Severe antibody-mediated rejection (AMR) is associated with chronic lung allograft dysfunction (CLAD) and death in lung transplant recipients. However, AMR diagnostics are imprecise, and new biomarkers are needed. We assessed whether airway inflammation gene signatures could distinguish AMR cases from controls. We analyzed small airway brush RNA sequencing from 16 AMR cases and 39 controls across 2 centers. We compared gene signatures for complement activation, molecular target of rapamycin (mTOR) signaling, and natural killer cell-mediated injury. Differential gene expression and pathway analyses sought AMR molecular features. Additionally, we evaluated the airway inflammation 2 (AI2) score, previously associated with CLAD and graft failure, in relation to AMR clinical features and survival outcomes. AMR airway brushes demonstrated transcriptional evidence of airway inflammation and upregulation of complement and natural killer cell pathways. The AI2 score was significantly elevated in AMR cases (P < .001) and was associated with AMR-compatible histology, donor-specific antibodies, complement binding, and acute graft dysfunction. Increasing AI2 scores were associated with worse retransplant-free survival, independent of other AMR features. In small airways, lung transplant AMR predominantly exhibits molecular features of cellular rejection rather than a distinct humoral rejection profile. Airway brush transcriptomics may provide a valuable tool for characterizing and prognosticating suspected AMR.

Chronic lung allograft dysfunction (CLAD) is the major barrier for long-term survival in lung transplant recipients (LTRs). CLAD remains a diagnosis of exclusion with poor responses to therapies. A molecular diagnostic for CLAD is needed to risk-stratify LTRs for prognosis and identify new targets to mitigate CLAD progression. We used weighted gene correlation network analysis on the airway brush-derived airway transcriptome to identify immune pathways and markers relevant to CLAD. Weighted gene correlation network analysis was performed on RNA sequencing from airway brushings of 37 LTRs with CLAD compared with 37 stable LTRs. We analyzed gene coexpression networks (modules) for their biological significance and association with CLAD. Three gene modules were positively correlated with CLAD, its severity, allograft dysfunction, and survival. These enriched components of the acute phase response, type 1 adaptive immunity, and innate immunity, respectively. A fourth module correlated with protection and was inversely correlated with the other modules. We validated our findings by identification of downstream protein and eicosanoid levels in the bronchoalveolar lavage, and an external validation cohort where module expression differentiated LTRs with CLAD and correlated with worse survival. The CLAD airway transcriptome enriches for coexpression networks associated with network modules that correlate with allograft dysfunction and 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.

As we have previously shown, idiopathic pulmonary fibrosis lung transplant recipients (IPF-LTRs) with short-telomere length are prone to develop significant cytopenias and poor tolerance to cell-cycle inhibitors, specifically mycophenolate mofetil (MMF), post transplant. We investigated the use of belatacept as an alternative immunosuppressive agent in a prospective, open-label cohort of 9 ST-IPF-LTRs at our institution. These patients were either challenged with MMF (majority) or immediately started on belatacept post transplant with the goal to bridge to everolimus, an mammalian target of rapamycin inhibitor that is commonly used post transplant. We describe outcomes in the first-year post transplant, including the incidence of acute cellular rejection, Epstein-Barr virus viremia, and 1 case of post-transplant lymphoproliferative disorder (PTLD) at 13 months. The use of belatacept postlung transplant may be an acceptable short-term alternative therapy to cell-cycle inhibitors in ST-IPF-LTRs with cytopenias but may lead to a higher risk of Epstein-Barr Virus viremia and PTLD when belatacept is used long term in these patients.

We examined innate and antibody responses in C3PO clinical trial participants of coronavirus disease 2019 (COVID-19) convalescent plasma to identify predictors of disease progression. We found severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viremia in 64% of participants at enrollment, and we could also quantify viremia in approximately half of those samples using an RT-PCR assay. Viremia was associated with increased risk of disease progression (OR, 3.0; 95% CI, 1.7-5.0). Participants with viremia at baseline had lower SARS-CoV-2 binding antibody levels and higher proinflammatory cytokine levels, including IP-10 (CXCL10), TNF-α, calprotectin, and CRP. Disease progression correlated with extracellular vesicle levels from multiple cell types in the convalescent but not acute phase of the disease. Male sex predicted worse disease outcome and was associated with higher baseline levels of several proinflammatory cytokines. Viremia's strong predictive value for disease progression argues for further study of its use to predict which patients with COVID-19 might require more intensive therapy or monitoring.

