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.

Heart failure and ischemic heart disease represent prevalent causes of death among cancer survivors. Despite extensive use of conventional chemotherapies, a limited understanding of how these agents affect the cardiac immune landscape exists. Using mouse models, we show that DNA-damaging agents selectively deplete cardiac-resident macrophages through activation of p53 signaling and resultant necroptosis and apoptosis. Genetic lineage tracing, transcriptomic profiling, and functional studies revealed that recruited monocytes progressively reconstitute the cardiac-resident macrophage compartment, were transcriptionally distinct from embryonic-derived cardiac-resident macrophages, and conferred protection from subsequent hypertensive and ischemic cardiac injury in mice. Monocyte-derived resident-like cardiac macrophages suppressed inflammation and attenuated adverse myocardial remodeling through a type I interferon-dependent mechanism. Collectively, these findings highlight unrecognized effects of DNA-damaging chemotherapies on the cardiac immune landscape and shed light on our understanding of monocyte plasticity and resident macrophage dynamics.

BACKGROUND:Although surgical resection is the standard of care for early-stage non-small cell lung cancer, frailty influences treatment decisions. We evaluated the prognostic utility of the Veterans Affairs Frailty Index, a claims-based assessment tool, among veterans undergoing resection for stage I non-small cell lung cancer. METHODS:We conducted a retrospective cohort study of veterans who underwent curative-intent surgery for stage I non-small cell lung cancer in the Veterans Health Administration from 2006 to 2020. Using the Veterans Affairs Frailty Index, frailty was categorized as follows: nonfrail (≤0.1), prefrail (0.1-0.2), mildly frail (0.2-0.3), moderately frail (0.3-0.4), and severely frail (>0.4). The primary outcome was overall survival, assessed using multivariable Cox regression. Secondary outcomes included major complications, prolonged hospitalization, 30-day readmission, 90-day mortality, and recurrence. RESULTS:Among 12,271 veterans, 7.7% were severely frail. Compared with nonfrail patients, severely frail patients were older (mean age 70.3 years vs 64.7 years; P < .001) and were more likely to undergo minimally invasive surgery (57.7% vs 37.8%) and sublobar resection (41.2% vs 22.3%) (all P < .001). Severe frailty was independently associated with higher risk of major complications (adjusted odds ratio 2.85, 95% confidence interval 2.18-3.71), prolonged hospitalization (adjusted odds ratio 2.67), 30-day readmission (adjusted odds ratio 1.76), 90-day mortality (adjusted odds ratio 2.87), and worse overall survival (adjusted hazard ratio 2.20, 95% confidence interval 1.97-2.46; all P < .001). Recurrence was not significantly associated with frailty (adjusted hazard ratio 0.87; P = .410). CONCLUSION:Frailty, as measured by the Veterans Affairs Frailty Index, independently predicts adverse postoperative and survival outcomes following resection for stage I non-small cell lung cancer. Preoperative frailty assessment may improve risk stratification and guide surgical decision making.

Inflammation contributes to the pathogenesis of myocardial infarction and heart failure and represents a viable therapeutic target. Monocytes and their progeny are highly abundant and display striking functional diversity, serving as key determinants of myocardial inflammation and tissue repair. Much remains to be learned regarding mechanisms and signaling events that instruct monocyte fate decisions. We devised a genetic lineage tracing strategy using Ccr2crERT2Rosa26LSL-tdTomato mice in combination with single cell RNA-seq to map the differentiation trajectories of monocytes that infiltrate the heart after reperfused myocardial infarction. Monocytes were recruited to the heart early after injury and gave rise to transcriptionally distinct and spatially restricted macrophage and dendritic cell-like subsets that were specified prior to extravasation and chronically persisted within the myocardium. Pseudotime analysis predicted 2 differentiation trajectories of monocyte-derived macrophages that are partitioned into the border and infarct zones, respectively. Among these trajectories, we demonstrated that macrophages expressing a type I interferon-responsive signature were an intermediate population that gave rise to MHC-IIhi macrophages, were localized within the border zone, induce regulatory T cells, and promote myocardial protection. Collectively, these data uncover complexities of monocyte differentiation in the infarcted heart and suggest that modulating monocyte fate decisions may have clinical implications.

