessive proliferation of granulation tissue and destructive fibrous scarring of small airways.5 Mauck and Hosenpud93 explored this concept ex vivo by demonstrating that human bronchial epithelial cells not only can elicit an allogeneic immune response but also can express several growth factors that could potentially play a role in the development of OB. These results were expanded by Jaramillo et al,94 who demonstrated that anti-HLA class I antibodies can activate airway epithelial cells to produce fibrogenic growth factors and induce epithelial cell apoptosis. In addition to the humoral response, a robust cellular response seems to be involved in airway epithelial cell injury. Smith et al96 demonstrated that airway

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epithelial cell-specific T cells can induce airway epithelial damage as well as the expression of the proapoptotic molecule Fas, and HLA class I and II, which may augment the allogeneic immune response. In 2004, Fernandez et al95 elegantly articulated the evolution of understanding that epithelial cells can initiate an immune response leading to OB toward the concept that epithelial cells may be essential in initiating the process of airway obliteration. It was demonstrated not only that orthotopic tracheal transplants undergo reepithelialization with recipient-derived epithelium but also that this epithelium is the primary target of allograft rejection in murine obliterative airway disease.95 These results, combined with the previous ex vivo data, provide strong evidence that epithelial cell injury and inflammation lead to excessive fibroproliferation and finally OB (Fig 2).97 Interestingly, the recent study that found antibodies to the epithelial specific protein, Ka1 tubulin, in patients with BOS demonstrated that sera positive for the antibodies induced profibrotic growth factors from airway epithelial cells.15 The response to injury and inflammation of the epithelium may not only impact the ongoing alloimmune response but also initiate an autoimmune response. Both mechanisms may be deleterious to the epithelium and promote fibrosis in the lung. Endothelium. Although acute vascular rejection is a risk factor for the development of OB, currently no accepted mechanism links these two disease processes. However, a general mechanistic outline has been proposed.98 In the nascent stages of chronic lung allograft rejection, it is recognized that OB is associated with a decrease in the microvascular blood supply to the small airways. Although evidence of neovascularization exists, it does not stop the progress of small airway obliteration.99 Yousem et al100 have proposed that the vascular changes in lung allografts are immune mediated and are directed toward vascular endothelial cells, which causes an endothelialitis and eventually the fibrointimal narrowing of arterioles and venules. Damage to vascular endothelial cells may interrupt the microvasculature and cause an ischemic injury, which compromises the ability of the small airways to self-repair and remain patent and functional. This hypothesis has been tested in animal models.101,102 The alloimmune response directed toward the vascular endothelium initiates a cascade of inflammatory mediators, including upregulation of transforming growth factor-b, which subsequently leads to both the increased production of fibronectin and procollagen and the downregulation of collagenases and proteases. The result is increased deposition of extracellular matrix and the promotion of fibrosis.103-105 In addition, growing evidence indicates that within the developing scar tissue

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of OB, fibroblasts release nitric oxide, which promotes the transcription of vascular endothelial growth factor (VEGF).106-108 VEGF, which has been associated with both acute and chronic allograft rejection, potentiates the genesis of fibrotic lesions in animal models and is believed to be instrumental in the formation of obliterative airway disease.109,111 Furthermore, the blockade of VEGF, along with platelet-derived growth factor, has been shown to prevent luminal occlusion of tracheal allografts.111 However, VEGF is not a straightforward target for therapy. Data suggests that VEGF may both maintain the epithelial barrier by increasing early epithelial cell proliferation as well as increase chemotaxis of mononuclear inflammatory cells and promote luminal scarring and the progression of OB.111 Thus, the exact role of VEGF, as well as the connection between acute vascular rejection and lung epithelial destruction has yet to be fully elucidated. The relationship between microvascular injury and epithelial damage was recently tested meticulously by Babu et al.112 Murine orthotopic tracheal transplants, which were analyzed by tissue oximetry and morphometric analysis, demonstrated that tracheal allografts underwent a time-dependent loss of vascular perfusion that corresponded to the presence and extent of tissue hypoxia. A correlation between tissue hypoxia and subsequent allograft rejection was found. Immunosuppression of the alloimmune response rescued the tracheal allografts from destruction but only if the vascular endothelium remained undamaged. This finding was demonstrated by showing that damage to the tracheal allograft microvasculature elicited ineffective revascularization and subsequent fibrosis. These experiments demonstrated a direct connection between microvascular injury and epithelial damage in a model of OB (Fig 2). Despite the evident link between acute vascular rejection and epithelial injury found by Babu et al,112 the exact relationship has yet to be completely defined. Despite the severe vascular rejection and resultant necrosis observed in the orthotopic murine lung transplant model, allograft airway epithelial cells remained undamaged.34,87 It was proposed that the preservation of the epithelial cells was caused by the increased expression of the antiapoptotic protein Bcl-2 in airway epithelial cells.87 Nonetheless, these paradoxical results reveal that our understanding of the mechanisms by which vascular injury leads to epithelial damage is still incomplete. Okazaki et al87 proposed that vascular rejection alone is not sufficient to elicit epithelial damage and that an additional insult to the epithelium, possibly from bile reflux or infection, may also be required. Data suggest that gastroesophageal and bile salt reflux are risk factors for BOS (Fig 2).113,114 These insults have been associated with decreased concentrations of pulmonary surfactant

