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Ischemia/reperfusion injury-mediated (IRI-mediated) primary graft dysfunction (PGD) adversely affects both short- and long-term outcomes after lung transplantation, a procedure that remains the only treatment option for patients suffering from end-stage respiratory failure. While B cells are known to regulate adaptive immune responses, their role in lung IRI is not well understood. Here, we demonstrated by intravital imaging that B cells are rapidly recruited to injured lungs, where they extravasate into the parenchyma. Using hilar clamping and transplant models, we observed that lung-infiltrating B cells produce the monocyte chemokine CCL7 in a TLR4-TRIF-dependent fashion, a critical step contributing to classical monocyte (CM) recruitment and subsequent neutrophil extravasation, resulting in worse lung function. We found that synergistic BCR-TLR4 activation on B cells is required for the recruitment of CMs to the injured lung. Finally, we corroborated our findings in reperfused human lungs, in which we observed a correlation between B cell infiltration and CM recruitment after transplantation. This study describes a role for B cells as critical orchestrators of lung IRI. As B cells can be depleted with currently available agents, our study provides a rationale for clinical trials investigating B cell-targeting therapies., Introduction Primary graft dysfunction (PGD) is a common early complication after lung transplantation that adversely affects both shortand long-term outcomes. This includes both early morbidity and mortality as well as [...]

Lung transplantation, a cure for a number of end-stage lung diseases, continues to have the worst long-term outcomes when compared with other solid organ transplants. Preclinical modeling of the most common and serious lung transplantation complications are essential to better understand and mitigate the pathophysiological processes that lead to these complications. Various animal and in vitro models of lung transplant complications now exist and each of these models has unique strengths. However, significant issues, such as the required technical expertise as well as the robustness and clinical usefulness of these models, remain to be overcome or clarified. The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop in March 2016 to review the state of preclinical science addressing the three most important complications of lung transplantation: primary graft dysfunction (PGD), acute rejection (AR), and chronic lung allograft dysfunction (CLAD). In addition, the participants of the workshop were tasked to make consensus recommendations on the best use of these complimentary models to close our knowledge gaps in PGD, AR, and CLAD. Their reviews and recommendations are summarized in this report. Furthermore, the participants outlined opportunities to collaborate and directions to accelerate research using these preclinical models.

Tertiary lymphoid organs are aggregates of immune and stromal cells including high endothelial venules and lymphatic vessels that resemble secondary lymphoid organs and can be induced at nonlymphoid sites during inflammation. The function of lymphatic vessels within tertiary lymphoid organs remains poorly understood. During lung transplant tolerance, [Foxp3.sup.+] cells accumulate in tertiary lymphoid organs that are induced within the pulmonary grafts and are critical for the local downregulation of alloimmune responses. Here, we showed that tolerant lung allografts could induce and maintain tolerance of heterotopic donor-matched hearts through pathways that were dependent on the continued presence of the transplanted lung. Using lung retransplantation, we showed that [Foxp3.sup.+] cells egressed from tolerant lung allografts via lymphatics and were recruited into donor-matched heart allografts. Indeed, survival of the heart allografts was dependent on lymphatic drainage from the tolerant lung allograft to the periphery. Thus, our work indicates that cellular trafficking from tertiary lymphoid organs regulates immune responses in the periphery. We propose that these findings have important implications for a variety of disease processes that are associated with the induction of tertiary lymphoid organs., Introduction Tertiary lymphoid organs can be induced in nonlymphoid tissues during states of chronic inflammation or antigen exposure and are structurally similar to secondary lymphoid organs. One such example is [...]

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1[beta]-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective., Introduction Primary graft dysfunction (PGD) affects over 50% of recipients following lung transplantation and has emerged as the principal risk factor for both short-term mortality as well as long-term graft [...]

