ABSTRACT: Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by a hypersensitivity to beryllium and characterized by the accumulation of beryllium-specific CD4(+) T cells in the lung. Genetic susceptibility to beryllium-induced disease is strongly associated with HLA-DP alleles possessing a glutamic acid at the 69th position of the ?-chain (?Glu69). The structure of HLA-DP2, the most prevalent ?Glu69-containing molecule, revealed a unique solvent-exposed acidic pocket that includes ?Glu69 and represents the putative beryllium-binding site. The delineation of mimotopes and endogenous self-peptides that complete the ??TCR ligand for beryllium-specific CD4(+) T cells suggests a unique role of these peptides in metal ion coordination and the generation of altered self-peptides, blurring the distinction between hypersensitivity and autoimmunity.
Project description:Chronic beryllium disease (CBD) is a granulomatous lung disorder that is associated with the accumulation of beryllium (Be)-specific CD4(+) T cells into the lung. Genetic susceptibility is linked to HLA-DPB1 alleles that possess a glutamic acid at position 69 (?Glu69), and HLA-DPB1*02:01 is the most prevalent ?Glu69-containing allele. Using HLA-DP2 transgenic (Tg) mice, we developed a model of CBD that replicates the major features of the human disease. Here we characterized the T-cell receptor (TCR) repertoire of Be-responsive CD4(+) T cells derived from the lungs of Be oxide-exposed HLA-DP2 Tg mice. The majority of Be-specific T-cell hybridomas expressed TCR V?6, and a subset of these hybridomas expressed identical or nearly identical ?-chains that were paired with different ?-chains. We delineated mimotopes that bind to HLA-DP2 and form a complex recognized by Be-specific CD4(+) T cells in the absence of Be. These Be-independent peptides possess an arginine at p5 and a tryptophan at p7 that surround the Be-binding site within the HLA-DP2 acidic pocket and likely induce charge and conformational changes that mimic those induced by the Be(2+) cation. Collectively, these data highlight the interplay between peptides and Be in the generation of an adaptive immune response in metal-induced hypersensitivity.
Project description:Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4? T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4? T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4? T cells specific for these ligands in all HLADP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4? T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.
Project description:Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed , the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work . In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade.
Project description:Susceptibility to chronic beryllium disease (CBD) is linked to certain HLA-DP molecules, including HLA-DP2. To elucidate the molecular basis of this association, we exposed mice transgenic (Tg) for HLA-DP2 to beryllium oxide (BeO) via oropharyngeal aspiration. As opposed to WT mice, BeO-exposed HLA-DP2 Tg mice developed mononuclear infiltrates in a peribronchovascular distribution that were composed of CD4(+) T cells and included regulatory T (Treg) cells. Beryllium-responsive, HLA-DP2-restricted CD4(+) T cells expressing IFN-? and IL-2 were present in BeO-exposed HLA-DP2 Tg mice and not in WT mice. Using Be-loaded HLA-DP2-peptide tetramers, we identified Be-specific CD4(+) T cells in the mouse lung that recognize identical ligands as CD4(+) T cells derived from the human lung. Importantly, a subset of HLA-DP2 tetramer-binding CD4(+) T cells expressed forkhead box P3, consistent with the expansion of antigen-specific Treg cells. Depletion of Treg cells in BeO-exposed HLA-DP2 Tg mice exacerbated lung inflammation and enhanced granuloma formation. These findings document, for the first time to our knowledge, the development of a Be-specific adaptive immune response in mice expressing HLA-DP2 and the ability of Treg cells to modulate the beryllium-induced granulomatous immune response.
Project description:T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.
Project description: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 V?5.1(+) CD4(+) T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed V?5.1 with a highly conserved CDR3? motif. Interestingly, Be-specific, V?5.1-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 CDR3? motif. TCR V?-chain analysis of purified V?5.1(+) CD4(+) T cells based on differential tetramer-binding intensity showed differing TCR V?-chain pairing requirements, with the high-affinity population having promiscuous V?-chain pairing and the low-affinity subset requiring restricted V?-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, V?5.1-expressing public T cell repertoire in the lungs of HLA-DP2-expressing CBD patients using promiscuous V?-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.
Project description:Susceptibility to chronic beryllium (Be) disease is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the β-chain (βGlu69), with the most prevalent βGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2 transgenic (Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2 mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.
Project description:Metal-induced hypersensitivity is driven by dendritic cells (DCs) that migrate from the site of exposure to the lymph nodes, upregulate costimulatory molecules, and initiate metal-specific CD4+ T cell responses. Chronic beryllium disease (CBD), a life-threatening metal-induced hypersensitivity, is driven by beryllium-specific CD4+ Th1 cells that expand in the lung-draining lymph nodes (LDLNs) after beryllium exposure (sensitization phase) and are recruited back to the lung, where they orchestrate granulomatous lung disease (elicitation phase). To understand more about how beryllium exposures impact DC function during sensitization, we examined the early events in the lung and LDLNs after pulmonary exposure to different physiochemical forms of beryllium. Exposure to soluble or crystalline forms of beryllium induced alveolar macrophage death/release of IL-1? and DNA, enhanced migration of CD80hi DCs to the LDLNs, and sensitized HLA-DP2 transgenic mice after single low-dose exposures, whereas exposures to insoluble particulate forms beryllium did not. IL-1? and DNA released by alveolar macrophages upregulated CD80 on immature BMDC via IL-1R1 and TLR9, respectively. Intrapulmonary exposure of mice to IL-1R and TLR9 agonists without beryllium was sufficient to drive accumulation of CD80hi DCs in the LDLNs, whereas blocking both pathways prevented accumulation of CD80hi DCs in the LDLNs of beryllium-exposed mice. Thus, in contrast to particulate forms of beryllium, which are poor sensitizers, soluble or crystalline forms of beryllium promote death of alveolar macrophages and their release of IL-1? and DNA, which act as damage-associated molecular pattern molecules to enhance DC function during beryllium sensitization.
Project description:Chronic beryllium (Be) disease is a granulomatous lung disorder that results from Be exposure in a genetically susceptible host. The disease is characterized by the accumulation of Be-responsive CD4(+) T cells in the lung, and genetic susceptibility is primarily linked to HLA-DPB1 alleles possessing a glutamic acid at position 69 of the ?-chain. Recent structural analysis of a Be-specific TCR interacting with a Be-loaded HLA-DP2-peptide complex revealed that Be is coordinated by amino acid residues derived from the HLA-DP2 ?-chain and peptide and showed that the TCR does not directly interact with the Be(2+) cation. Rather, the TCR recognizes a modified HLA-DP2-peptide complex with charge and conformational changes. Collectively, these findings provide a structural basis for the development of this occupational lung disease through the ability of Be to induce posttranslational modifications in preexisting HLA-DP2-peptide complexes, resulting in the creation of neoantigens.
Project description:Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.