Small airway brush gene expression predicts chronic lung allograft dysfunction and mortality.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) limits survival following lung transplant, but substantial lung damage occurs before diagnosis by traditional methods. We hypothesized that small airway gene expression patterns could identify CLAD risk before spirometric diagnosis and predict subsequent graft failure. METHODS: Candidate genes from 4 rejection-associated transcript sets were assessed for associations with CLAD or graft failure in a derivation cohort of 156 small airway brushes from 45 CLAD cases and 37 time-matched controls with >1-year stable lung function. Candidate genes not associated with CLAD and time to graft failure were excluded, yielding the Airway Inflammation 2 (AI2) gene set. Area under the receiver operating curve (AUC) for CLAD and competing risks of death or graft failure were assessed in an independent validation cohort of 37 CLAD cases and 37 controls. RESULTS: Thirty-two candidate genes were associated with CLAD and graft failure, comprising the AI2 score, which clustered into 3 subcomponents. The AI2 score identified CLAD before its onset, in early and late post-CLAD brushes, as well as in the validation cohort (AUC 0.69-0.88). The AI2 score association with CLAD was independent of positive microbiology, CLAD stage, or CLAD subtype. However, transcripts most associated with CLAD evolved over time from CLAD onset. The AI2 score predicted time to graft failure and retransplant-free survival in both cohorts (p ≤ 0.03). CONCLUSIONS: This airway inflammation gene score is associated with CLAD development, graft failure, and death. Future studies defining the molecular heterogeneity of airway inflammation could lead to endotype-targeted therapies.

CD94+ natural killer cells potentiate pulmonary ischaemia-reperfusion injury.

BACKGROUND: Pulmonary ischaemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centred natural killer (NK) cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. METHODS: We hypothesised that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. RESULTS: We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing, respectively. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. CONCLUSIONS: Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.

Innate and adaptive effector immune drivers of cytomegalovirus disease in lung transplantation: a double-edged sword.

Up to 90% of the global population has been infected with cytomegalovirus (CMV), a herpesvirus that remains latent for the lifetime of the host and drives immune dysregulation. CMV is a critical risk factor for poor outcomes after solid organ transplant, though lung transplant recipients (LTR) carry the highest risk of CMV infection, and CMV-associated comorbidities compared to recipients of other solid organ transplants. Despite potent antivirals, CMV remains a significant driver of chronic lung allograft dysfunction (CLAD), re-transplantation, and death. Moreover, the extended utilization of CMV antiviral prophylaxis is not without adverse effects, often necessitating treatment discontinuation. Thus, there is a critical need to understand the immune response to CMV after lung transplantation. This review identifies key elements of each arm of the CMV immune response and highlights implications for lung allograft tolerance and injury. Specific attention is paid to cellular subsets of adaptive and innate immune cells that are important in the lung during CMV infection and reactivation. The concept of heterologous immune responses is reviewed in depth, including how they form and how they may drive tissue- and allograft-specific immunity. Other important objectives of this review are to detail the emerging role of NK cells in CMV-related outcomes, in addition to discussing perturbations in CMV immune function stemming from pre-existing lung disease. Finally, this review identifies potential mechanisms whereby CMV-directed treatments may alter the cellular immune response within the allograft.

Comparative analysis of patellar tendon, achilles tendon and plantar fascia structure in indoor and outdoor football players: a novel cross-sectional pilot study.

Different sport modalities were associate with tendon adaptation or even tendon disturbances, such as volleyball, soccer or basketball. Purpose: the aim of the present study was to determine de difference between indoor and outdoor football players on patellar tendon (PT), Achilles tendon (AT), plantar fascia (FP) and Hoffa's fat pad thickness assessed with ultrasound imaging (USI). A cross-sectional study was developed with a total sample of 30 soccer players divided in two groups: outdoor group (n = 15) and indoor group (n = 15). The thickness of PT, AT, PF and Hoffa's fat pad has been assessed with USI. Hoffa's fat pad reported significant differences for the left side between groups (P = 0.026). The rest of variables did not show any significant difference (P < 0.05). The ultrasonography assessment of the thickness of the PT, AT and PF did not show differences between outdoor and indoor football players. Hoffa's fat pad resulted showed a significant decrease for outdoor soccer players with respect futsal players. Thus, it can be considered that the load stimuli received in both soccer players were not enough to produce structural adaptations in PT, AT and PF tissues.

