The textures of sarcoidosis: quantifying lung disease through variograms.

Objective: Sarcoidosis is a granulomatous disease affecting the lungs in over 90% of patients. Qualitative assessment of chest CT by radiologists is standard clinical practice and reliable quantification of disease from CT would support ongoing efforts to identify sarcoidosis phenotypes. Standard imaging feature engineering techniques such as radiomics suffer from extreme sensitivity to image acquisition and processing, potentially impeding generalizability of research to clinical populations. In this work, we instead investigate approaches to engineering variogram-based features with the intent to identify a robust, generalizable pipeline for image quantification in the study of sarcoidosis. Approach: For a cohort of more than 300 individuals with sarcoidosis, we investigated 24 feature engineering pipelines differing by decisions for image registration to a template lung, empirical and model variogram estimation methods, and feature harmonization for CT scanner model, and subsequently 48 sets of phenotypes produced through unsupervised clustering. We then assessed sensitivity of engineered features, phenotypes produced through unsupervised clustering, and sarcoidosis disease signal strength to pipeline. Main results: We found that variogram features had low to mild association with scanner model and associations were reduced by image registration. For each feature type, features were also typically robust to all pipeline decisions except image registration. Strength of disease signal as measured by association with pulmonary function testing and some radiologist visual assessments was strong (optimistic AUC ≈ 0.9, p ≪ 0.0001 in models for architectural distortion, conglomerate mass, fibrotic abnormality, and traction bronchiectasis) and fairly consistent across engineering approaches regardless of registration and harmonization for CT scanner. Significance: Variogram-based features appear to be a suitable approach to image quantification in support of generalizable research in pulmonary sarcoidosis.

Compartment-specific protein interactions in beryllium lung disease.

The study provides insights into proteins that may be relevant in BeS and CBD. It provides a framework to investigate the global changes in lung compartment-specific inflammatory cells to better understand the potential interplay of proteins in CBD. https://bit.ly/3PLNTXC.

Multi-Omic Signatures of Sarcoidosis and Progression in Bronchoalveolar Lavage Cells.

Introduction: Sarcoidosis is a heterogeneous, granulomatous disease that can prove difficult to diagnose, with no accurate biomarkers of disease progression. Therefore, we profiled and integrated the DNA methylome, mRNAs, and microRNAs to identify molecular changes associated with sarcoidosis and disease progression that might illuminate underlying mechanisms of disease and potential genomic biomarkers. Methods: Bronchoalveolar lavage cells from 64 sarcoidosis subjects and 16 healthy controls were used. DNA methylation was profiled on Illumina HumanMethylationEPIC arrays, mRNA by RNA-sequencing, and miRNAs by small RNA-sequencing. Linear models were fit to test for effect of diagnosis and phenotype, adjusting for age, sex, and smoking. We built a supervised multi-omics model using a subset of features from each dataset. Results: We identified 46,812 CpGs, 1,842 mRNAs, and 5 miRNAs associated with sarcoidosis versus controls and 1 mRNA, SEPP1 - a protein that supplies selenium to cells, associated with disease progression. Our integrated model emphasized the prominence of the PI3K/AKT1 pathway in sarcoidosis, which is important in T cell and mTOR function. Novel immune related genes and miRNAs including LYST, RGS14, SLFN12L, and hsa-miR-199b-5p, distinguished sarcoidosis from controls. Our integrated model also demonstrated differential expression/methylation of IL20RB, ABCC11, SFSWAP, AGBL4, miR-146a-3p, and miR-378b between non-progressive and progressive sarcoidosis. Conclusions: Leveraging the DNA methylome, transcriptome, and miRNA-sequencing in sarcoidosis BAL cells, we detected widespread molecular changes associated with disease, many which are involved in immune response. These molecules may serve as diagnostic/prognostic biomarkers and/or drug targets, although future testing will be required for confirmation.

Occupational and environmental exposures in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study.