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Fig 2. The innate and adaptive immune responses of the lung contribute to OB. Alloimmune and nonimmune factors contribute to chronic rejection and fibrosis of the small airways. A, Endothelial damage induced hypoxia. Activated T cells may damage the epithelial microvasculature directly, which results in tissue hypoxia. B, Immunemediated epithelial damage. Activated allogeneic and autoreactive T cells, as well as alloantibodies and autoantibodies, may induce epithelial and interstitial damage. C, Development of OB. Other immune and nonimmune factors, such as bile salts, acid reflux, and infections, may promote epithelial damage characterized by loss of microvilli, flattening of cuboidal cells, as well as activation of macrophages and myofibroblasts. The end result is luminal occlusion and obliteration of the small airways. (Color version of figure is available online.)

collectin proteins and phospholipids, which are components of innate immunity in the lung.115 Because of the link between vascular injury and epithelial damage, it has been speculated that the effects from OB may be mitigated or even prevented via bronchial artery revascularization after lung transplantation.10,11,112 Nørgaard and colleagues compared data from their institution with data from Stanford pertaining to bronchial artery revascularization at the time of lung transplantation.11 This small study suggested that direct bronchial artery revascularization may postpone the onset of BOS and OB. Unfortunately, stringent statistical analysis was not performed because of the small sample size. Nonetheless, these results do provide support for the causative relationship between vascular injury and epithelial damage. A more recent study by Langenbach et al116 evaluated the hypothesis that a lack of bronchial artery revascularization would predispose to increased angiogenesis and BOS.116 However, the authors failed to find a significant difference of airway vascularity between stable and BOS lung transplant patients. Others have demonstrated that vascular remodeling is a component of OB/BOS in both an animal model and in humans.117 Furthermore, in a retrospective analysis of over 50 lung transplant recipients, pulmonary hypertension and vascular remodeling were found to be more prevalent in patients with OB/BOS.118 Although ischemia and vascular remodeling have been linked to the pathogenesis of OB/BOS, the benefit of bronchial artery

vascularization in abrograting these processes remains to be established. Since OB can develop after allogeneic hematopoietic stem cell transplantation in humans, ischemia may not be an absolute requirement.119 FUTURE DIRECTIONS

New models are constantly being developed to determine the mechanisms of OB more accurately. The ideal model for studying lung alloreactivity would be one that is not technically prohibitive and that closely resembles both the natural pulmonary physiology, as well as the pathophysiology of lung transplantation rejection both acute and chronic. Currently, none of the available models fulfill these criteria. The limitations of the current models have provided an incentive to investigators to develop novel models that are technically feasible and accurately recapitulate the physiology and the pathophysiology of OB. One model was recently described by Panoskaltsis-Mortari et al.120 Lethally irradiated wild-type C57Bl/6 mice received allogeneic T cells and bone marrow cells. The authors found pulmonary perivascular and peribronchiolar inflammation, as well as airway obstruction similar to OB. Notably, minimal graft-versus-host disease was observed in other organs. Although this method does not model physiologic conditions caused by the lack of a true transplant, it allows study of the mechanisms by which alloreactive T cells promote OB in an intact lung. Another exciting approach uses transgenic mice that express ovalbumin exclusively

