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1.  Identification of Multiple Public T Cell Receptor Repertoires in Chronic Beryllium Disease1 
Chronic beryllium disease (CBD) is a granulomatous lung disease characterized by the accumulation of beryllium (Be)-specific CD4+ T cells in bronchoalveolar lavage (BAL). These expanded CD4+ T cells are composed of oligoclonal T cell subsets, suggesting their recruitment to the lung in response to conventional antigen. In the present study, we noted that all BAL-derived T cell lines from HLA-DP2-expressing CBD patients contained an expansion of Be-responsive Vβ5.1+ CD4+ T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed Vβ5.1with a highly conserved CDR3β motif. Interestingly, Be-specific, Vβ5.1-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 CDR3β motif. TCR Vα chain analysis of purified Vβ5.1+ 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, Vβ5.1-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.
PMCID: PMC4011988  PMID: 24719461
Human; T cells; MHC; Lung; T cell receptors
2.  Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease1 
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.
PMCID: PMC4347847  PMID: 18250483
3.  Exposure and genetics increase risk of beryllium sensitisation and chronic beryllium disease in the nuclear weapons industry 
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-DPβ chain (DPβE69). However, the nature of the relationship between exposure and carriage of the DPβE69 genotype has not been well studied. The goal of this study was to determine the relationship between DPβE69 and exposure in BeS and CBD.
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.
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 DPβE69 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 0.1μg/m3. Those with both risk factors had higher increased odds (OR 24.1, 95% CI 4.77 to 122).
DPβE69 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 DPβE69 alone appears to be similar.
PMCID: PMC4347849  PMID: 21460389
4.  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.
PMCID: PMC4347851  PMID: 22972925
5.  Beryllium Lymphocyte Proliferation Test Surveillance Identifies Clinically Significant Beryllium Disease 
Workplace surveillance identifies chronic beryllium disease (CBD) but it remains unknown over what time frame mild CBD will progress to a more severe form.
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.
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.
Medical surveillance for CBD identifies individuals at significant risk of disease progression and impairment with sufficient time since first exposure.
PMCID: PMC3063521  PMID: 19681064
beryllium; chronic beryllium disease; medical surveillance
6.  Beryllium-Specific CD4+ T Cells in Blood as a Biomarker of Disease Progression 
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, Th1-type cytokine-secreting T cells have been characterized. Compared to beryllium (Be)-sensitized subjects, an increased number of Be-responsive T cells are present in the blood of CBD patients.
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.
Using IFN-γ ELISPOT and proliferation-based assays, we determined the frequency and proliferative capacity of Be-responsive T cells in blood.
Compared with the Be lymphocyte proliferation test which detected an abnormal Be-induced proliferative response in 11 of 260 (4.2%) workers from a Be-machining facility, IFN-γ ELISPOT detected a sensitization rate of 10% (χ2 = 55.7; P < 0.0001). A significant positive correlation was also noted between the number of Be-responsive CD4+ T cells in blood and lung of CBD patients. Importantly, the transition from Be sensitization to CBD was associated with an increased number of antigen-specific T cells in blood.
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.
Clinical implications
These findings suggest that the number of Be-responsive T cells in the circulation can serve as a biomarker of disease progression and as an estimate of the severity of Be-induced lung inflammation.
PMCID: PMC3205205  PMID: 21943943
Human; Lung; CD4-Positive T-Lymphocytes; Beryllium; Cytokines; Granuloma; ELISPOT
7.  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.
PMCID: PMC3136994  PMID: 21471109
berylliosis; genetics; case–control studies; occupational exposure; HLA-DP antigens

Results 1-7 (7)