INTRODUCTION: Sarcoidosis is a granulomatous disorder thought to be caused by exposures in genetically susceptible individuals. This study investigated whether specific exposures were associated with different sarcoidosis phenotypes. METHODS: Extensive demographic, occupational and environmental exposure data was analyzed from subjects enrolled in the NHLBI Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. RESULTS: In patients with sarcoidosis, radiation exposure was significantly associated with an increased risk of cardiac sarcoidosis versus non-cardiac sarcoidosis. No exposures were significantly associated with pulmonary only disease versus extrapulmonary disease with or without pulmonary involvement, Scadding Stage II/III/IV versus Scadding Stage 0/I, acute or remitting disease versus non-acute or non-remitting disease, nor chronic versus non-chronic disease. Although not reaching statistically significance after adjustment for multiple comparisons, there were a number of exposures associated with specific disease phenotypes, including exposures where relationships to sarcoidosis have previously been described such as rural exposures and pesticide exposures. CONCLUSIONS: Radiation exposure may be a risk factor for cardiac sarcoidosis. Other exposures may also be associated with specific phenotypes and should be further explored. The study was limited by small groups of exposed subjects for individual exposures and multiple comparisons. The development of novel and innovative exposure assessment tools is needed.

Realistic biomarkers from plasma extracellular vesicles for detection of beryllium exposure.

PURPOSE: Exposures related to beryllium (Be) are an enduring concern among workers in the nuclear weapons and other high-tech industries, calling for regular and rigorous biological monitoring. Conventional biomonitoring of Be in urine is not informative of cumulative exposure nor health outcomes. Biomarkers of exposure to Be based on non-invasive biomonitoring could help refine disease risk assessment. In a cohort of workers with Be exposure, we employed blood plasma extracellular vesicles (EVs) to discover novel biomarkers of exposure to Be. METHODS: EVs were isolated from plasma using size-exclusion chromatography and subjected to mass spectrometry-based proteomics. A protein-based classifier was developed using LASSO regression and validated by ELISA. RESULTS: We discovered a dual biomarker signature comprising zymogen granule protein 16B and putative protein FAM10A4 that differentiated between Be-exposed and -unexposed subjects. ELISA-based quantification of the biomarkers in an independent cohort of samples confirmed higher expression of the signature in the Be-exposed group, displaying high predictive accuracy (AUROC = 0.919). Furthermore, the biomarkers efficiently discriminated high- and low-exposure groups (AUROC = 0.749). CONCLUSIONS: This is the first report of EV biomarkers associated with Be exposure and exposure levels. The biomarkers could be implemented in resource-limited settings for Be exposure assessment.

HLA-DPB1 E69 genotype and exposure in beryllium sensitisation and disease.

OBJECTIVES: Human leukocyte antigen-DP beta 1 (HLA-DPB1) with a glutamic acid at the 69th position of the ß chain (E69) genotype and inhalational beryllium exposure individually contribute to risk of chronic beryllium disease (CBD) and beryllium sensitisation (BeS) in exposed individuals. This retrospective nested case-control study assessed the contribution of genetics and exposure in the development of BeS and CBD. METHODS: Workers with BeS (n=444), CBD (n=449) and beryllium-exposed controls (n=890) were enrolled from studies conducted at nuclear weapons and primary beryllium manufacturing facilities. Lifetime-average beryllium exposure estimates were based on workers' job questionnaires and historical and industrial hygienist exposure estimates, blinded to genotype and case status. Genotyping was performed using sequence-specific primer-PCR. Logistic regression models were developed allowing for over-dispersion, adjusting for workforce, race, sex and ethnicity. RESULTS: Having no E69 alleles was associated with lower odds of both CBD and BeS; every additional E69 allele increased odds for CBD and BeS. Increasing exposure was associated with lower odds of BeS. CBD was not associated with exposure as compared to controls, yet the per cent of individuals with CBD versus BeS increased with increasing exposure. No evidence of a gene-by-exposure interaction was found for CBD or BeS. CONCLUSIONS: Risk of CBD increases with E69 allele frequency and increasing exposure, although no gene by environment interaction was found. A decreased risk of BeS with increasing exposure and lack of exposure response in CBD cases may be due to the limitations of reconstructed exposure estimates. Although reducing exposure may not prevent BeS, it may reduce CBD and the associated health effects, especially in those carrying E69 alleles.

Genomic biomarkers in chronic beryllium disease and sarcoidosis.