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in the small airways of the lung.121 This approach has been used previously to elucidate mechanisms of tissue-specific injury in models of cardiac, islet, and intestinal autoimmune disease.122-124 The advantage of this model is that transgenic T cells specific for ovalbumin can then be manipulated and adoptively transferred into the mice. Medoff et al121 found that ova-specific CD81 T cells mediate an acute bronchiolitis similar to the acute rejection found in humans and that T-cell deficiency of the leukotriene B4 receptor, BLT1, attenuated the bronchiolitis. BLT1 may be a target for therapy to prevent acute rejection. Currently, we are using these mice in our laboratory to understand the role of CD41 T cells in mediating acute and chronic rejection. We hope to develop a model relevant for human OB/BOS. Lung transplantation research will benefit from novel and creative approaches to understanding chronic rejection in the laboratory. SUMMARY

The lung is a complex organ involved in mucosal defense and gas exchange in which the immune system has evolved to protect both barriers. The response to chronic alloimmune mediated and acute I/R injury including PGD, infectious, and environmental insults and their effects on the adaptive immune response are only beginning to be defined (Fig 2). The future of lung transplantation depends on dissecting the deleterious immune response to these insults and on determining the mechanisms promoting a regulatory immune response. From studies in humans and animal models, new insights into the types of T cells involved and their link to OB/ BOS have developed. An early Th1-type response seems to prevail, but the persistent T-cell response that may be dominated by Th17 cells remains to be well characterized. Recent data demonstrate that autoimmunity complicates lung transplantation and is an exciting new area of research for therapies to prevent or modulate OB/BOS. Just as pathogen defense cannot rely on antibiotics alone, it seems that lung transplantation cannot move forward with only generalized immunosuppressive drugs. Specific mechanisms of tolerance in the lung need to be elucidated and exploited. It follows that the continued development of novel tools in the laboratory and innovative approaches to studies in humans are necessary to improve outcomes after lung transplantation. We thank Drs. J. Cannon, A. Chong, and M. Alegre for critical reading of the manuscript and helpful discussions.