Macrophage and CD8 T cell discordance are associated with acute lung allograft dysfunction progression.

BACKGROUND: Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. METHODS: We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. RESULTS: BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p < 0.05 for each). Recipients with discordance between these cells had a nearly 5-fold increased risk of severe graft dysfunction or death (HR 4.6, 95% CI 1.1-19.2, adjusted p = 0.03). We validated these key findings in 2 public lung transplant genomic datasets. CONCLUSIONS: BAL anti-inflammatory macrophages may protect against CLAD by suppressing CD8 T cells. These populations merit functional and longitudinal assessment in additional cohorts.

Disturbed sleep after lung transplantation is associated with worse patient-reported outcomes and chronic lung allograft dysfunction.

Many lung transplant recipients fail to derive the expected improvements in functioning, HRQL, or long-term survival. Sleep may represent an important, albeit rarely examined, factor influencing lung transplant outcomes. Within a larger cohort study, 141 lung transplant recipients completed the Medical Outcomes Study (MOS) Sleep Scale along with a broader survey of patient-reported outcome (PRO) measures and frailty assessment. MOS Sleep yields the Sleep Problems Index (SPI); we also derived an insomnia-specific subscale. Potential perioperative predictors of disturbed sleep and time to chronic lung allograft dysfunction (CLAD) and death were derived from medical records. We investigated associations between perioperative predictors on SPI and Insomnia and associations between SPI and Insomnia on PROs and frailty by linear regressions, adjusting for age, sex, and lung function. We evaluated the associations between SPI and Insomnia on time to CLAD and death using Cox models, adjusting for age, sex, and transplant indication. Post-transplant hospital length of stay >30 days was associated with worse sleep by SPI and insomnia (SPI: p=0.01; Insomnia p=0.02). Worse sleep by SPI and insomnia was associated with worse depression, cognitive function, HRQL, physical disability, health utilities, and Fried Frailty Phenotype frailty (all p<0.01). Those in the worst quartile of SPI and insomnia exhibited increased risk of CLAD (HR 2.18; 95%CI: 1.22-3.89 ; p=0.01 for SPI and HR 1.96; 95%CI 1.09-3.53; p=0.03 for insomnia). Worsening in SPI but not insomnia was also associated with mortality (HR: 1.29; 95%CI: 1.05-1.58; p=0.01). Poor sleep after lung transplant may be a novel predictor of patient reported outcomes, frailty, CLAD, and death with potentially important screening and treatment implications.

MICB Genomic Variant Is Associated with NKG2D-mediated Acute Lung Injury and Death.

Rationale: Acute lung injury (ALI) carries a high risk of mortality but has no established pharmacologic therapy. We previously found that experimental ALI occurs through natural killer (NK) cell NKG2D receptor activation and that the cognate human ligand, MICB, was associated with ALI after transplantation. Objectives: To investigate the association of a common missense variant, MICBG406A, with ALI. Methods: We assessed MICBG406A genotypes within two multicenter observational study cohorts at risk for ALI: primary graft dysfunction (N = 619) and acute respiratory distress syndrome (N = 1,376). Variant protein functional effects were determined in cultured and ex vivo human samples. Measurements and Main Results: Recipients of MICBG406A-homozygous allografts had an 11.1% absolute risk reduction (95% confidence interval [CI], 3.2-19.4%) for severe primary graft dysfunction after lung transplantation and reduced risk for allograft failure (hazard ratio, 0.36; 95% CI, 0.13-0.98). In participants with sepsis, we observed 39% reduced odds of moderately or severely impaired oxygenation among MICBG406A-homozygous individuals (95% CI, 0.43-0.86). BAL NK cells were less frequent and less mature in participants with MICBG406A. Expression of missense variant protein MICBD136N in cultured cells resulted in reduced surface MICB and reduced NKG2D ligation relative to wild-type MICB. Coculture of variant MICBD136N cells with NK cells resulted in less NKG2D activation and less susceptibility to NK cell killing relative to the wild-type cells. Conclusions: These data support a role for MICB signaling through the NKG2D receptor in mediating ALI, suggesting a novel therapeutic approach.

