Cardiac allograft vasculopathy (CAV) is a fibroproliferative form of transplant rejection with limited treatment options other than retransplantation. In this issue, See and colleagues examined human explanted allografts with CAV. They found that a high proportion of intragraft plasma cells produce antibodies that recognize the heme catabolic end product, bilirubin. Clonotypic profiling revealed that bilirubin-reactive antibody-producing plasma cells develop from graft-infiltrating innate-like B cells, a subset often characterized by their rapid production of polyreactive natural antibodies as an early defense against infection. CAV but not nonrejecting graft tissue contained bilirubin deposits along with macrophages that expressed genes involved in heme catabolism. These findings raise the intriguing possibility that graft-derived bilirubin-specific antibodies target local heme catabolism to promote CAV.

BACKGROUND:Lung allograft rejection is determined by pathologist grading of transbronchial biopsies (TBBx) according to criteria established by the International Society of Heart and Lung Transplantation (ISHLT). Lymphocytic bronchiolitis (LB), described as mononuclear cells in bronchiolar submucosa, exhibits significant histologic overlap with bronchus-associated lymphoid tissue (BALT) leading to diagnostic uncertainty. Additionally, the role of BALT in tolerance and rejection is debated. We sought to characterize lymphoid aggregates in ISHLT acute cellular rejection (ACR) grades A0 or A3 biopsies to better understand the spectrum of BALT and rejection lesions. METHODS:TBBx were reviewed for the presence of BALT. Representative A0 and A3 biopsies were selected for spatial transcriptomics and multiplex immunofluorescence microscopy. RESULTS:Spatial transcriptomics of allograft biopsies enabled unbiased identification of BALT and rejection lesions via neighborhood analysis. CXCL9/10+ T and PDL1/L2+ myeloid cell clusters were enriched in A0 BALT, while an NKG7+CD8+ T cell cluster was enriched in A3 BALT. Higher expression of CXCL9 and CXCL10 in the BALT correlated with A0 grade. Computational methods identified occult areas of lymphoid aggregates in rejecting lung parenchyma with similarity to rejection lesions. A3 BALT and rejection lesions showed substantial similarities. Immunofluorescence confirmed the key transcriptomics findings. CONCLUSIONS:This study suggests a relationship between the immune microenvironment in BALT and graft rejection. Additionally, lymphoid infiltration in allografts graded A3 may be more widespread than apparent on Hematoxylin and Eosin (H&E) stain.

TGF-β-activating integrins promote solid-organ fibrosis, suggesting their use as a molecular marker of disease. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-β activation. However, the expression patterns of TGF-β-activating integrins remain undefined in lung transplants. Single-cell RNA sequencing in a mouse CLAD model revealed high levels of the TGF-β-activating integrin αvβ6, which was mainly localized to fibrosis-associated Krt8+ transitional alveolar cells (AT1/2), while tolerant transplants lacked both αvβ6 expression and Krt8+AT1/2 cells. Molecular imaging with a newly developed positron emission tomography radiotracer specific for αvβ6, [64Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD versus tolerant transplants. In contrast, [64Cu]Cu-DOTA-A20-K16R allograft uptake was reduced by treatments that lowered αvβ6 expression and CLAD severity. Finally, [64Cu]Cu-DOTA-A20-K16R autoradiographic analysis on human explanted lungs with CLAD showed elevated activity that correlated with αvβ6 expression. Collectively, these findings demonstrate the potential utility of αvβ6 molecular imaging to detect CLAD pathogenesis. ### Competing Interest Statement The authors have declared no competing interest.

The consequence of lymphatic disruption during transplantation of solid organs remains unknown. Long-term survival after organ transplantation is limited by chronic rejection, a poorly understood process involving fibrotic remodeling and functional decline of the graft. Here, we found that transplanted human lungs and hearts with chronic rejection exhibited fibrosis distributed along dysmorphic lymphatics in areas densely concentrated with hyaluronan, an interstitial glycosaminoglycan that depends on lymphatic drainage for clearance. We illustrated similar findings in transplanted mouse lungs and hearts, which were accompanied by lymphographic findings of graft lymphedema. Using unsupervised clustering, we found a subset of stromal cells present in fibrotic syngeneic mouse lung grafts and human lung and heart grafts with chronic rejection that coexpressed hyaluronan synthase 1 and interleukin-1 receptor 1. Shortly after reperfusion of syngeneic mouse lung grafts, we identified neutrophilic expression of interleukin-1β (Il1b) as a driver of hyaluronan synthase 1 up-regulation. We found interleukin-1-mediated hyaluronan accumulation as a mechanism driving fibrosis that occurred independent of alloimmunity in the setting of lymphatic disruption after transplantation. Development of fibrotic remodeling in transplanted mouse lungs was inhibited by preventing hyaluronan synthesis through the administration of 4-methylumbilliferone, accelerating lymphangiogenesis with pharmacologic activation of VEGF (vascular endothelial growth factor) receptor-3, or inhibiting interleukin-1 receptor 1 signaling in the graft. These therapeutic interventions lay the foundation for future clinical strategies to prevent chronic rejection.