REFERENCES

1. Carrel A, Guthrie CC. Functions of a transplanted kidney. Science 1905;22:473.

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2. Starzl TE, Marchioro TL, Waddell WR. The reversal of rejection in human renal homografts with subsequent development of homograft tolerance. Surg Gynecol Obstet 1963;117:385–95. 3. The Organ Procurement and Transplant Network. Available at: http://www.optn.org/data. 4. Christie JD, Edwards LB, Aurora P, et al. Registry of the international society for heart and lung transplantation: twenty-fifth official adult lung and heart/lung transplantation report— 2008. J Heart Lung Transplant 2008;27:957–69. 5. Nicod LP. Mechanisms of airway obliteration after lung transplantation. Proc Am Thorac Soc 2006;3:444–9. 6. Wilkes DS, Egan TM, Reynolds HY. Lung transplantation: opportunities for research and clinical advancement. Am J Respir Crit Care Med 2005;172:944–55. 7. McDyer JF. Human and murine obliterative bronchiolitis in transplant. Proc Am Thorac Soc 2007;4:37–43. 8. Husain AN, Siddiqui MT, Holmes EW, et al. Analysis of risk factors for the development of bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 1999;159:829–33. 9. Glanville AR, Aboyoun CL, Havryk A, Plit M, Rainer S, Malouf MA. Severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation. Am J Respir Crit Care Med 2008;177:1033–40. 10. Baudet EM, Dromer C, Dubrez J, et al. Intermediate-term results after en bloc double-lung transplantation with bronchial arterial revascularization. Bordeaux Lung and Heart-Lung Transplant Group. J Thorac Cardiovasc Surg 1996;112:1292–300. 11. Nørgaard MA, Andersen CB, Pettersson G. Does bronchial artery revascularization influence results concerning bronchiolitis obliterans syndrome and/or obliterative bronchiolitis after lung transplantation? Eur J Cardiothorac Surg 1998;14:311–8. 12. Daud SA, Yusen RD, Meyers BF, et al. Impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 2007;175:507–13. 13. Huang HJ, Yusen RD, Meyers BF, et al. Late primary graft dysfunction after lung transplantation and bronchiolitis obliterans syndrome. Am J Transplant 2008;8:2454–62. 14. Burlingham WJ, Love RB, Jankowska-Gan E, et al. IL-17-dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants. J Clin Invest 2007; 117:349–506. 15. Goers TA, Ramachandran S, Aloush A, Trulock E, Patterson GA, Mohanakumar T. De novo production of K-alpha1 tubulin-specific antibodies: role in chronic lung allograft rejection. J Immunol 2008;180:4487–94. 16. Christie JD, Carby M, Bag R, Corris P, Hertz M, Weill D. Report of the ISHLT Working group on primary lung graft dysfunction part II: definition. A consensus statement of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant 2005;24:1454–9. 17. King RC, Binns OA, Rodriguez F, et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann Thorac Surg 2000;69:1681–5. 18. Christie JD, Kotloff RM, Ahya VN, et al. The effect of primary graft dysfunction on survival after lung transplantation. Am J Respir Crit Care Med 2005;171:1312–6. 19. Haque MA, Mizobuchi T, Yasufuku K, et al. Evidence for immune responses to a self-antigen in lung transplantation: role of type V collagen-specific T cells in the pathogenesis of lung allograft rejection. J Immunol 2002;169:1542–9. 20. Yoshida S, Haque A, Mizobuchi T, et al. Anti-type V collagen lymphocytes that express IL-17 and IL-23 induce rejection pathology in fresh and well-healed lung transplants. Am J Transplant 2006;6:724–35.

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21. Mizobuchi T, Yasufuku K, Zheng Y, et al. Differential expression of Smad7 transcripts identifies the CD41CD45RChigh regulatory T cells that mediate type V collagen-induced tolerance to lung allografts. J Immunol 2003;171:1140–7. 22. Bobadilla JL, Love RB, Jankowska-Gan E, et al. Th-17, monokines, collagen type V, and primary graft dysfunction in lung transplantation. Am J Respir Crit Care Med 2008;177:660–8. 23. Iwata T, Philipovskiy A, Fisher AJ, et al. Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction. J Immunol 2008;181:5738–47. 24. Marck KW, Wildevuur CR. Lung transplantation in the rat: I. Technique and survival. Ann Thorac Surg 1982;34:74–80. 25. Marck KW, Piers DA, Wildevuur CR. Lung transplantation in the rat: II. lung perfusion scintigraphy in normal and left lung-transplanted rats. Ann Thorac Surg 1982;34:81–8. 26. Marck KW, Prop J, Wildevuur CR. Lung transplantation in the rat. III. Functional studies in iso- and allografts. J Surg Res 1983;35:149–58. 27. Yasufuku K, Heidler KM, Woods KA, et al. Prevention of bronchiolitis obliterans in rat lung allografts by type V collageninduced oral tolerance. Transplantation 2002;73:500–5. 28. Hirschburger M, Greschus S, Kuchenbuch T, et al. Lung transplantation in the Fischer 344–.Wistar Kyoto rat strain combination is not suitable to study bronchiolitis obliterans. J Heart Lung Transplant 2007;26:390–8. 29. Allan JS, Wain JC, Schwarze ML, Houser SL, Madsen JC, Sachs DH. Obliterative bronchiolitis develops in miniature swine transplanted across a minor histocompatibility barrier. Transplant Proc 2001;33:358–9. 30. Hertz MI, Jessurun J, King MB, Savik SK, Murray JJ. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993;142:1945–51. 31. Genden EM, Boros P, Liu J, Bromberg JS, Mayer L. Orthotopic tracheal transplantation in the murine model. Transplantation 2002;73:1420–5. 32. Dutly AE, Andrade CF, Verkaik R, et al. A novel model for posttransplant obliterative airway disease reveals angiogenesis from the pulmonary circulation. Am J Transplant 2005;5:248–54. 33. Sato M, Liu M, Anraku M, et al. Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling. Am J Transplant 2008;8:517–28. 34. Okazaki M, Krupnick AS, Kornfeld CG, et al. A mouse model of orthotopic vascularized aerated lung transplantation. Am J Transplant 2007;7:1672–9. 35. Felix NJ, Allen PM. Specificity of T-cell alloreactivity. Nat Rev Immunol 2007;7:942–53. 36. Benichou G. Direct and indirect antigen recognition: the pathways to allograft immune rejection. Front Biosci 1999;4: D476–80. 37. Game DS, Lechler RI. Pathways of allorecognition: implications for transplantation tolerance. Transpl Immunol 2002;10:101–8. 38. Lechler RI, Batchelor JR. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J Exp Med 1982;155:31–41. 39. Gokmen MR, Lombardi G, Lechler RI. The importance of the indirect pathway of allorecognition in clinical transplantation. Curr Opin Immunol 2008;20:568–74. 40. Stanford RE, Ahmed S, Hodson M, Banner NR, Rose ML. A role for indirect allorecognition in lung transplant recipients with obliterative bronchiolitis. Am J Transplant 2003;3:736–42. 41. SivaSai KS, Smith MA, Poindexter NJ, et al. Indirect recognition of donor HLA class I peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Transplantation 1999;67: 1094–8.