Objectives Although the long-term prognosis after lung transplantation has improved recently, primary graft dysfunction (PGD) remains the major cause of early mortality. The aim of this study was to elucidate trends in PGD incidence and short-term mortality following lung transplantation in the contemporary era. Methods We analyzed a single-center database of lung transplantations performed across three periods (Era 1: 2009–2013, Era 2: 2014–2017, and Era 3: 2018–2021). PGD was graded according to the 2016 International Society for Heart and Lung Transplantation definition, and PGD grade 3 within T0–T72 was used as the primary outcome. Trends in PGD incidence, factors associated with PGD, and early mortality rates after lung transplantation were identified. Results This study included 856 lung transplants: 277 in Era 1, 296 in Era 2, and 283 in Era 3. PGD grade 3 incidence decreased significantly over time: 35.9% (99 cases) in Era 1, 26.4% (78 cases) in Era 2, and 18.4% (52 cases) in Era 3 (P<0.001). During the study period, the lung allocation score (LAS) and intraoperative cardiopulmonary bypass (CPB) use decreased, whereas the use of intraoperative nitric oxide and extracorporeal membrane oxygenation increased. Logistic multivariate modeling identified era, recipient sex (male), underlying disease, race, and blood transfusion as factors associated with PGD. No significant difference was observed in 30-day hospital mortality across the three eras (2.9%, 1.4%, and 1.4% for Era 1, Era 2, and Era 3, respectively; P=0.313). Conclusion This study demonstrated a significant reduction in PGD incidence over time, which coincided with a decrease in LAS and intraoperative CPB use. However, no significant changes were observed in short-term mortality after lung transplantation.

BACKGROUND:Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality in the United States, but survival outcomes are shifting with evolving therapies and screening. Recent findings suggest that survival improvements vary across health-care systems, with favorable trends observed in the Veterans Health Administration (VHA). We sought to compare NSCLC survival trends across US health systems. METHODS:We conducted a retrospective cohort study of patients with NSCLC (2007-2019) using data from the VHA and the National Cancer Database (NCDB), representing the non-VHA US general population. The primary exposure was health system (VHA vs non-VHA), and the primary outcome was 3-year all-cause mortality. Multivariable Cox models estimated annual adjusted overall survival (OS), with interaction terms for health system and stage. RESULTS:Among 1 463 996 patients (VHA n = 79 027; NCDB n = 1 38 4 969), adjusted 3-year OS increased from 24% to 51% (VHA) and from 24% to 41% (non-VHA) between 2007 and 2019. The VHA survival advantage persisted in analyses stratified by stage (all P-values <.0001) and when analyses were limited to NCDB patients with Medicare or private insurance (51% vs 42% in 2019; P < .0001). CONCLUSIONS:In this nationally representative study, 3-year OS among patients with NSCLC improved between 2007 and 2019, with larger and more rapid gains observed within the VHA compared with non-VHA settings. These findings suggest that lung cancer care delivered within an integrated, publicly funded system is associated with greater and more rapidly improving survival than care delivered across a heterogeneous mix of US health-care delivery settings represented in the NCDB.

BACKGROUND:Thoracic organ procurement procedures have been standardized for decades, and serious organ injury during procurement is considered rare. However, when injuries do occur, they may result in irreversible loss of transplantable organs. CASE SUMMARY:We report a rare case of unintentional donor heart injury caused by transdiaphragmatic liver core needle biopsy using a Tru-Cut-type device during multi-organ procurement. Two puncture injuries were identified on the inferior wall of the heart adjacent to the posterior descending coronary artery. Although there was no active bleeding and gross ventricular function appeared preserved, the proximity to a coronary branch raised concern for potential coronary injury and re-bleeding following systemic heparinization. After multidisciplinary discussion among the cardiac procurement team, recipient transplant team, and the organ procurement organization, the donor heart was declined intraoperatively. CONCLUSION:This case highlights a preventable mechanism of procurement-related cardiac injury and underscores the importance of coordination, timing, and situational awareness among procurement teams to minimize avoidable donor organ loss.

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.

