Frequency of beryllium-specific, central memory CD4+ T cells in blood determines proliferative response

Fontenot, Andrew P.; Palmer, Brent E.; Sullivan, Andrew; Joslin, Fenneke G.; Wilson, Cara C.; Maier, Lisa A.; Newman, Lee S.; Kotzin, Brian L.

doi:10.1172/jci24908

Cited by 55 publications

(62 citation statements)

References 35 publications

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“…Proliferation assays were performed using PBMCs and BAL cells (2 ϫ 10 6 cells/well) labeled with 2.0 M CFSE (Molecular Probes) (31,32). The CFSE-labeled cells were cultured at 37°C for 5 days in a humidified 5% CO 2 atmosphere in RPMI 1640 supplemented with 10% heat-inactivated human serum, 20 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen Life Technologies) with the addition of either medium, 10 g/ml platebound anti-CD3, or 100 M BeSO 4 .…”

Section: Proliferation Assaymentioning

confidence: 99%

“…Biexponential scaling was used in all dot plots. Because the frequency of beryllium-specific CD4 ϩ T cells in blood tends to be low, we only examined the expression of PD-1 on cytokine-producing cells with frequencies Ն0.04% to ensure an adequate number of events for analysis as previously described (31)(32)(33). The CFSE-blocking experiments were acquired using a FACSCalibur flow cytometer (BD Immunocytometry Systems), and CellQuest Pro software was used to analyze the data.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…IFN-␥-producing T cells were initially analyzed because we had previously shown that beryllium-specific CD4 ϩ T cells in both blood and BAL were skewed toward poorly proliferating, IFN-␥-producing T EM cells (32). Polychromatic flow cytometry was used to measure PD-1 expression on Ag-specific CD4 ϩ (CD3 ϩ /CD4 ϩ /CD8 Ϫ ) T cells.…”

Section: Pd-1 Expression Is Up-regulated On Beryllium-specific Cd4 ϩ mentioning

confidence: 99%

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Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease

Palmer¹,

Mack²,

Martin³

et al. 2008

The Journal of Immunology

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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.

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Section: Proliferation Assaymentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

Section: Pd-1 Expression Is Up-regulated On Beryllium-specific Cd4 ϩ mentioning

confidence: 99%

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Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease

Palmer¹,

Mack²,

Martin³

et al. 2008

The Journal of Immunology

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“…We utilized Delta Proportion of Cells that Divided (delta PD) marker as sensitive and more acceptable in clinical measurements than previously reported Proliferating Ratio (PD) (17). We demonstrated that PHA and Candida stimulated both CD4 þ and CD8 þ T cells, while only CD4 þ T cells respond to beryllium in vitro because of MHC class II HLA-DP Glu69 mutation (5,33,34). Determination of specific subpopulation of T cells, responding to beryllium, makes this CFSE proliferation technique compatible to BeLPT, and would have great potential to be developed further for early diagnosis of sensitization to beryllium.…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis between CFSE flow cytometric and tritiated thymidine incorporation tests for beryllium sensitivity

Milovanova

2007

Cytometry Part B Clinical

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Background: In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium.Methods: Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3 þ CD4 þ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT.Results: Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (j 5 0.845, P < < 0.0001). We determined 6.8% positive subjects in the berylliumexposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4 þ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT.Conclusions: Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD41 T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity. q 2007 Clinical Cytometry Society

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“…Streptavidin-PE was used as a second-step reagent for TCR staining. The lymphocyte population was identified using forward and 90-degree light scatter patterns, and fluorescence intensity was analyzed using a FACSCalibur cytometer (BD Immunocytometry Systems) (30,31).…”

Section: Flow Cytometry and Immunofluorescence Analysismentioning

confidence: 99%

Regulatory Role of γδ T Cells in the Recruitment of CD4+ and CD8+ T Cells to Lung and Subsequent Pulmonary Fibrosis

Simonian

Roark

Valle

et al. 2006

The Journal of Immunology

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The mechanisms by which T cells accumulate in the lungs of patients with pulmonary fibrosis are poorly understood. Because the lung is continually exposed to microbial agents from the environment, we repeatedly exposed C57BL/6 mice to the ubiquitous microorganism, Bacillus subtilis, to determine whether chronic exposure to an inhaled microorganism could lead to T cell accumulation in the lungs and subsequent pulmonary fibrosis. C57BL/6 mice repeatedly treated with B. subtilis for 4 consecutive weeks developed a 33-fold increase in the number of CD4+ T cells and a 354-fold increase in γδ T cells in the lung. The γδ T cells consisted almost entirely of Vγ6/Vδ1+ cells, a murine subset bearing an invariant TCR the function of which is still unknown. Treatment of C57BL/6 mice with heat-killed vs live B. subtilis resulted in a 2-fold increase in the number of CD4+ T cells in the lung but no expansion of γδ T cells indicating that γδ cells accumulate in response to live microorganisms. In addition, mice treated with heat-killed B. subtilis developed significantly increased pulmonary fibrosis compared with mice treated with the live microorganism. Mice deficient in Vγ6/Vδ1+ T cells when treated with B. subtilis had a 231-fold increase in lung CD4+ T cells and significantly increased collagen deposition compared with wild-type C57BL/6 mice, consistent with an immunoregulatory role for the Vγ6/Vδ1 T cell subset. These findings indicate that chronic inhalation of B. subtilis can result in T cell accumulation in the lung and fibrosis, constituting a new model of immune-mediated pulmonary fibrosis.

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Frequency of beryllium-specific, central memory CD4+ T cells in blood determines proliferative response

Cited by 55 publications

References 35 publications

Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease

Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease

Comparative analysis between CFSE flow cytometric and tritiated thymidine incorporation tests for beryllium sensitivity

Regulatory Role of γδ T Cells in the Recruitment of CD4+ and CD8+ T Cells to Lung and Subsequent Pulmonary Fibrosis

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