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-[beta] bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-[beta] and reduce TGF-[beta] bioavailability through secretion of the TGF-[beta] antagonist decorin. In untreated recipients, high airway TGF-[beta] activity stimulated AMs to express CCL2, leading to [CCR2.sup.+] monocyte-driven BOS development. Moreover, we found TGF-[beta] receptor 2-dependent differentiation of [CCR2.sup.+] monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8* T cells that inflicted airway injury through Blimp-1-mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-[beta]-dependent network that couples [CCR2.sup.+] monocyte recruitment and differentiation to alloimmunity and BOS., Introduction Bronchiolitis obliterans syndrome (BOS) is the most common form of chronic lung allograft dysfunction (CLAD) and the leading cause of rejection after the first year of transplantation (1). The [...]

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAMs) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor NCMs. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAMs. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or coreceptor CD14 on donor TRAMs prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia/reperfusion-primed donor TRAMs., Introduction Prolonged mechanical ventilation, aspiration of gastric contents into the lungs around the time of death, and immune dysfunction after brain death all contribute to increased rates of bacterial infections [...]

Primary graft dysfunction (PGD) is the leading cause of postoperative mortality in lung transplant recipients and the most important risk factor for development of chronic lung allograft dysfunction. The mechanistic basis for the variability in the incidence and severity of PGD between lung transplant recipients is not known. Using a murine orthotopic vascularized lung transplant model, we found that redundant activation of Toll-like receptors 2 and 4 (TLR2 and -4) on nonclassical monocytes activates MyD88, inducing the release of the neutrophil attractant chemokine CXCL2. Deletion of Itgam (encodes CD11b) in nonclassical monocytes enhanced their production of CXCL2 and worsened PGD, while a CD11b agonist, leukadherin-1, administered only to the donor lung prior to lung transplantation, abrogated CXCL2 production and PGD. The damageassociated molecular pattern molecule HMGB1 was increased in peripheral blood samples from patients undergoing lung transplantation after reperfusion and induced CXCL2 production in nonclassical monocytes via TLR4/MyD88. An inhibitor of HMGB1 administered to the donor and recipient prior to lung transplantation attenuated PGD. Our findings suggest that CD11b acts as a molecular brake to prevent neutrophil recruitment by nonclassical monocytes following lung transplantation, revealing an attractive therapeutic target in the donor lung to prevent PGD in lung transplant recipients., Introduction Primary graft dysfunction (PGD) is a syndrome of acute lung injury that develops in 20% to 30% of recipients in the first 72 hours following lung transplantation (1-3). PGD [...]

Nonapoptotic forms of cell death can trigger sterile inflammation through the release of damage-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation, the mechanisms that initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the levels of pro-ferroptotic hydroperoxy-arachidonoyl- phosphatidylethanolamine, reduces cardiomyocyte cell death, and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation-induced myocardial ischemia reperfusion injury (IRI), in which inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular (LV) systolic function, and reduced LV remodeling. Using intravital imaging of cardiac transplants, we show that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/Trif/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif- dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients who are vulnerable to IRI following restoration of coronary blood flow., Introduction Ischemia reperfusion injury (IRI) following heart transplantation is a clinically significant form of sterile inflammation. IRI-mediated primary graft dysfunction complicates the postoperative course of up to 28% of human [...]

Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that [Foxp3.sup.+] T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus- associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident [Foxp3.sup.+] T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident [Foxp3.sup.+] T lymphocytes resulted in prolonged interactions between B and [CD4.sup.+] T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory [Foxp3.sup.+] T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses., Introduction Lung transplantation is the only treatment available for numerous patients suffering from end-stage pulmonary failure. Compared with outcomes for other organ transplants, that after lung transplantation remains far worse, [...]

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that [CCR2.sup.+] classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1[beta] production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic., Introduction Ischemia-reperfusion injury (IRI) following solid organ transplantation is mediated by host neutrophils that, upon extravasation, initiate NETosis and induce allograft damage (1, 2). Following lung transplantation, IRI leads to [...]