Grossman and Shilling

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42. Reznik SI, Jaramillo A, SivaSai KS, et al. Indirect allorecognition of mismatched donor HLA class II peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Am J Transplant 2001;1:228–35. 43. Lee RS, Grusby MJ, Glimcher LH, Winn HJ, Auchincloss H Jr. Indirect recognition by helper cells can induce donor-specific cytotoxic T lymphocytes in vivo. J Exp Med 1994;179:865–72. 44. Smyth LA, Afzali B, Tsang J, Lombardi G, Lechler RI. Intercellular transfer of MHC and immunological molecules: molecular mechanisms and biological significance. Am J Transplant 2007;7:1442–9. 45. Richards DM, Dalheimer SL, Hertz MI, Mueller DL. Trachea allograft class I molecules directly activate and retain CD81 T cells that cause obliterative airways disease. J Immunol 2003;171: 6919–28. 46. Romaniuk A, Prop J, Petersen AH, Wildevuur CR, Nieuwenhuis P. Expression of class II major histocompatibility complex antigens by bronchial epithelium in rat lung allografts. Transplantation 1987;44:209–14. 47. Burke CM, Glanville AR, Theodore J, Robin ED. Lung immunogenicity, rejection, and obliterative bronchiolitis. Chest 1987;92: 547–9. 48. Herrera OB, Golshayan D, Tibbott R, et al. A novel pathway of alloantigen presentation by dendritic cells. J Immunol 2004; 173:4828–37. 49. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr.. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:394–7. 50. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. 51. Tesar BM, Jiang D, Liang J, Palmer SM, Noble PW, Goldstein DR. The role of hyaluronan degradation products as innate alloimmune agonists. Am J Transplant 2006;6:2622–35. 52. Chen L, Wang T, Zhou P, et al. TLR engagement prevents transplantation tolerance. Am J Transplant 2006;6:2282–91. 53. Garantziotis S, Palmer SM, Snyder LD, et al. Alloimmune lung injury induced by local innate immune activation through inhaled lipopolysaccharide. Transplantation 2007;84:1012–9. 54. Shimamoto A, Pohlman TH, Shomura S, Tarukawa T, Takao M, Shimpo H. Toll-like receptor 4 mediates lung ischemia-reperfusion injury. Ann Thorac Surg 2006;82:2017–23. 55. Palmer SM, Burch LH, Trindade AJ, et al. Innate immunity influences long-term outcomes after human lung transplant. Am J Respir Crit Care Med 2005;171:780–5. 56. Stockinger B, Veldhoen M. Differentiation and function of Th17 T cells. Curr Opin Immunol 2007;19:281–6. 57. Kuo E, Bharat A, Goers T, et al. Respiratory viral infection in obliterative airway disease after orthotopic tracheal transplantation. Ann Thorac Surg 2006;82:1043–50. 58. Kuo E, Bharat A, Shih J, et al. Role of airway epithelial injury in murine orthotopic tracheal allograft rejection. Ann Thorac Surg 2006;82:1226–33. 59. Sharples LD, McNeil K, Stewart S, Wallwork J. Risk factors for bronchiolitis obliterans: a systematic review of recent publications. J Heart Lung Transplant 2002;21:271–81. 60. Goldstein DR, Palmer SM. Role of Toll-like receptor-driven innate immunity in thoracic organ transplantation. J Heart Lung Transplant 2005;24:1721–9. 61. Alegre ML, Goldstein DR, Chong AS. Toll-like receptor signaling in transplantation. Curr Opin Organ Transplant 2008;13: 358–65. 62. Cook DN, Bottomly K. Innate immune control of pulmonary dendritic cell trafficking. Proc Am Thorac Soc 2007;4:234–9.

