BACKGROUND:Lung transplantation represents a potential life-extending therapy for patients with advanced CTD-ILD. This study aims to characterize lung transplant listing outcomes among CTD-ILD patients over a 20-year period using the Organ Procurement and Transplantation Network (OPTN) national database. METHODS:Data analyzed from the OPTN between 2003-2023 included adults ≥18 years of age with CTD-ILD listed for lung transplantation. Patients were categorized into six diagnoses: scleroderma, lupus, rheumatoid arthritis (RA), myositis, Sjögren's, and "Other" (including mixed connective tissue disease, CTD, etc.). Disease and patient specific data were obtained. Trends in listing and outcomes were analyzed in four time periods across the 20 years and among various diagnoses. We used descriptive summary statistics to characterize the sample, and univariate and multivariable logistic regression models to identify factors associated with undergoing lung transplantation. RESULTS:A total of 1977 CTD-ILD patients were listed. Scleroderma constituted the majority (47%). Listings increased fourfold (185 to 744) from the first to last time periods. Listings for all diagnoses increased with time, with rising representation of non-White patients. Trend noted towards listing patients with more advanced lung disease with time. Transplant rates rose, while wait times, and waitlist mortality declined overtime. All diseases received transplants at comparable rates. Older age, lower lung allocation scores, and male sex were associated with higher odds of transplantation, female sex with lower odds. CONCLUSION:Over two decades, CTD-ILD transplant listings have increased in volume, matched with substantially improved outcomes. This reflects the evolution of listing practices and a growing confidence in lung transplantation as a viable option for CTD-ILD.

Lung tissue-resident CD8+ T cells facilitate viral clearance and protective immunity to influenza viruses in animal models. Their role during acute human infection is not clear. Here we use bronchoalveolar lavage samples collected from human subjects naturally infected with influenza B virus to show that influenza-specific CD8+ T cells are expanded in the lower airways during acute infection and target only a few epitopes from phylogenetically conserved internal influenza virus proteins. The lower airway influenza-specific CD8+ T-cell immunodominance hierarchy is different from the hierarchy observed in matched blood samples. Transcriptional and protein-level analyses using HLA class I tetramers reveal a tissue-resident profile and less expression of cytotoxic effector molecules in lower airway influenza-specific CD8+ T cells. Collectively, our data show that high-frequency influenza-specific CD8+ T cells with a tissue-resident phenotype are found in the lower airways during viral clearance. These cells recognize a handful of conserved viral epitopes and exhibit a functional phenotype different from cells found in blood. These cells may play a role in controlling human influenza infection.

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.

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.

The availability and structure of donor care units (DCUs) at the Organ Procurement Organization (OPO) level may influence lung utilization and transplant outcomes. Adult first-time lung transplant recipients and donors with lung disposition data from January 1, 2018, through August 31, 2025, were abstracted from the Scientific Registry of Transplant Recipients database. Mixed-effects logistic regression and frailty-adjusted Cox models were performed to examine lung utilization and graft survival, respectively, across no DCU available (DCU-negative), hospital-based DCU available (DCU-hospital available), and independent DCU available (DCU-independent available) groups. An OPO performance visualization tool was developed (http://opolung2025.com/). Among 108 502 donors, lung utilization increased stepwise by available DCU structure. Donation after brain death donors recovered by OPOs with DCU-hospital available (odds ratio = 1.11; 95% confidence interval (CI), 1.01-1.22) and DCU-independent available (odds ratio = 1.21; 95% CI, 1.08-1.36) had higher odds of utilization than donation after brain death donors recovered by DCU-negative OPOs, whereas no significant DCU association was observed among donation after circulatory death donors. Among 18 271 lung transplant recipients, graft survival did not differ between DCU-positive and DCU-negative groups (hazard ratio = 1.03; 95% CI, 0.96-1.11) or between DCU-independent-available and DCU-hospital-available groups within the DCU-positive cohort (hazard ratio = 1.00; 95% CI, 0.87-1.15). DCU availability was associated with higher donation after brain death donor lung utilization but not recipient graft survival.