CCR5 drives NK cell-associated airway damage in pulmonary ischemia-reperfusion injury.

Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.

Short airway telomeres are associated with primary graft dysfunction and chronic lung allograft dysfunction.

Primary graft dysfunction (PGD) is a major risk factor for chronic lung allograft dysfunction (CLAD) following lung transplantation, but the mechanisms linking these pathologies are poorly understood. We hypothesized that the replicative stress induced by PGD would lead to erosion of telomeres, and that this telomere dysfunction could potentiate CLAD. In a longitudinal cohort of 72 lung transplant recipients with >6 years median follow-up time, we assessed tissue telomere length, PGD grade, and freedom from CLAD. Epithelial telomere length and fibrosis-associated gene expression were assessed on endobronchial biopsies taken at 2 to 4 weeks post-transplant by TeloFISH assay and nanoString digital RNA counting. Negative-binomial mixed-effects and Cox-proportional hazards models accounted for TeloFISH staining batch effects and subject characteristics including donor age. Increasing grade of PGD severity was associated with shorter airway epithelial telomere lengths (p = 0.01). Transcriptomic analysis of fibrosis-associated genes showed alteration in fibrotic pathways in airway tissue recovering from PGD, while telomere dysfunction was associated with inflammation and impaired remodeling. Shorter tissue telomere length was in turn associated with increased CLAD risk, with a hazard ratio of 1.89 (95% CI 1.16-3.06) per standard deviation decrease in airway telomere length, after adjusting for subject characteristics. PGD may accelerate telomere dysfunction, potentiating immune responses and dysregulated repair. Epithelial cell telomere dysfunction may represent one of several mechanisms linking PGD to CLAD.

Decreased Lymphocytic Bronchitis Severity in the Era of Azithromycin Prophylaxis.

Large-airway lymphocytic inflammation (LB), assessed on endobronchial biopsies, has been associated with acute cellular rejection and chronic lung allograft dysfunction (CLAD). Azithromycin (AZI) prophylaxis has been used to prevent airway inflammation and subsequent CLAD, with inconsistent results. We hypothesized that AZI prophylaxis would be associated with reduced LB, changes in bronchoalveolar lavage (BAL) immune cell populations, and improved CLAD-free survival. Methods: We compared frequencies of LB from endobronchial biopsies before (N = 1856) and after (N = 975) protocolized initiation of AZI prophylaxis at our center. LB was classified as none, minimal, mild, or moderate by histopathologic analysis. LB grades were compared using ordinal mixed-model regression. Corresponding automated BAL leukocyte frequencies were compared using mixed-effects modeling. The effect of AZI prophylaxis on CLAD-free survival was assessed by a Cox proportional hazards model adjusted for age, sex, ethnicity, transplant indication, and cytomegalovirus serostatus. Results: Biopsies in the pre-AZI era had 2-fold increased odds (95% confidence interval, 1.5-2.7; P < 0.001) of higher LB grades. LB was associated with BAL neutrophilia in both eras. However, there was no difference in risk for CLAD or death between AZI eras (hazard ratio 1.3; 95% confidence interval, 0.7-2.0; P = 0.45). Conclusions: Decreased airway inflammation in the era of AZI prophylaxis may represent a direct effect of AZI therapy or reflect other practices or environmental changes. In this cohort, AZI prophylaxis was not associated with improved CLAD-free survival.

The Association Between Frailty and Chronic Lung Allograft Dysfunction After Lung Transplantation.