164

Grossman and Shilling

63. Solari MG, Thomson AW. Human dendritic cells and transplant outcome. Transplantation 2008;85:1513–22. 64. Holt PG. Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract. Proc Am Thorac Soc 2005;2:116–20. 65. Reinhardt RL, Kang SJ, Liang HE, Locksley RM. T helper cell effector fates—who, how and where? Curr Opin Immunol 2006;18:271–7. 66. Hammad H, Lambrecht BN. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Rev Immunol 2008;8:193–204. 67. Yamada A, Konishi K, Cruz GL, et al. Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation 2000;69:743–9. 68. Rumbley CA, Silver SJ, Phillips SM. Dependence of murine obstructive airway disease on CD40 ligand. Transplantation 2001;72:1616–25. 69. Tikkanen JM, Lemstrom KB, Koskinen PK. Blockade of CD28/ B7-2 costimulation inhibits experimental obliterative bronchiolitis in rat tracheal allografts: suppression of helper T cell type1dominated immune response. Am J Respir Crit Care Med 2002;165:724–9. 70. Gelman AE, Okazaki M, Lai J, et al. CD41 T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants. J Immunol 2008;180:4754–62. 71. Hertz MI, Henke CA, Nakhleh RE, et al. Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor. Proc Natl Acad Sci U S A 1992;89:10385–9. 72. Oyaizu T, Okada Y, Shoji W, et al. Reduction of recipient macrophages by gadolinium chloride prevents development of obliterative airway disease in a rat model of heterotopic tracheal transplantation. Transplantation 2003;76:1214–20. 73. Uehara S, Chase CM, Colvin RB, Russell PS, Madsen JC. Further evidence that NK cells may contribute to the development of cardiac allograft vasculopathy. Transplant Proc 2005;37:70–1. 74. Uehara S, Chase CM, Kitchens WH, et al. NK cells can trigger allograft vasculopathy: the role of hybrid resistance in solid organ allografts. J Immunol 2005;175:3424–30. 75. Fildes JE, Yonan N, Tunstall K, et al. Natural killer cells in peripheral blood and lung tissue are associated with chronic rejection after lung transplantation. J Heart Lung Transplant 2008;27: 203–7. 76. Fildes JE, Yonan N, Leonard CT. Natural killer cells and lung transplantation, roles in rejection, infection, and tolerance. Transpl Immunol 2008;19:1–11. 77. Lu LF, Lind EF, Gondek DC, et al. Mast cells are essential intermediaries in regulatory T-cell tolerance. Nature 2006;442: 997–1002. 78. Galli SJ, Grimbaldeston M, Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat Rev Immunol 2008;8:478–86. 79. Jaramillo A, Smith MA, Phelan D, et al. Development of ELISAdetected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation. Transplantation 1999;67:1155–61. 80. Sundaresan S, Mohanakumar T, Smith MA, et al. HLA-A locus mismatches and development of antibodies to HLA after lung transplantation correlate with the development of bronchiolitis obliterans syndrome. Transplantation 1998;65:648–53.