Ischemia-reperfusion injury (IRI) and acute cellular rejection (ACR) are common postoperative complications that lead to poor outcomes in lung transplant recipients. Transient receptor potential vanilloid 4 (TRPV4) is a plasma membrane Ca2+ channel that controls vascular tone and integrity. We examined whether TRPV4 influences IRI and alloimmune responses in a mouse model of orthotopic lung transplantation. Recipients receiving syngeneic lungs briefly treated with a TRPV4-specific inhibitor or engrafted with syngeneic lungs with an endothelial cell-specific TRPV4 gene ablation (TRPV4ΔEC) had better graft function, reduced endothelial cell adhesion molecule expression, and lower inflammatory cytokine expression and neutrophil recruitment. TRPV4ΔEC lungs transplanted into immunosuppressed, major histocompatibility complex-mismatched recipients had more intragraft CD25+ Foxp3+ CD4+ T cells and fewer IFN-γ+ CD8+ T cells and were more resistant to IRI-induced ACR than wildtype allografts. Finally, pharmacologic inhibition of TRPV4 reduced donor antigen-triggered production of IFN-γ and IL-17A by intragraft T cells and prevented ACR despite IRI. Our findings show that pulmonary endothelial cell TRPV4 activity couples ischemic tissue injury to T cell alloimmunity, highlighting the potential of therapies that enhance vascular integrity to promote transplant tolerance.

Chronic lung allograft dysfunction (CLAD) is characterized by fibrotic graft remodeling and limits long-term survival after pulmonary transplantation. Despite clinical evidence that myeloid cells drive conditions that increase the risk of CLAD development, contemporary immunosuppression primarily targets lymphocytes. We evaluated an mTOR-inhibiting nanobiologic (mTORi-NB) targeting myeloid cells and their progenitors in a semiallogeneic mouse lung transplant model of CLAD. We found that the mTORi-NB preferentially targeted myeloid and endothelial cells in the allografts. Brief perioperative therapy with the mTORi-NBs reduced early macrophage graft infiltration and inhibited acute inflammation. In mTORi-NB-treated lung recipients, transcriptomic analysis revealed downregulation of fibrotic genes in macrophages and type I and type II (AT2) alveolar epithelial cells, known drivers of pulmonary fibrogenesis. We also observed upregulation of antifibrotic genes in macrophages after treatment with mTORi-NBs, as well as reduction of allograft fibrosis, associated with preservation of club and AT2 cells. Our findings suggest that myeloid-avid nanobiologics may be a promising immunosuppressive strategy for inhibiting CLAD.

Ischemia-reperfusion injury (IRI) contributes to deleterious outcomes after lung transplantation. Although we have shown a unique protective role for eosinophils in both establishing and maintaining lung allograft tolerance, their role in IRI remains unclear. Based on previous research demonstrating a protective role for eosinophils in liver IRI, we hypothesized that they might play a similar function in the lung. In this study, we show that donor-, but not recipient-derived, eosinophils worsen injury of syngeneic and allogeneic lung grafts rather than protect from it. Eosinophils in the lungs stored in a low potassium dextran-based extracellular preservation solution become activated, degranulate, and die during rewarming. This damage correlates with decreased oxygenation and increased tissue injury upon reperfusion. In vitro studies confirm that eosinophils exposed to a clinically used low-potassium dextran-based preservation solution, but not to other preservation solutions, experience oxidative stress. Supplementing such a solution with the antioxidant glutathione, which is present in solutions used to preserve other solid organs, reduces activation and injury. While IRI has been attributed solely to graft damage mediated by recipient-derived cells entering the graft upon reperfusion, our new findings uncover a previously unrecognized role for donor-derived leukocytes in this process and open unexplored avenues to improve graft function.

Complement component 3 (C3) is crucial for host defense against bacteria. While the liver is the primary source of circulating C3, local C3 production at barrier surfaces such as the lung is key in early responses. Yet, how local complement-mediated responses are initiated at mucosal barriers is unknown. This study investigates the kinetics and necessity of lung-derived C3 during the initial hours of an infection. Using models of bacterial pneumonia in ex vivo-perfused human lungs and mice deficient in liver-derived C3, we demonstrate that intrapulmonary C3 production and activation precedes the accumulation of circulating C3 into the bronchoalveolar space. Utilizing mice deficient in lung-derived C3, we demonstrate that epithelial cell-derived C3 is required for early neutrophil recruitment in pneumonia. Transcriptomic and proteomic analyses reveal that neutrophil chemotactic pathways such as C5a and CXCL2 depend on lung epithelial cell-derived C3. These findings demonstrate how lung epithelial-derived C3 influences early mucosal responses to infection via both canonical (direct) and non-canonical (indirect) pathways.