BACKGROUND: After lung transplantation, both frailty and chronic lung allograft dysfunction (CLAD) commonly develop, and when they do, are associated with poorer outcomes. Given their potential shared mechanisms, we sought to explore the temporal relationship between frailty and CLAD onset. METHODS: In a single center, we prospectively measured frailty by the short physical performance battery (SPPB) repeatedly after transplant. Because of the nature of the relationship between frailty and CLAD is unknown, we tested the association between frailty, modeled as a time-dependent predictor, and CLAD development as well as CLAD development, modeled as a time-dependent predictor, and frailty development. To do so, we used Cox proportional cause-specific hazards and conditional logistic regression models adjusted for age, sex, race, diagnosis, cytomegalovirus serostatus, posttransplant body mass index, and acute cellular rejection episodes as time-dependent covariates. We tested SPPB frailty as a binary (≤9 points) and continuous predictor (12-point scale); as an outcome, we defined frailty as SPPB ≤9. RESULTS: The 231 participants were a mean age of 55.7 y (SD 12.1). After adjusting for covariates, the development of frailty within 3 y after lung transplant was associated with cause-specific CLAD risk (adjusted cause-specific hazard ratio: 1.76; 95% confidence interval [CI], 1.05-2.92 when defining frailty as SPPB ≤9 and adjusted cause-specific hazard ratio: 1.10, 95% CI, 1.03-1.18 per 1-point worsening in SPPB). CLAD onset did not appear to be a risk factor for subsequent frailty (odds ratio, 4.0; 95% CI, 0.4-197.0). CONCLUSIONS: Studying the mechanisms underlying frailty and CLAD could provide new insights into the pathobiology of both and potential targets for intervention.

Design and implementation of a digital health home spirometry intervention for remote monitoring of lung transplant function.

BACKGROUND: We developed an automated, chat-based, digital health intervention using Bluetooth-enabled home spirometers to monitor for complications of lung transplantation in a real-world application. METHODS: A chat-based application prompted patients to perform home spirometry, enter their forced expiratory volume in 1 second (FEV1), answer symptom queries, and provided patient education. The program alerted patients and providers to substantial FEV1 decreases and concerning symptoms. Data was integrated into the electronic health record (EHR) system and dashboards were developed for program monitoring. RESULT: Between May 2020 and December 2021, 544 patients were invited to enroll, of whom 427 were invited remotely and 117 were enrolled in-person. 371 (68%) participated by submitting ≥1 FEV1 values. Overall engagement was high, with an average of 197 unique patients submitting FEV1 data per month. In-person enrollees submitted an average of 4.6 FEV1 values per month and responded to 55% of scheduled chats. Home and laboratory FEV1 values correlated closely (rho = 0.93). There was an average of 133 ± 59 FEV1 decline alerts and 59 ± 23 symptom alerts per month. 72% of patients accessed education modules, and the program had a high net promoter score (53) amongst users. CONCLUSIONS: We demonstrate that a novel, automated, chat-based, and EHR-integrated home spirometry intervention is well accepted, generates reliable assessments of graft function, and can deliver automated feedback and education resulting in moderately-high adherence rates. We found that in-person onboarding yields better engagement and adherence. Future work will aim to demonstrate the impact of remote care monitoring on early detection of lung transplant complications.

Development of the Lung Transplant Frailty Scale (LT-FS).

BACKGROUND: Existing measures of frailty developed in community dwelling older adults may misclassify frailty in lung transplant candidates. We aimed to develop a novel frailty scale for lung transplantation with improved performance characteristics. METHODS: We measured the short physical performance battery (SPPB), fried frailty phenotype (FFP), Body Composition, and serum Biomarkers representative of putative frailty mechanisms. We applied a 4-step established approach (identify frailty domain variable bivariate associations with the outcome of waitlist delisting or death; build models sequentially incorporating variables from each frailty domain cluster; retain variables that improved model performance ability by c-statistic or AIC) to develop 3 candidate "Lung Transplant Frailty Scale (LT-FS)" measures: 1 incorporating readily available clinical data; 1 adding muscle mass, and 1 adding muscle mass and research-grade Biomarkers. We compared construct and predictive validity of LT-FS models to the SPPB and FFP by ANOVA, ANCOVA, and Cox proportional-hazard modeling. RESULTS: In 342 lung transplant candidates, LT-FS models exhibited superior construct and predictive validity compared to the SPPB and FFP. The addition of muscle mass and Biomarkers improved model performance. Frailty by all measures was associated with waitlist disability, poorer HRQL, and waitlist delisting/death. LT-FS models exhibited stronger associations with waitlist delisting/death than SPPB or FFP (C-statistic range: 0.73-0.78 vs. 0.57 and 0.55 for SPPB and FFP, respectively). Compared to SPPB and FFP, LT-FS models were generally more strongly associated with delisting/death and improved delisting/death net reclassification, with greater improvements with increasing LT-FS model complexity (range: 0.11-0.34). For example, LT-FS-Body Composition hazard ratio for delisting/death: 6.0 (95%CI: 2.5, 14.2), SPPB HR: 2.5 (95%CI: 1.1, 5.8), FFP HR: 4.3 (95%CI: 1.8, 10.1). Pre-transplant LT-FS frailty, but not SPPB or FFP, was associated with mortality after transplant. CONCLUSIONS: The LT-FS is a disease-specific physical frailty measure with face and construct validity that has superior predictive validity over established measures.