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81. Kuo E, Maruyama T, Fernandez F, Mohanakumar T. Molecular mechanisms of chronic rejection following transplantation. Immunol Res 2005;32:179–85. 82. Higuchi T, Jaramillo A, Kaleem Z, Patterson GA, Mohanakumar T. Different kinetics of obliterative airway disease development in heterotopic murine tracheal allografts induced by CD41 and CD81 T cells. Transplantation 2002;74:646–51. 83. Duncan SR, Leonard C, Theodore J, et al. Oligoclonal CD4(1) T cell expansions in lung transplant recipients with obliterative bronchiolitis. Am J Respir Crit Care Med 2002;165:1439–44. 84. DeBruyne LA, Lynch JP 3rd, Baker LA, et al. Restricted V beta usage by T cells infiltrating rejecting human lung allografts. J Immunol 1996;156:3493–500. 85. Kuo E, Bharat A, Dharmarajan S, Fernandez F, Patterson GA, Mohanakumar T. Animal models for bronchiolitis obliterans syndrome following human lung transplantation. Immunol Res 2005;33:69–81. 86. Neuringer IP, Mannon RB, Coffman TM, et al. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998;19:379–86. 87. Okazaki M, Gelman AE, Tietjens JR, et al. Maintenance of airway epithelium in acutely rejected orthotopic vascularized mouse lung transplants. Am J Respir Cell Mol Biol 2007;37:625–30. 88. Bharat A, Kuo E, Steward N, et al. Immunological link between primary graft dysfunction and chronic lung allograft rejection. Ann Thorac Surg 2008;86:189–97. 89. Sumpter TL, Wilkes DS. Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection. Am J Physiol Lung Cell Mol Physiol 2004;286:L1129–39. 90. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 2008;28:454–67. 91. Fietta AM, Meloni F. Lung transplantation: the role of azithromycin in the management of patients with bronchiolitis obliterans syndrome. Curr Med Chem 2008;15:716–23. 92. Chatenoud L. Immune therapies of autoimmune diseases: are we approaching a real cure? Curr Opin Immunol 2006;18:710–7. 93. Mauck KA, Hosenpud JD. The bronchial epithelium: a potential allogeneic target for chronic rejection after lung transplantation. J Heart Lung Transplant 1996;15:709–14. 94. Jaramillo A, Smith CR, Maruyama T, Zhang L, Patterson GA, Mohanakumar T. Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome. Hum Immunol 2003;64:521–9. 95. Fernandez FG, Jaramillo A, Chen C, et al. Airway epithelium is the primary target of allograft rejection in murine obliterative airway disease. Am J Transplant 2004;4:319–25. 96. Smith CR, Jaramillo A, Duffy BF, Mohanakumar T. Airway epithelial cell damage mediated by antigen-specific T cells: implications in lung allograft rejection. Hum Immunol 2000;61: 985–92. 97. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group. J Heart Lung Transplant 1996;15:1–15. 98. Luckraz H, Goddard M, McNeil K, Atkinson C, Sharples LD, Wallwork J. Is obliterative bronchiolitis in lung transplantation associated with microvascular damage to small airways? Ann Thorac Surg 2006;82:1212–8. 99. Luckraz H, Goddard M, McNeil K, et al. Microvascular changes in small airways predispose to obliterative bronchiolitis after lung transplantation. J Heart Lung Transplant 2004;23:527–31.