OBJECTIVE:Lung transplantation is the definitive treatment for end-stage pulmonary disease, but ongoing challenges remain in long-term survival. We report our single-center experience of 2000 adult lung transplants over a nearly 35-year period and assess trends in patient demographics, intraoperative management, and perioperative and long-term outcomes. METHODS:We retrospectively reviewed 2000 lung transplants performed between 1988 and 2023 at our center. Recipients and donors were separated into 3 eras: Era 1 (1988-2000), Era 2 (2001-2011), and Era 3 (2012-2023). Recipient outcomes were compared among the eras. RESULTS:There were differences in recipient demographics across the eras. Over time, we have increasingly performed transplantation in patients with restrictive lung disease. Overall graft survival has improved, with median graft survival increasing from 5.5 years (Era 1) to 9.0 years (Era 3) (P < .0001). We observed similar trends when patients were stratified by transplant indication. The incidence of primary graft dysfunction grade 3 has remained stable at 28.7% in Era 2 and 26.7% in Era 3 (P = .4892). The median freedom from chronic lung allograft dysfunction has improved from 3.2 years (Era 2) to 3.4 years (Era 3) (P = .0001). CONCLUSIONS:Lung graft survival has improved over time at our institution due to advances in perioperative and long-term management. However, primary graft dysfunction grade 3 rates have not changed, and chronic lung allograft dysfunction is commonly diagnosed within the first 4 years after transplant. Further research is necessary to understand these disease processes and to generate new treatment strategies to address them.

Antibody-mediated rejection (AMR) is an increasingly recognized form of rejection and cause of graft failure after lung transplantation. AMR has been the focus of extensive research over the past decade. Despite growing awareness and recent advances in our understanding of AMR, outcomes remain dismal with a 2-year survival of only 20%. The International Society for Heart and Lung Transplantation convened a multidisciplinary workgroup of experts in AMR to review the most up-to-date research and clinical experience and to update the 2016 definition. The workgroup was divided into 9 subgroups covering a broad range of topics pertaining to AMR and used the modified Delphi method to synthesize a cohesive summary of the literature. A multidimensional definition was developed to enhance precision by reporting the specific presenting features. This Graft, Antibody, and Pathology (GAP) definition is based on the presence of Graft dysfunction, the presence and characteristics of Antibodies, and Pathological findings. The workgroup emphasized that identifying better treatments for AMR is a critical unmet need and proposed that a more precise definition might allow better management by providing a platform for testing and developing new therapies.

Proinflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor-α, and IL-6, have been linked to graft rejection, and protocols have been developed to target these in solid organ transplant recipients. Pathways regulating rejection and tolerance differ across organs and remain poorly understood in the lungs. Here, we used a mouse lung transplantation model to examine the role of inflammatory cytokines in graft acceptance. We found that recipient IL-1β drove systemic granulocyte colony-stimulating factor release early after transplantation and promoted allograft infiltration with neutrophils that express immunosuppressive gene signatures. IL-1β promoted inducible nitric oxide (NO) synthase expression in neutrophils and accumulation of NO in allografts, a pathway that is critical for the acceptance of transplanted lungs. We observed that human and mouse neutrophils from lung transplant recipients suppress T cell responses in an NO-dependent fashion. Finally, recipients deficient in Il1b, but not Tnfa or Il6, acutely rejected lung allografts despite receiving immunosuppression that results in tolerance when administered to wild-type hosts. Thus, contrary to the widely held notion that proinflammatory cytokines promote deleterious outcomes after transplantation, our findings uncovered a previously unknown role for recipient IL-1β as a critical mediator of pulmonary allograft acceptance. Our findings inform future studies developing lung-specific immunosuppressive strategies.