Background Previous gene expression studies have identified genes IFNγ, TNFα, RNase 3, CXCL9, and CD55 as potential biomarkers for sarcoidosis and/or chronic beryllium disease (CBD). We hypothesized that differential expression of these genes could function as diagnostic biomarkers for sarcoidosis and CBD, and prognostic biomarkers for sarcoidosis. Study Design/Methods We performed RT-qPCR on whole blood samples from CBD (n = 132), beryllium sensitized (BeS) (n = 109), and sarcoidosis (n = 99) cases and non-diseased controls (n = 97) to determine differential expression of target genes. We then performed logistic regression modeling and generated ROC curves to determine which genes could most accurately differentiate: 1) CBD versus sarcoidosis 2) CBD versus BeS 3) sarcoidosis versus controls 4) non-progressive versus progressive sarcoidosis. Results CD55 and TNFα were significantly upregulated, while CXCL9 was significantly downregulated in CBD compared to sarcoidosis (p < 0.05). The ROC curve from the logistic regression model demonstrated high discriminatory ability of the combination of CD55, TNFα, and CXCL9 to distinguish between CBD and sarcoidosis with an AUC of 0.98. CD55 and TNFα were significantly downregulated in sarcoidosis compared to controls (p < 0.05). The ROC curve from the model showed a reasonable discriminatory ability of CD55 and TNFα to distinguish between sarcoidosis and controls with an AUC of 0.86. There was no combination of genes that could accurately differentiate between CBD and BeS or sarcoidosis phenotypes. Interpretation CD55, TNFα and CXCL9 expression levels can accurately differentiate between CBD and sarcoidosis, while CD55 and TNFα expression levels can accurately differentiate sarcoidosis and controls.

Chronic Beryllium Disease: Update on a Moving Target.

Beryllium exposure remains an ongoing occupational health concern for workers worldwide. Since the initial Occupational Safety and Health Administration (OSHA) ruling on a permissible exposure limit (PEL) for beryllium in 1971, our understanding of the risks of beryllium sensitization and chronic beryllium disease (CBD) has evolved substantially. A new OSHA ruling released in early 2017 and implemented in late 2018 reduced the PEL for beryllium, increased requirements for medical screening and monitoring, and may ultimately enhance worker protection. This review highlights advances in our understanding of the pathway from beryllium exposure to sensitization and progression to CBD that guided the development of this OSHA ruling. Screening workers exposed to beryllium and management of CBD will also be discussed. Finally, we will discuss the role of beryllium as a cause of morbidity and mortality among exposed workers in this potentially preventable occupational lung disease.

Polymorphism of FCGR3A gene in chronic beryllium disease.

Previously we showed that alveolar macrophages (AMs) from patients with chronic beryllium disease (CBD) and beryllium sensitization (BeS) demonstrated significantly greater cell surface CD16 (encoded by the FCGR3A gene) than controls. We hypothesized that these differences were related to polymorphisms in the FCGR3A gene. This study was to determine the association between FCGR3A polymorphisms in CBD, BeS versus controls as well as clinical data, providing potential information about disease pathogenesis, risk, and activity. A total of 189 CBD/154 BeS/150 controls (92 Be-exposed non-diseased and 58 healthy controls) were included in this study. Sequence-specific primers polymerase chain reaction (PCR-SSP) was used to determine FCGR3A 158V/F polymorphisms. We found significantly higher frequencies of the 158V allele (OR: 1.60 (CI: 1.17-2.19), p = 0.004) and 158VV homozygotes (OR: 2.97 (CI: 1.48-5.97) p = 0.007) in CBD versus controls. No differences were found in the frequencies of FCGR3A alleles or genotypes between BeS versus controls and CBD versus BeS. Average changes in exercise testing maximum workload (Wlm), maximum oxygen consumption (VO2m), and diffusion capacity of carbon monoxide (DLCO) demonstrated greater decline over time in those CBD cases with the 158VV gene, modeled between 10 and 40 years from first beryllium exposure. The FCGR3A V158F polymorphism is associated with CBD compared to BeS and controls and may impact lung function in CBD.

Effect of inhaled corticosteroids on lung function in chronic beryllium disease.

BACKGROUND: The clinical effects of inhaled corticosteroids (ICS) on chronic beryllium disease (CBD) are unknown. Although frequently used for symptoms or disease not requiring systemic therapy, the clinical course of patients on ICS has not been evaluated. METHODS: In a retrospective cohort study, forty-eight subjects with CBD, diagnosed by granulomas on lung biopsy and treated with inhaled corticosteroids, were matched to sixty-eight subjects with CBD who were not treated. Pulmonary function testing, exercise tolerance, blood BeLPT, BAL cell count, and symptoms were evaluated. RESULTS: Treated patients showed no significant change over time in pulmonary function, when compared to controls, by forced vital capacity (FVC, p = 0.28) or diffusion capacity (DLCO, p = 0.45) or in exercise tolerance testing. However, symptoms of cough significantly improved in 58% (compared to 17% in controls) and dyspnea improved in 26% after ICS treatment (compared to 0 in controls). Symptoms of cough were improved in patients with a lower baseline FEV1 and FEV1/FVC ratio. Subgroup analysis showed significant lung function response in cases with lower baseline FEV1/FVC and higher residual volume (RV). CONCLUSION: Although FVC and DLCO did not improve in the ICS treated group, we saw no difference in decline compared to matched controls. Symptoms of dyspnea and cough improved with ICS especially in those with obstruction and air trapping suggesting that these should be considered an indication of ICS use in CBD patients.