Introduction: We have recently shown that short telomere length (STL) are common in pulmonary fibrosis lung transplant recipients (STLPF-LTRs). Patients with STL are prone to develop significant cytopenias and poor tolerance to cell cycle inhibitors, specifically Mycophenolate mofetil (MMF), post-transplant. We investigated the use of Belatacept as an alternative immunosuppressant agent in a retrospective cohort of 9 STLPF-LTRs. Methods: Post-transplant data were extracted on a retrospective cohort of 9 STLPF-LTRs (median age 54 respectively). Lymphocyte and granulocyte TL were measured using flow cytometry and fluorescence in-situ hybridization (flowFISH). Genetic assessment was done using Whole Genome Sequencing or a targeted sequence panel. Categorical variables were compared using chi-square test. Results: A small cohort of 9 STLPF-LTRs who underwent double lung transplant between 2018 and 2024 was identified retrospectively at our institution, in which flowFISH revealed that all 9 STLPF-LTRs had short-TL. In lymphocytes, 44% (4/9) had TL <1st percentile, 22% (2/9) at the 1st percentile and 33% (3/9) between the 1st-10th percentile. Granulocytes were measured in 8/9 with 88% (7/8) <1st percentile and 1/8 (11%) between the 1st-10th percentile. 7 of the 9 (77%) underwent genetic characterization for the telomere maintenance genes with 5/7 (71%) having a rare gene variant: 2 in RTEL1, 2 in TERT and 1 in TINF2. This entire cohort received Belatacept post-transplant as an alternative to cell cycle inhibitor, MMF. These patients were either challenged with a cell cycle inhibitor or immediately started on Belatacept post-transplant. Only one STLPF-LTR was not challenged with MMF due to myelodysplastic syndrome diagnosed pre-lung transplant, while 8 received MMF the day after transplant as standard of care. The median time of stopping MMF for cytopenia's was 4 days post-transplant. The 8 that failed the MMF challenge, started Belatacept with the goal to bridge to Everolimus, an mTOR inhibitor, commonly used post lung transplant. 63% (5/8) were successfully transitioned to Everolimus with median time of Belatecept use being 8 months, while 38% continued Belatacept as part of there IS regimens, along with a calcineurin inhibitor and steroid. Two STLPF-LTRs required discontinuation of Belatacept due to development of low level EBV viremia. EBV and CMV PCRs are monitored weekly. There was no incidence of post-transplant lymphoproliferative disorder in this cohort. Conclusions: The use of Belatacept post-lung transplant may be a well-tolerated alternative to a cell cycle inhibitor in STLPF-LTRs who develop, or at risk to develop post-transplant cytopenias.

PURPOSE OF REVIEW:Median survival after lung transplantation is 5.7 years, which lags behind other solid organ transplants, such as heart, liver, and kidney. The major barrier to long-term survival in lung transplant recipients is chronic lung allograft dysfunction (CLAD). This review discusses the challenge of CLAD as a barrier to tolerance and identifies key areas in the field that require further development. RECENT FINDINGS:CLAD is a heterogenous disease in its kinetics of onset and severity and remains a clinical diagnosis of exclusion, based on a decline in allograft function. While acute cellular rejection and antibody-mediated rejection are major risk-factors for CLAD, other barriers to long-term allograft acceptance are aspiration and primary graft dysfunction. However infections, particularly respiratory viral infections and Cytomegalovirus (CMV) remain the most significant risks for CLAD. Additionally, the lung transplant field is limited by a lack of molecular diagnostic assays for CLAD. Further, new targets are needed for precision immunosuppression, and more studies are needed to develop novel interventions to extend allograft acceptance. SUMMARY:This review discusses new lines of study to address important unmet needs necessary to extend lung allograft acceptance. Other studies, such as tandem lung transplant and bone marrow transplant in select patients with primary immunodeficiency may provide additional lessons on how to potentially establish tolerance. However, tolerance in lung transplant is extremely rare, and further studies are needed to pursue this ultimate goal.