Subphenotypes of frailty in lung transplant candidates.

Heterogeneous frailty pathobiology might explain the inconsistent associations observed between frailty and lung transplant outcomes. A Subphenotype analysis could refine frailty measurement. In a 3-center pilot cohort study, we measured frailty by the Short Physical Performance Battery, body composition, and serum biomarkers reflecting causes of frailty. We applied latent class modeling for these baseline data. Next, we tested class construct validity with disability, waitlist delisting/death, and early postoperative complications. Among 422 lung transplant candidates, 2 class model fit the best (P = .01). Compared with Subphenotype 1 (n = 333), Subphenotype 2 (n = 89) was characterized by systemic and innate inflammation (higher IL-6, CRP, PTX3, TNF-R1, and IL-1RA); mitochondrial stress (higher GDF-15 and FGF-21); sarcopenia; malnutrition; and lower hemoglobin and walk distance. Subphenotype 2 had a worse disability and higher risk of waitlist delisting or death (hazards ratio: 4.0; 95% confidence interval: 1.8-9.1). Of the total cohort, 257 underwent transplant (Subphenotype 1: 196; Subphenotype 2: 61). Subphenotype 2 had a higher need for take back to the operating room (48% vs 28%; P = .005) and longer posttransplant hospital length of stay (21 days [interquartile range: 14-33] vs 18 days [14-28]; P = .04). Subphenotype 2 trended toward fewer ventilator-free days, needing more postoperative extracorporeal membrane oxygenation and dialysis, and higher need for discharge to rehabilitation facilities (P ≤ .20). In this early phase study, we identified biological frailty Subphenotypes in lung transplant candidates. A hyperinflammatory, sarcopenic Subphenotype seems to be associated with worse clinical outcomes.

CD16+ natural killer cells in bronchoalveolar lavage are associated with antibody-mediated rejection and chronic lung allograft dysfunction.

Acute and chronic rejections limit the long-term survival after lung transplant. Pulmonary antibody-mediated rejection (AMR) is an incompletely understood driver of long-term outcomes characterized by donor-specific antibodies (DSAs), innate immune infiltration, and evidence of complement activation. Natural killer (NK) cells may recognize DSAs via the CD16 receptor, but this complement-independent mechanism of injury has not been explored in pulmonary AMR. CD16+ NK cells were quantified in 508 prospectively collected bronchoalveolar lavage fluid samples from 195 lung transplant recipients. Associations between CD16+ NK cells and human leukocyte antigen mismatches, DSAs, and AMR grade were assessed by linear models adjusted for participant characteristics and repeat measures. Cox proportional hazards models were used to assess CD16+ NK cell association with chronic lung allograft dysfunction and survival. Bronchoalveolar lavage fluid CD16+ NK cell frequency was associated with increasing human leukocyte antigens mismatches and increased AMR grade. Although NK frequencies were similar between DSA+ and DSA- recipients, CD16+ NK cell frequencies were greater in recipients with AMR and those with concomitant allograft dysfunction. CD16+ NK cells were associated with long-term graft dysfunction after AMR and decreased chronic lung allograft dysfunction-free survival. These data support the role of CD16+ NK cells in pulmonary AMR.