Clinical tool for disease phenotyping in granulomatous lung disease.

BACKGROUND: Exposure to beryllium may lead to granuloma formation and fibrosis in those who develop chronic beryllium disease (CBD). Although disease presentation varies from mild to severe, little is known about CBD phenotypes. This study characterized CBD disease phenotypes using longitudinal measures of lung function. METHODS: Using a case-only study of 207 CBD subjects, subject-specific trajectories over time were estimated from longitudinal pulmonary function and exercise-tolerance tests. To estimate linear combinations of the 30-year values that define underlying patterns of lung function, we conducted factor analysis. Cluster analysis was then performed on all the predicted lung function values at 30 years. These estimates were used to identify underlying features and subgroups of CBD. RESULTS: Two factors, or composite measures, explained nearly 70% of the co-variation among the tests; one factor represented pulmonary function in addition to oxygen consumption and workload during exercise, while the second factor represented exercise tests related to gas exchange. Factors were associated with granulomas on biopsy, exposure, steroid use and lung inflammation. Three clusters of patients (n = 53, n = 59 and, n = 95) were identified based on the collection of test values. Lower levels of each of the factor composite scores and cluster membership were associated with baseline characteristics of patients. CONCLUSIONS: Using factor analysis and cluster analysis, we identified disease phenotypes that were associated with baseline patient characteristics, suggesting that CBD is a heterogeneous disease with varying severity. These clinical tools may be used in future basic and clinical studies to help define the mechanisms and risk factors for disease severity.

Identification of multiple public TCR repertoires in chronic beryllium disease.

Chronic beryllium disease (CBD) is a granulomatous lung disease characterized by the accumulation of beryllium (Be)-specific CD4(+) T cells in bronchoalveolar lavage. These expanded CD4(+) T cells are composed of oligoclonal T cell subsets, suggesting their recruitment to the lung in response to conventional Ag. In the current study, we noted that all bronchoalveolar lavage-derived T cell lines from HLA-DP2-expressing CBD patients contained an expansion of Be-responsive Vß5.1(+) CD4(+) T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed Vß5.1 with a highly conserved CDR3ß motif. Interestingly, Be-specific, Vß5.1-expressing CD4(+) T cells displayed differential HLA-DP2-peptide tetramer staining intensity, and sequence analysis of the distinct tetramer-binding subsets showed that the two populations differed by a single conserved amino acid in the CDR3ß motif. TCR Vα-chain analysis of purified Vß5.1(+) CD4(+) T cells based on differential tetramer-binding intensity showed differing TCR Vα-chain pairing requirements, with the high-affinity population having promiscuous Vα-chain pairing and the low-affinity subset requiring restricted Vα-chain usage. Importantly, disease severity, as measured by loss of lung function, was inversely correlated with the frequency of tetramer-binding CD4(+) T cells in the lung. Our findings suggest the presence of a dominant Be-specific, Vß5.1-expressing public T cell repertoire in the lungs of HLA-DP2-expressing CBD patients using promiscuous Vα-chain pairing to recognize an identical HLA-DP2-peptide/Be complex. Importantly, the inverse relationship between expansion of CD4(+) T cells expressing these public TCRs and disease severity suggests a pathogenic role for these T cells in CBD.

Chronic beryllium disease, HLA-DPB1, and the DP peptide binding groove.