Donor-derived cell-free DNA (dd-cfDNA) is a validated, highly sensitive, plasma molecular biomarker of allograft injury after solid organ transplantation. Robust experiences with dd-cfDNA testing after kidney and heart transplantation have generated interest in this biomarker within the lung transplantation (LTx) community. A growing body of evidence now provides increased insight into dd-cfDNA utility for molecular monitoring of lung allograft health after transplantation. The expanding understanding of lung allograft injury to appropriately frame the advancing role of dd-cfDNA in the evolution of the diagnostic approach after LTx is described. Performance characteristics of both laboratory-based shotgun-sequenced testing from the Genome Transplant Dynamics (GTD) and Genomic Research Alliance for Transplantation (GRAfT) consortia, as well as commercially available central lab-based algorithmic next-generation sequenced dd-cfDNA tests for lung transplant recipients (LTR) (AlloSure, CareDx and Prospera, Natera) are described. Kinetics of dd-cfDNA in LTRs over time, in multiple different clinical scenarios, from several investigator groups are aggregated. Phenotypes of lung allograft injury, such as acute lung allograft dysfunction, and associated dd-cfDNA patterns and performance are identified in alignment with established definitions and evolving molecular injury insights. Certain patterns of molecular injury that may predict long-term outcomes including chronic lung allograft dysfunction and mortality are examined. Lastly, clinical approaches to testing and interpretation of dd-cfDNA results in LTRs, a practical approach to using dd-cfDNA, and a rational framework for interpreting dd-cfDNA results in LTRs are presented.

OBJECTIVE:The use of lungs from brain-dead donors is low partly as a result of the lack of reliable donor assessment criteria. The validated Lung Donor (LUNDON) score predicts lung acceptance for transplantation by using 9 clinically relevant variables, including the presence of an abnormality on radiograph of the chest. Because most organ donor evaluations now include routine computed tomography (CT) of the chest, we aimed to assess whether the addition of CT findings impacts the LUNDON model's performance. METHODS:Data including CT findings were collected for adult brain-dead donors from 3 organ procurement organizations from 2014 to 2020. The primary outcome was lung acceptance for transplantation. We collated all CT findings into a weighted CT composite score, with greater scores representing more CT abnormalities, and calculated the score for each donor. RESULTS:The lung acceptance rate was 40.4% among 2454 donors with CTs of the chest and 22.3% among 1980 donors without CTs of the chest. Emphysema, pulmonary edema, and traumatic lung injury on CT were associated with a lower likelihood lung acceptance. The LUNDON model's performance was comparable between use of the original radiograph of the chest variable, the CT composite score, or both variables together (C-statistics 0.883, 0.887, 0.890, respectively). All 3 iterations of the model were predictive of 1-year graft survival. CONCLUSIONS:Undergoing CT was independently associated with donor lung acceptance. The incorporation of highly granular findings from CT of the chest to the previously established LUNDON model maintained, but did not meaningfully improve, its excellent baseline ability to predict lung use and its association with graft survival.

IL-33 is a key driver of type 2 inflammation and implicated in pathology of chronic obstructive pulmonary disease (COPD) and asthma. However, the mechanism for IL-33 secretion and regulation in the context of chronic airway disease is poorly understood. We previously reported an airway disease-associated isoform IL-33Δ34 that escapes nuclear sequestration and is tonically secreted from epithelial cells. Here, we describe how this IL-33Δ34 isoform interacts with HSP70 within cells and is targeted to secretory organelles through coordinated binding to phosphatidylserine (PS) and delivered to compartments for unconventional protein secretion (CUPS). Once secreted, extracellular HSP70 (eHSP70) in complex with IL-33Δ34 stabilizes the cytokine by inhibiting oxidation and degradation, which results in enhanced IL-33Δ34-receptor binding and activity. We further find evidence that IL-33 along with mediators of the proteostasis network HSP70, HSP90, and the Chaperonin Containing TCP1 (CCT) complex are dysregulated in human chronic airway disease. This phenomenon is reflected in the differential extracellular vesicle (EV) proteome in bronchial wash from COPD and asthma samples, which could mark disease activity and potentiate IL-33 function. This study confirms proteostasis intermediates, chiefly HSP70, as chaperones for noncanonical IL-33 secretion and activity that may be amenable for therapeutic targeting in the chronic airway diseases COPD and asthma.

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.

RATIONALE:IL-33 is a key driver of type 2 inflammation relevant to airway epithelial biology. However, the mechanisms for IL-33 secretion and regulation in the context of chronic airway disease is poorly understood. OBJECTIVES:We sought to define how a disease associated isoform IL-33d34 that escapes nuclear sequestration and is tonically secreted from epithelial cells can be recruited to non-canonical secretory pathways. METHODS:IL-33d34 interaction with HSP70 was assessed and validated by affinity purification, mass-spectrometry and miniTurboID proximity labeling. Secretion and activity reporter assays were used to probe the effect of HSP70 on epithelial IL-33d34 secretion and receptor binding. Human airway disease biospecimens were analyzed for dysregulation of heat shock pathways revealing modulation of TCP1 complex intermediates. MEASUREMENTS AND MAIN RESULTS:We confirmed that HSP70 interacts directly with IL-33d34, recruits the cytokine to a vesicular compartment and enhances stability upon secretion. IL-33, HSP70 and other key mediators of proteostasis were found to be dysregulated in airway disease biospecimens and secreted extracellular vesicles. The IL-33d34 interactome was characterized and novel secretion modulators were identified. CONCLUSIONS:This study confirms a role for HSP70 in non-canonical IL-33d34 secretion and function that may be amenable for therapeutic targeting in airway diseases.