Background Lung transplantation is commonly required for advanced lung disease in cystic fibrosis (CF). Long-term lung allograft survival is limited primarily by chronic lung allograft dysfunction (CLAD), and microbial factors have been implicated in CLAD development. However studies have not specifically investigated CF patients despite the unique microbe-rich nature of the CF respiratory tract. We investigated whether early post-transplantation lung microbiome features associate with CLAD development. Methods We investigated a longitudinal cohort of 23 CF patients undergoing lung transplantation. Lung lavage was collected from donor lungs, and from recipient allografts serially during the first year post-transplantation. Patients were followed for a median of 4.9 years. This was complemented by a case-control study of 8 CF patients sampled at incident CLAD along with non-CLAD CF transplant controls. Lung bacteria were enumerated by 16S rRNA gene sequencing and quantified by qPCR, and immune mediators investigated by multiplex assay. Results Cohort patients who developed CLAD had lower lung bacterial burden, lower relative abundances of classic CF lung microbiota, and lower mediator levels during the first-year post-transplantation than those remaining CLAD-free. In contrast, incident CLAD showed elevated lung immune mediators but no microbiome differences. Conclusions Low lung bacterial content and immune mediators during the first year post-transplantation for CF associate CLAD, whereas CLAD onset is characterized by elevated immune mediators but no lung microbiome differences. Whether airway bacteria early after transplantation for CF may protect against CLAD or serve as a biomarker merits further study.

Chronic lung allograft dysfunction (CLAD) substantially limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and nondiseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of CD8+ tissue-resident memory T cells (TRMs) with high cytotoxic potential, including upregulation of KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRMs accumulated around the CLAD airways and had a 100-fold increase in clonal overlap with lung-draining lymph nodes when compared with non-CLAD lungs. Using a murine model of orthotopic lung transplantation, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplantation alone. Furthermore, blocking NKG2D in vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell- mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ + T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ + T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-g) g ) responses along with elevated CD4+NKG7+ + NKG7 + and CD4+CD107a+ + CD107a + responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ + T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ + T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ + NKG7 + T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection. The Journal of Immunology, , 2024, 213: 898-905.- 905.

BACKGROUND:Primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after lung transplantation. Accurate prediction of PGD risk could inform donor approaches and perioperative care planning. We sought to develop a clinically useful, generalizable PGD prediction model to aid in transplant decision-making. METHODS:We derived a predictive model in a prospective cohort study of subjects from 2012 to 2018, followed by a single-center external validation. We used regularized (lasso) logistic regression to evaluate the predictive ability of clinically available PGD predictors and developed a user interface for clinical application. Using decision curve analysis, we quantified the net benefit of the model across a range of PGD risk thresholds and assessed model calibration and discrimination. RESULTS:The PGD predictive model included distance from donor hospital to recipient transplant center, recipient age, predicted total lung capacity, lung allocation score (LAS), body mass index, pulmonary artery mean pressure, sex, and indication for transplant; donor age, sex, mechanism of death, and donor smoking status; and interaction terms for LAS and donor distance. The interface allows for real-time assessment of PGD risk for any donor/recipient combination. The model offers decision-making net benefit in the PGD risk range of 10% to 75% in the derivation centers and 2% to 10% in the validation cohort, a range incorporating the incidence in that cohort. CONCLUSION:We developed a clinically useful PGD predictive algorithm across a range of PGD risk thresholds to support transplant decision-making, posttransplant care, and enrich samples for PGD treatment trials.

BackgroundCirculating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury. In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction (PGD) and graft rejection after lung transplant.MethodsA total of 40 subjects undergoing de-novo lung transplants at our institution were recruited in this study. Blood samples were collected at various time points before and after lung transplant for 1 year. Dd-cfDNA in samples was determined using AlloSure assay (CareDx Inc.). The correlation of the value of %dd-cfDNA was investigated with the incidence of PGD, acute cellular rejection (ACR), and donor-specific antibody.ResultsWe observed a rapid increase of %dd-cfDNA in the blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks. The peak was observed within 72 h after transplantation. The peak values of %dd-cfDNA varied among subjects and did not correlate with severe PGD incidence. We observed an association between levels of %dd-cfDNA from blood collected at the time of transbronchial biopsy and the histological diagnosis of ACR at 3 weeks.ConclusionOur data show that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD. Plasma levels of dd-cfDNA may be a less invasive tool to estimate graft rejection after lung transplantation however larger studies are still necessary to better identify thresholds.