Usefulness of gene expression profiling of bronchoalveolar lavage cells in acute lung allograft rejection.

BackgroundChronic lung allograft dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Because effective therapies are lacking, early identification and mitigation of risk factors is a pragmatic approach to improve outcomes. Acute cellular rejection (ACR) is the most pervasive risk factor for CLAD, but diagnosis requires transbronchial biopsy, which carries risks. We hypothesized that gene expression in the bronchoalveolar lavage (BAL) cell pellet (CP) could replace biopsy and inform on mechanisms of CLAD.MethodsWe performed RNA sequencing on BAL CPs from 219 lung transplant recipients with A-grade ACR (n = 61), lymphocytic bronchiolitis (n = 58), infection (n = 41), or no rejection/infection (n = 59). Differential gene expression was based on absolute fold difference >2.0 and Benjamini-adjusted p-value ≤0.05. We used the Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resource for pathway analyses. For classifier modeling, samples were randomly split into training (n = 154) and testing sets (n = 65). A logistic regression model using recursive feature elimination and 5-fold cross-validation was trained to optimize area under the curve (AUC).ResultsDifferential gene expression identified 72 genes. Enriched pathways included T-cell receptor signaling, natural killer cell-mediated cytotoxicity, and cytokine-cytokine receptor interaction. A 4-gene model (AUC = 0.72) and classification threshold defined in the training set exhibited fair performance in the testing set; accuracy was 76%, specificity 82%, and sensitivity 60%. In addition, classification as ACR was associated with worse CLAD-free survival (hazard ratio = 2.42; 95% confidence interval = 1.29-4.53).ConclusionsBAL CP gene expression during ACR is enriched for immune response pathways and shows promise as a diagnostic tool for ACR, especially ACR that is a precursor of CLAD

Gene Expression Profiling of Bronchoalveolar Lavage Cells Preceding a Clinical Diagnosis of Chronic Lung Allograft Dysfunction.

BackgroundChronic Lung Allograft Dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Although CLAD is usually not responsive to treatment, earlier identification may improve treatment prospects.MethodsIn a nested case control study, 1-year post transplant surveillance bronchoalveolar lavage (BAL) fluid samples were obtained from incipient CLAD (n = 9) and CLAD free (n = 8) lung transplant recipients. Incipient CLAD cases were diagnosed with CLAD within 2 years, while controls were free from CLAD for at least 4 years following bronchoscopy. Transcription profiles in the BAL cell pellets were assayed with the HG-U133 Plus 2.0 microarray (Affymetrix). Differential gene expression analysis, based on an absolute fold change (incipient CLAD vs no CLAD) >2.0 and an unadjusted p-value ≤0.05, generated a candidate list containing 55 differentially expressed probe sets (51 up-regulated, 4 down-regulated).ResultsThe cell pellets in incipient CLAD cases were skewed toward immune response pathways, dominated by genes related to recruitment, retention, activation and proliferation of cytotoxic lymphocytes (CD8+ T-cells and natural killer cells). Both hierarchical clustering and a supervised machine learning tool were able to correctly categorize most samples (82.3% and 94.1% respectively) into incipient CLAD and CLAD-free categories.ConclusionsThese findings suggest that a pathobiology, similar to AR, precedes a clinical diagnosis of CLAD. A larger prospective investigation of the BAL cell pellet transcriptome as a biomarker for CLAD risk stratification is warranted

Protection against Bronchiolitis Obliterans Syndrome Is Associated with Allograft CCR7+CD45RA− T Regulatory Cells

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA−, and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4+ and CD103− and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7+ Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7+ Treg frequency and inversely with BOS. Higher frequencies of CCR7+ CD3+CD4+CD25hiFoxp3+CD45RA− lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection

An Unorthodox Introduction to QCD

These are lecture notes presented at the 2017 CTEQ Summer School at the University of Pittsburgh and the 2018 CTEQ Summer School at the University of Puerto Rico, Mayaguez. The title is a reference to hep-th/0309149 and introduces perturbative QCD and its application to jet substructure from a bottom-up perspective based on the approximation of QCD as a weakly-coupled, conformal field theory. Using this approach, a simple derivation of the Sudakov form factor with soft gluon emission modeled as a Poisson process is presented. Topics of the identification and discrimination of quark- versus gluon-initiated jets and jet grooming are also discussed.Comment: 16 pages, 18 figures. Comments welcome!, v2: updated to include both lectures from the 2018 CTEQ schoo

Inflammasomes and IL-1 biology in the pathogenesis of allograft dysfunction

Inflammasomes are high-molecular-weight cytosolic complexes that mediate the activation of caspases. There are many inflammasomes, and each is influenced by a unique pattern-recognition receptor response. Two signals are typically involved in the inflammasome pathways. Signal one involves recognition of pathogen-associated molecular patterns (PAMPs), such as LPS or other colonizing/invading microbes, that interact with TLRs, which induce the downstream production of pro-IL-1β. This is followed by signal two, which involves recognition of PAMPs or damage-associated molecular patterns (DAMPs), such as uric acid or ATP, via NLRP3, which leads to caspase-1-dependent cleavage of pro-IL-1β to active IL-1β and pyroptosis. Ultimately, these two signals cause the release of multiple proinflammatory cytokines. Both PAMPs and DAMPs can be liberated by early insults to the allograft, including ischemia/reperfusion injury, infections, and rejection. The consequence of inflammasome activation and IL-1 expression is the upregulation of adhesion molecules and chemokines, which leads to allograft neutrophil sequestration, mononuclear phagocyte recruitment, and T cell activation, all of which are key steps in the continuum from allograft insult to chronic allograft dysfunction