Rationale: IL-33 is a key driver of type 2 inflammation and relevant to epithelial biology. However, the mechanisms for IL-33 secretion and regulation in the context of chronic airway disease is still not understood. Objectives: We sought to define how a disease associated IL-33 isoform that escapes nuclear sequestration and is continuously secreted from epithelial cells is co-opted by a non-canonical secretion mechanism driven by HSP70. Methods: IL-33Δ34 and HSP70 interaction was assessed by affinity purification, mass-spec, miniTurboID proximity labeling. ELISA assays were used to probe the effect of HSP70 inhibition/activation on IL-33Δ34 secretion as well as receptor binding. Measurements and Main Results: We confirmed that HSP70 binds to IL-33Δ34 within and outside of the cell. In the cytoplasm, HSP70 facilitates IL-33Δ34 recruitment to a vesicular compartment for secretion while outside of the cell it protects it from inactivation by oxidation. IL-33, HSP70 and other key mediators of proteo-stasis in human lung tissue were measured by RT-qPCR. COPD EV proteins and the IL-33Δ34 interactome were identified by mass spectrometry. Conclusions: This study confirms a non-canonical role for HSP70 in IL-33Δ34 secretion and function that may be amenable for therapeutic targeting. Key Words: IL-33; Type 2 inflammation; HSP70; Extracellular Vesicles; ST2; RAGE; COPD

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.

BACKGROUND:The spirometric response to standard-of-care (SOC) immunosuppressive therapy for the management of bronchiolitis obliterans syndrome (BOS) has been sparsely reported in the literature. Data from a Medicare-approved Registry were analyzed to characterize the effectiveness/durability of a wide range of SOC interventions to manage the decline of lung function and to validate the study spirometric criteria for initiation of rescue therapy. METHODS:Lung transplant recipients with refractory BOS at 21 US collaborating centers were enrolled in the Registry. Data included both nonspirometric (eg, demographic, Immunosuppressive Regimens for management of BOS) and spirometric parameters (ie, forced expiratory volume in 1 s [FEV 1 ] measurements and derived indices). The utility of study FEV 1 criteria for treatment (ie, statistically significant rate of FEV 1 decline >30 mL/mo) was evaluated by comparing the spirometric course between participants who met or did not meet this criterion. RESULTS:Only 21% of participants treated with SOC therapy had >50% decrease (76 ± 25% decrease) in the rate of FEV 1 decline. Although 51% of participants had a partial response (rate of FEV 1 decline decreased on average 71%), 49% of participants had a substantial increase (mean increase 224%). The FEV 1 criterion for treatment was able to identify 19% of participants (48/258) who achieved durable stabilization (ie, nonsignificant rate of FEV 1 <30 mL/mo) with SOC therapy. CONCLUSIONS:Patients with BOS have a widely variable response to SOC therapy. Our findings support the use of FEV 1 rate of decline to assess response to SOC therapy and to ensure appropriate assignment of participants with refractory BOS to rescue therapy treatment cohorts.

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.

A greater understanding of chronic lung allograft dysfunction (CLAD) pathobiology, the primary cause of death after lung transplantation (LTx), is needed to improve outcomes. The complement system links innate to adaptive immune responses and is activated early after lung transplantation to form C3 convertase, a critical enzyme that cleaves the central complement component C3. We hypothesized that LTx recipients with a genetic predisposition to enhanced complement activation have worse CLAD-free survival mediated through increased adaptive alloimmunity. We interrogated a known functional C3 polymorphism (C3 R102G) that increases complement activation through impaired C3 convertase inactivation in 2 independent LTx recipient cohorts. C3 R102G, identified in at least 1 of 3 LTx recipients, was associated with worse CLAD-free survival, particularly in the subset of recipients who developed donor-specific antibodies (DSAs). In a mouse orthotopic LTx model, impaired recipient complement regulation led to B cell-dependent CLAD pathology despite moderate differences in graft-infiltrating effector T cells. Dysfunctional complement regulation promoted intragraft accumulation of memory B cells and Ab-secreting cells, leading to increased local and circulating DSA levels in mice. In summary, genetic predisposition to complement activation is associated with an increased humoral response and worse CLAD-free survival.