Multiple epidemiologic studies demonstrate associations between chronic beryllium disease (CBD), beryllium sensitization (BeS), and HLA-DPB1 alleles with a glutamic acid residue at position 69 (E69). Results suggest that the less-frequent E69 variants (non-*0201/*0202 alleles) might be associated with greater risk of CBD. In this study, we sought to define specific E69-carrying alleles and their amino acid sequences in the DP peptide binding groove, as well as their relationship to CBD and BeS risk, using the largest case control study to date. We enrolled 502 BeS/CBD subjects and 653 beryllium-exposed controls from three beryllium industries who gave informed consent for participation. Non-Hispanic white cases and controls were frequency-matched by industry. HLA-DPB1 genotypes were determined using sequence-specific primer PCR. The E69 alleles were tested for association with disease individually and grouped by amino acid structure using logistic regression. The results show that CBD cases were more likely than controls to carry a non-*02 E69 allele than an *02 E69, with odds ratios (95% confidence interval) ranging from 3.1 (2.1-4.5) to 3.9 (2.6-5.9) (p < 0.0001). Polymorphic amino acids at positions 84 and 11 were associated with CBD: DD versus GG, 2.8 (1.8-4.6), p < 0.0001; GD versus GG, 2.1 (1.5-2.8), p < 0.0001; LL versus GG, 3.2 (1.8-5.6), p < 0.0001; GL versus GG, 2.8 (2.1-3.8), p < 0.0001. Similar results were found within the BeS group and CBD/BeS combined group. We conclude that the less frequent E69 alleles confer more risk for CBD than does *0201. Recent studies examining how the composition and structure of the binding pockets influence peptide binding in MHC genes, as well of studies showing the topology of the TCR to likely bind DPB1 preferentially, give plausible biological rationale for these findings.

Beryllium-specific CD4+ T cells in blood as a biomarker of disease progression.

BACKGROUND: CD4(+) T cells are responsible for the progressive lung damage seen in patients with chronic beryllium disease (CBD), a granulomatous lung disorder in which antigen-specific, T(H)1-type, cytokine-secreting T cells have been characterized. Compared with those seen in beryllium (Be)-sensitized subjects, increased numbers of Be-responsive T cells are present in the blood of patients with CBD. OBJECTIVE: The aim of this study was to determine whether the number of Be-specific T cells in blood predicted the development of CBD in a cohort of Be-exposed subjects. METHODS: Using IFN-γ ELISpot and proliferation-based assays, we determined the frequency and proliferative capacity of Be-responsive T cells in blood. RESULTS: Compared with the Be lymphocyte proliferation test, which detected an abnormal Be-induced proliferative response in 11 (4.2%) of 260 workers from a Be-machining facility, the IFN-γ ELISpot detected a sensitization rate of 10% (χ(2) = 55.7, P < .0001). A significant positive correlation was also noted between the number of Be-responsive CD4(+) T cells in the blood and lung tissue of patients with CBD. Importantly, the transition from Be sensitization to CBD was associated with an increased number of antigen-specific T cells in blood. CONCLUSION: These findings have important implications for Be-induced disease and potentially other immune-mediated disorders, suggesting that the frequency of antigen-specific T cells in blood can serve as a noninvasive biomarker to predict disease development and severity of the Be-specific CD4(+) T-cell alveolitis.

Risk of chronic beryllium disease by HLA-DPB1 E69 genotype and beryllium exposure in nuclear workers.

RATIONALE: Beryllium sensitization (BeS) and chronic beryllium disease (CBD) are determined by at least one genetic factor, a glutamic acid at position 69 (E69) of the HLA-DPB1 gene, and by exposure to beryllium. The relationship between exposure and the E69 genotype has not been well characterized. OBJECTIVES: The study goal was to define the relationship between beryllium exposure and E69 for CBD and BeS. METHODS: Workers (n = 386) from a U.S. nuclear weapons facility were enrolled into a case-control study (70 BeS, 61 CBD, and 255 control subjects). HLA-DPB1 genotypes were determined by sequence-specific primer-polymerase chain reaction. Beryllium exposures were reconstructed on the basis of worker interviews and historical exposure measurements. MEASUREMENTS AND MAIN RESULTS: Any E69 carriage increased odds for CBD (odds ratio [OR], 7.61; 95% confidence interval [CI], 3.66-15.84) and each unit increase in lifetime weighted average exposure increased the odds for CBD (OR, 2.27; 95% CI, 1.26-4.09). Compared with E69-negative genotypes, a single E69-positive *02 allele increased the odds for BeS (OR, 12.01; 95% CI, 4.28-33.71) and CBD (OR, 3.46; 95% CI, 1.42-8.43). A single non-*02 E69 allele further increased the odds for BeS (OR, 29.54; 95% CI, 10.33-84.53) and CBD (OR, 11.97; 95% CI, 5.12-28.00) and two E69 allele copies conferred the highest odds for BeS (OR, 55.68; 95% CI, 14.80-209.40) and CBD (OR, 22.54; 95% CI, 7.00-72.62). CONCLUSIONS: E69 and beryllium exposure both contribute to the odds of CBD. The increased odds for CBD and BeS due to E69 appear to be differentially distributed by genotype, with non-*02 E69 carriers and E69 homozygotes at higher odds than those with *02 genotypes.