BACKGROUND:Multiple studies have demonstrated excellent survival for patients with advanced non-small cell lung cancer who undergo induction chemoimmunotherapy followed by surgery. However, up to 22% of patients in these studies did not undergo surgical resection. National Veterans Health Administration data outlining the uptake, utilization, and dropout in this cohort are limited. We sought to describe utilization, non-progression to surgery, and outcomes in this cohort. METHODS:Using the United States Veterans Health Administration database, we identified patients with clinical stage (cStage) IB-III-N2 non-small cell lung cancer who underwent chemoimmunotherapy (2021-2024). All patients received at least 2 cycles CIO. Exclusion criteria included radiation during chemoimmunotherapy, clinical N3 disease, >4 cycles chemoimmunotherapy, or cStage IV disease. Clinicopathological characteristics and treatment patterns were described. Overall survival (OS) was estimated using the Kaplan-Meier method. RESULTS:In total, 86 patients met final inclusion criteria: 60 (69.8%) underwent surgery and 26 (30.2%) did not. The groups had similar cStage, age, and comorbidities. Among patients who did not undergo surgery, 46.2% developed distant metastatic disease during induction therapy. The 2-year OS was 80.2% for the entire cohort (86.7% surgical; 65.8% non-surgical). CONCLUSIONS:In our nationally representative cohort, patients undergoing chemoimmunotherapy with intent for surgical resection had favorable intermediate-term survival. however, nearly one-third of patients did not undergo surgery, most commonly due to disease progression. These findings highlight the need to better identify patients who do not progress to resection and define management strategies for these patients.

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.

OBJECTIVES:Phantom lymph node collection, defined as intraoperative sampling of tissue presumed to be nodal that, on pathologic review, proves to be non-nodal, is a previously understudied outcome in thoracic surgery. This study aimed to quantify its incidence, identify associated factors, and assess its relationship with cancer-specific outcomes. METHODS:We conducted a retrospective cohort study using a meticulously curated Veterans Health Administration dataset of patients undergoing curative-intent non-small cell lung cancer resection between 2018 and 2024. Our primary outcomes included frequency of phantom lymph node collection, along with covariates independently associated with it. Secondary outcomes were overall survival and recurrence-free survival. RESULTS:A total of 2972 veterans were included in this cohort. Phantom lymph node collection occurred in 327 (11.0%) patients. On multivariable analysis, phantom lymph node collection was more likely with lower-lobe tumors (odds ratio, 1.45; 95% CI, 1.13-1.88), and less likely among patients who received guideline-concordant nodal sampling (≥3 N2 and ≥1 N1 nodal stations) (odds ratio, 0.56; 95% CI, 0.43-0.74). Notably, phantom lymph node collection was not associated with overall (hazard ratio, 0.96; 95% CI, 0.77-1.19) or recurrence-free survival (hazard ratio, 0.86; 95% CI, 0.68-1.08). CONCLUSIONS:Phantom lymph node collection is common during curative-intent resection of early-stage lung cancer. Although not prognostic, its association with guideline-concordant sampling highlights it as a potential target for surgical quality improvement initiatives.

There is a critical need for the development of noninvasive biomarkers of acute cellular rejection (ACR) in lung transplantation. We hypothesized that ACR induces changes in small extracellular vesicle (sEV) secretory output of T cells into the peripheral circulation, thereby providing a noninvasive method of diagnosis. First, we investigated this hypothesis in a mouse lung transplant model. In line with dense infiltration of lung allograft by T cells during ACR, peripheral blood total T cell and CD8+ T cell counts, as well as T cell sEV RNA cargoes (Cd3, Cd8, Ifng, Tcrb, and microRNA (Mir)21a) were significantly upregulated in mice receiving allogeneic compared with syngeneic lungs at the postoperative day 7 time point. Second, translating these findings to clinical lung transplantation, we analyzed circulating T cell sEV RNA cargoes with time-matched surveillance transbronchial allograft biopsies in 20 patients. In 8 patients with >A1 ACR, 8 candidate RNA biomarkers (CD8, TCR, IFNG, HLA-DRA, CD38, MIR21, MIRLET7I, MIR101) were significantly upregulated compared with 12 patients without ACR. Collectively, these findings support further investigation of T cell sEVs as a novel biomarker for ACR diagnosis in lung transplantation.

Aging strongly impacts CD8+ T cells, including the loss of naive cells and the emergence of age-associated GZMK+CD8+ T cells (TAA cells). Although TAA cells constitute a major population in aged mice, the pathway underlying their differentiation remains unknown. Here, we demonstrate that TAA cell development is cell extrinsic and requires antigen exposure within aged non-lymphoid tissues. Using a TNFΔ69AU/+ mouse model, we show that low-grade inflammation accelerates CD8+ T cell aging and promotes early accumulation of TAA cells. Analysis of TAA cell heterogeneity further identifies a progenitor subpopulation enriched in the aged adipose tissue. Finally, heterochronic transplantation experiments suggest that the aged adipose tissue can serve as a systemic source of TAA cells and contribute to the conversion of young CD8+ T cells into the aged phenotype. Together, these findings indicate that aged non-lymphoid tissues actively drive CD8+ T cell remodeling and identify adipose tissue as an important niche shaping immune aging.