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100. Yousem SA, Burke CM, Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation. Hum Pathol 1985;16:911–23. 101. Adams BF, Brazelton T, Berry GJ, Morris RE. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000;69:661–4. 102. Ikonen T, Uusitalo M, Taskinen E, et al. Small airway obliteration in a new swine heterotopic lung and bronchial allograft model. J Heart Lung Transplant 1998;17:945–53. 103. Bergmann M, Tiroke A, Schafer H, Barth J, Haverich A. Gene expression of profibrotic mediators in bronchiolitis obliterans syndrome after lung transplantation. Scand Cardiovasc J 1998; 32:97–103. 104. El-Gamel A, Sim E, Hasleton P, et al. Transforming growth factor beta (TGF-beta) and obliterative bronchiolitis following pulmonary transplantation. J Heart Lung Transplant 1999;18:828–37. 105. Elssner A, Jaumann F, Dobmann S, et al. Elevated levels of interleukin-8 and transforming growth factor-beta in bronchoalveolar lavage fluid from patients with bronchiolitis obliterans syndrome: proinflammatory role of bronchial epithelial cells. Munich Lung Transplant Group. Transplantation 2000;70:362–7. 106. Mason NA, Springall DR, Pomerance A, Evans TJ, Yacoub MH, Polak JM. Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis. J Heart Lung Transplant 1998;17:710–4. 107. Gabbay E, Walters EH, Orsida B, et al. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide levels and epithelial inducible nitric oxide synthase. Am J Respir Crit Care Med 2000;162:2182–7. 108. Reinders ME, Fang JC, Wong W, Ganz P, Briscoe DM. Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection. Transplantation 2003; 76:224–30. 109. Krebs R, Tikkanen JM, Nykanen AI, et al. Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis. Am J Respir Crit Care Med 2005;171:1421–9. 110. Ozdemir BH, Ozdemir FN, Haberal N, Emiroglu R, Demirhan B, Haberal M. Vascular endothelial growth factor expression and cyclosporine toxicity in renal allograft rejection. Am J Transplant 2005;5:766–74. 111. Tikkanen JM, Hollmen M, Nykanen AI, Wood J, Koskinen PK, Lemstrom KB. Role of platelet-derived growth factor and vascular endothelial growth factor in obliterative airway disease. Am J Respir Crit Care Med 2006;174:1145–52.

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112. Babu AN, Murakawa T, Thurman JM, et al. Microvascular destruction identifies murine allografts that cannot be rescued from airway fibrosis. J Clin Invest 2007;117:3774–85. 113. Blondeau K, Mertens V, Vanaudenaerde BA, et al. Gastro-oesophageal reflux and gastric aspiration in lung transplant patients with or without chronic rejection. Eur Respir J 2008;31:707–13. 114. D’Ovidio F, Mura M, Tsang M, et al. Bile acid aspiration and the development of bronchiolitis obliterans after lung transplantation. J Thorac Cardiovasc Surg 2005;129:1144–52. 115. D’Ovidio F, Mura M, Ridsdale R, et al. The effect of reflux and bile acid aspiration on the lung allograft and its surfactant and innate immunity molecules SP-A and SP-D. Am J Transplant 2006;6:1930–8. 116. Langenbach SY, Zheng L, McWilliams T, et al. Airway vascular changes after lung transplant: potential contribution to the pathophysiology of bronchiolitis obliterans syndrome. J Heart Lung Transplant 2005;24:1550–6. 117. Belperio JA, Keane MP, Burdick MD, et al. Role of CXCR2/ CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome. J Clin Invest 2005;115:1150–62. 118. Saggar R, Ross DJ, Saggar R, et al. Pulmonary hypertension associated with lung transplantation obliterative bronchiolitis and vascular remodeling of the allograft. Am J Transplant 2008;8:1921–30. 119. Soubani AO, Uberti JP. Bronchiolitis obliterans following haematopoietic stem cell transplantation. Eur Respir J 2007;29: 1007–19. 120. Panoskaltsis-Mortari A, Tram KV, Price AP, Wendt CH, Blazar BR. A new murine model for bronchiolitis obliterans post-bone marrow transplant. Am J Respir Crit Care Med 2007;176:713–23. 121. Medoff BD, Seung E, Wain JC, et al. BLT1-mediated T cell trafficking is critical for rejection and obliterative bronchiolitis after lung transplantation. J Exp Med 2005;202:97–110. 122. Grabie N, Delfs MW, Westrich JR, et al. IL-12 is required for differentiation of pathogenic CD81 T cell effectors that cause myocarditis. J Clin Invest 2003;111:671–80. 123. Liu Z, Lefrancois L. Intestinal epithelial antigen induces mucosal CD8 T cell tolerance, activation, and inflammatory response. J Immunol 2004;173:4324–30. 124. Eggena MP, Walker LS, Nagabhushanam V, Barron L, Chodos A, Abbas AK. Cooperative roles of CTLA-4 and regulatory T cells in tolerance to an islet cell antigen. J Exp Med 2004; 199:1725–30.