Exposure and genetics increase risk of beryllium sensitisation and chronic beryllium disease in the nuclear weapons industry.

OBJECTIVES: Beryllium sensitisation (BeS) and chronic beryllium disease (CBD) are caused by exposure to beryllium with susceptibility affected by at least one well-studied genetic host factor, a glutamic acid residue at position 69 (E69) of the HLA-DPß chain (DPßE69). However, the nature of the relationship between exposure and carriage of the DPßE69 genotype has not been well studied. The goal of this study was to determine the relationship between DPßE69 and exposure in BeS and CBD. METHODS: Current and former workers (n=181) from a US nuclear weapons production facility, the Y-12 National Security Complex (Oak Ridge, Tennessee, USA), were enrolled in a case-control study including 35 individuals with BeS and 19 with CBD. HLA-DPB1 genotypes were determined by PCR-SSP. Beryllium exposures were assessed through worker interviews and industrial hygiene assessment of work tasks. RESULTS: After removing the confounding effect of potential beryllium exposure at another facility, multivariate models showed a sixfold (OR 6.06, 95% CI 1.96 to 18.7) increased odds for BeS and CBD combined among DPßE69 carriers and a fourfold (OR 3.98, 95% CI 1.43 to 11.0) increased odds for those exposed over an assigned lifetime-weighted average exposure of 0.1 µg/m(3). Those with both risk factors had higher increased odds (OR 24.1, 95% CI 4.77 to 122). CONCLUSION: DPßE69 carriage and high exposure to beryllium appear to contribute individually to the development of BeS and CBD. Among workers at a beryllium-using facility, the magnitude of risk associated with either elevated beryllium exposure or carriage of DPßE69 alone appears to be similar.

Beryllium lymphocyte proliferation test surveillance identifies clinically significant beryllium disease.

BACKGROUND: Workplace surveillance identifies chronic beryllium disease (CBD) but it remains unknown over what time frame mild CBD will progress to a more severe form. METHODS: We examined physiology and treatment in 229 beryllium sensitization (BeS) and 171 CBD surveillance-identified cases diagnosed from 1982 to 2002. Never smoking CBD cases (81) were compared to never smoking BeS patients (83) to assess disease progression. We compared CBD machinists to non-machinists to examine effects of exposure. RESULTS: At baseline, CBD and BeS cases did not differ significantly in exposure time or physiology. CBD patients were more likely to have machined beryllium. Of CBD cases, 19.3% went on to require oral immunosuppressive therapy. At 30 years from first exposure, measures of gas exchange were significantly worse and total lung capacity was lower for CBD subjects. Machinists had faster disease progression as measured by pulmonary function testing and gas exchange. CONCLUSIONS: Medical surveillance for CBD identifies individuals at significant risk of disease progression and impairment with sufficient time since first exposure.

Up-regulation of programmed death-1 expression on beryllium-specific CD4+ T cells in chronic beryllium disease.

Chronic beryllium disease (CBD) is caused by workplace exposure to beryllium and is characterized by the accumulation of memory CD4+ T cells in the lung. These cells respond vigorously to beryllium salts in culture by producing proinflammatory Th1-type cytokines. The presence of these inflammatory cytokines leads to the recruitment of alveolar macrophages, alveolitis, and subsequent granuloma development. It has been shown that chronic exposure to conventional Ags leads to up-regulation in the expression of negative regulators of T cells such as programmed death-1 (PD-1). Due to the persistence of beryllium in the lung after the cessation of exposure, aberrant regulation of the PD-1 pathway may play an important role in CBD development. In the present study, PD-1 expression was measured on blood and bronchoalveolar lavage (BAL) CD4+ T cells from beryllium-sensitized and CBD subjects. PD-1 expression was significantly higher on BAL CD4+ T cells compared with those cells in blood, with the highest expression on the beryllium-specific T cell subset. In addition, the expression of PD-1 on BAL CD4+ T cells directly correlated with the severity of the T cell alveolitis. Increased expression of the PD-1 ligands, PD-L1 and PD-L2, on BAL CD14+ cells compared with blood was also seen. The addition of anti-PD-1 ligand mAbs augmented beryllium-induced CD4+ T cell proliferation, and an inverse correlation was seen between PD-1 expression on beryllium-specific CD4+ T cells and beryllium-induced proliferation. Thus, the PD-1 pathway is active in beryllium-induced disease and plays a key role in controlling beryllium-induced T cell proliferation.