Research Symposium: Basic Mechanisms and Human Health
Session 1. Immunopathology of Beryllium-Induced Granulomatous Disease
Characterization of Beryllium-Specific Memory CD4+ T Cells in Patients with Chronic Beryllium Disease
Andrew P. Fontenot, Scott J. Canavera, Laia Gharavi, Lee S. Newman,
and Brian L. Kotzin
Evidence suggest that CD4+ T cells play a critical role in the development of chronic beryllium disease (CBD). Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4+ T cells in the lungs of 10 CBD patients ranged from 1.4-29% (mean 18.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO4). Few, if any, beryllium-specific CD8+ T cells were identified. In contrast, the frequency of beryllium-responsive CD4+ T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive BAL CD4+ T cells detected with intracellular cytokine staining and lymphocyte proliferation in culture after BeSO4 exposure. The number of BeSO4-responsive CD4+ T cells appeared to reflect the industry of beryllium exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4+ cells in lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.
HLA-DP-Unrestricted TNF-a Release in Be-Stimulated PBMC from Berylliosis Patients
Massimo Amicosante1, Floriana Berretta2, Alberto Franchi3,
Paola Rogliani2, Chiara Dotti2, Monica Losi4,
Raed Dweik5, and Cesare Saltini2
Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ Th1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: the HLA-DP gene whose allelic variants carrying glutamate in position 69 of the b chain (HLA-DPGlu69), which play a central role as they can bind Be directly and present it to IFN-g releasing Th1 T-cell clones from patients with berylliosis, and the cytokine gene TNF-a which has been shown to increase berylliosis risk independent of HLA-DPGlu69. In order to determine whether TNF-a release was triggered by TH1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, we quantified the proliferation and IFN-g, TNF-a, Rantes, GM-CSF, IL-4, IL-6, IL-8, IL-10 and IL-12 release by of BeSO4-stimulated blood mononuclear cells in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation.
While proliferation and IFN-g was completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP MoAb: 88+16% and 77+16% respectively; anti-HLA-DR, 29+38% and 14+10% respectively, p<0.05) the release of TNF-a was not (inhibition with anti-HLA-DP MoAb: 8.9+7.8); no other cytokine was detected at significant levels. Moreover, Be was able to induce TNF-a production in healthy no Be-exposed control in the absence of T-cell proliferation and IFN-g production. Altogether this data suggests that, consistently with the finding that the TNFA2 and the HLA-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk, the TNF-a response of mononuclear cells is independent of the activation of Be-specific HLA-DP restricted T-cells.
Design, Engineering and Production of Human Recombinant T Cell Receptor Ligands Derived from HLA-DP
Gregory G. Burrows, Roberto Meza-Romero, Jeff Mooney, Justin W. Chang,
Hans Peter Bachinger, and Jianya Huan
Chronic beryllium disease (CBD) is a lung disease similar clinically to other granulomatous diseases, such as sarcoidosis, schistosomiasis and tuberculosis. Approximately 800,000 individuals are currently at risk for developing the disease, which is caused by metal and relatively insoluble compounds of beryllium. The disease process begins as a sensitizing cell-mediated immune response to beryllium antigen which develops into a non-caseating granuloma. Evidence strongly suggests that CD4+ T cells and the MHC class II allele HLA-DPB1*0201 are important in the immunopathogenesis of CBD. How the T cell receptor (TCR) on the T cells interacts with beryllium and the MHC, and the mechanism that gives rise to the pathogenesis of CBD, remains unknown. We have developed a family of novel recombinant HLA-DP-derived T cell receptor ligands (RTLs) to test critically the hypothesis that a specific MHC class II allele interacts with beryllium and induces T cell responses that contribute directly to the pathogenesis of CBD. Development and characterization of these novel constructs will provide the opportunity to identify unique points of intervention for controlling T cells and in turn the T cell immune response and repertoire. These molecules may provide a template for engineering a novel treatment of CBD.
A Flow Cytometric Assay for Beryllium Sensitization: Screening And Mechanistic Applications
A.J. Jabbour1, R.A. Ponce1, M.T. Rosato1,
T.J. Kavanagh1, L.S. Newman2, T.K.
Takaro1, and E.M. Faustman1
Beryllium inhalation in humans can result in chronic beryllium disease (CBD). Medical monitoring relies on the in vitro assessment of beryllium sensitization using the lymphocyte proliferation test (LPT) that measures tritiated thymidine incorporation into the DNA of proliferating cells. In this study, we demonstrate the utility of a flow cytometry-based method to test for beryllium sensitization and provide information on the mechanism of beryllium disease at the cellular and molecular levels through multiparametric analysis of cellular DNA and proteins. The four-color flow cytometry assay allows the simultaneous measurement of cumulative cell proliferation and surface marker expression for the purpose of identifying the types of cells responding to beryllium exposure. Peripheral blood mononuclear cells (PBMC) from known CBD patients and normal controls were isolated and tested in culture for their response to beryllium. Increased cell proliferation to beryllium was only seen with blood cells from sensitized individuals but not normal controls. Beryllium elicited a positive proliferative response in cultured PBMC from known CBD patients that ranged from 2-7% at day 4 and 3-10% at day 6 of culture. The lymphocyte composition of resting (G0/G1) and proliferating subpopulations was determined by immunolabeling specific markers of T (CD4, CD3) and B (CD19) cells. The resting subpopulation included 40-60% CD4+ T cells, 60-80% CD3+ T cells, and 5-10% B lymphocytes. Beryllium caused a selective stimulation of CD4+ T cells since the majority of proliferating cells were CD4+ T cells. The versatility of this assay may improve our ability to detect beryllium health effects and advance our understanding on the causes of CBD.
(Supported by Department of Energy Cooperative Agreement #DE-FCO1-95EW55084).
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