How pathogens inhibit transplant tolerance remains unclear. Here, we found that Pseudomonas aeruginosa infection, but not other common bacterial respiratory infections, increases antibody-mediated rejection (AMR) risk in recipients of lung transplants. To explore this relationship, we performed orthotopic lung transplants in mice, infected recipients with P. aeruginosa, and observed for the development of AMR. Intravital two-photon microscopy showed that P. aeruginosa rapidly invaded bronchial-associated lymphoid tissues, which resulted in acute lymphocytotoxicity, including the death of forkhead box P3 (Foxp3)+CD4+ T cells that are required to suppress AMR. P. aeruginosa-mediated AMR required expression of the type III secretion system (T3SS), which injects exotoxins into the cell cytoplasm. Through a combination of mutagenesis and epitope tagging experiments, we revealed that T3SS exotoxin T ADP ribosyl-transferase activity was sufficient for graft-resident Foxp3+CD4+ T cell apoptosis, leading to myeloid differentiation primary response 88 (Myd88)-dependent generation of T-box expressed in T cells (T-bet)- and C-X-C motif chemokine receptor 3 (CXCR3)-positive germinal center and memory B cells with high donor antigen avidity. We also found that T-bet+ and CXCR3+ B cells were elevated in biopsies from recipients of lung transplants who were diagnosed with AMR. In mice, CXCR3 deficiency restricted to B cells or CXCR3 blockade prevented AMR despite P. aeruginosa infection. Our work has identified a previously unrecognized role of bacterial virulence in lung allograft rejection and suggests potential strategies to prevent AMR for those at high risk of P. aeruginosa infection after transplant.

Tolerance after lung transplantation is associated with the induction of Foxp3+ regulatory T cell-enriched bronchus-associated lymphoid tissue, which suppresses local and systemic alloimmune responses. How this tolerogenic graft environment shapes responses to respiratory viral infections, a known contributor to adverse outcomes after lung transplantation, remains unknown. Using a mouse model of a seasonally circulating parainfluenza virus, we found that acute infection of tolerant lung allografts results in temporary reductions of both bronchus-associated lymphoid tissue size and abundance of graft-resident Foxp3+ cells but does not trigger rejection. At late time points, pathologic chronic type 2 inflammatory responses characteristic of severe parainfluenza virus infection decreased and Krt5+ and Krt8+ lesions were reduced in tolerant allografts when compared with infected native lungs or syngeneic grafts. This reduction in dysplastic alveolar regeneration in tolerant allografts was associated with an increase in amphiregulin-expressing Foxp3+ cells. Furthermore, type II alveolar epithelial cells in lung allografts upregulated genes related to injury when recipient Foxp3+ cells were deficient in amphiregulin. These findings shed new light on how immune pathways that are established in tolerant lung allografts, in addition to preventing rejection, protect against aberrant tissue repair after a clinically relevant respiratory viral infection.

Introduction:Many fundamental discoveries have occurred using primary cells from deceased donor lungs. These cells respond differently to injury when there are underlying co-morbidities like diabetes mellitus, hypertension, aging and exposures to cigarette smoke, cocaine and chronic alcohol use. However, the prevalence of these characteristics in donor lungs utilized for research is currently unknown. Methods:This retrospective cohort study procured data of lung transplant donors from Mid-America Transplant from January 2017 until July 2023. The donors were characterized based on lung utilization into three groups - lungs used for research, lungs used for transplant, and lungs not recovered from donors for either research or transplantation. Results:The mean age of donors whose lungs were utilized for research was 41±18 years. 25% of them were expanded criteria donors (ECD) while 10% of the donors in the transplant cohort were ECD. 14% of the donors whose lungs were utilized for research had history of diabetes compared to 8% of donors whose lungs were transplanted. A quarter of the research donor population had positive history of cigarette use within the preceding 20 years. At least 40% of donors had a positive history of non-intravenous drug use, of whom a majority had a history of continued non-intravenous drug use. Conclusions:No strict selection criteria or protocols exist when human donor lungs are obtained for ex-vivo research. There is a high prevalence of diabetes mellitus, history of smoking and non-intravenous drug use along with older age distribution in donors whose lungs used are for research.

Primary graft dysfunction (PGD) is a common complication after lung transplantation associated with poor outcomes. Although risk factors have been identified, the complex interactions between clinical variables affecting PGD risk are not well understood, which can complicate decisions about donor-lung acceptance. Previously, we developed a machine learning model to predict grade 3 PGD using donor and recipient electronic health record data, but it lacked granular information from donor-lung computed tomography (CT) scans, which are routinely assessed during offer review. In this study, we used a gated approach to determine optimal methods for analyzing donor-lung CT scans among patients receiving first-time, bilateral lung transplants at a single center over 10 years. We assessed 4 computer vision approaches and fused the best with electronic health record data at 3 points in the machine learning process. A total of 160 patients had donor-lung CT scans for analysis. The best imaging-only approach employed a 3D ResNet model, yielding median (interquar tile range) areas under the receiver operating characteristic and precision-recall curves of 0.63 (0.49-0.72) and 0.48 (0.35-0.6), respectively. Combining imaging with clinical data using late fusion provided the highest performance, with median areas under the receiver operating characteristic and precision-recall curves of 0.74 (0.59-0.85) and 0.61 (0.47-0.72), respectively.