Michael T. Falta

Crystal structure of HLA-DP2 and implications for chronic beryllium disease

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 β-chain (βGlu69). 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 α-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 β-chain α-helix and containing three glutamic acids from the β-chain, including βGlu69. 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.

Beryllium Presentation to CD4+ T Cells Is Dependent on a Single Amino Acid Residue of the MHC Class II β-Chain

Chronic beryllium disease (CBD) is characterized by a CD4+ T cell alveolitis and granulomatous inflammation in the lung. Genetic susceptibility to this disease has been linked with HLA-DP alleles, particularly those possessing a glutamic acid at position 69 (Glu69) of the β-chain. However, 15% of CBD patients do not possess a Glu69-containing HLA-DP allele, suggesting that other MHC class II alleles may be involved in disease susceptibility. In CBD patients without a Glu69-containing HLA-DP allele, an increased frequency of HLA-DR13 alleles has been described, and these alleles possess a glutamic acid at position 71 of the β-chain (which corresponds to position 69 of HLA-DP). Thus, we hypothesized that beryllium presentation to CD4+ T cells was dependent on a glutamic acid residue at the identical position of both HLA-DP and -DR. The results show that HLA-DP Glu69- and HLA-DR Glu71-expressing molecules are capable of inducing beryllium-specific proliferation and IFN-γ expression by lung CD4+ T cells. Using fibroblasts expressing mutated HLA-DP2 and -DR13 molecules, beryllium recognition was dependent on the glutamic acid at position 69 of HLA-DP and 71 of HLA-DR, suggesting a critical role for this amino acid in beryllium presentation to Ag-specific CD4+ T cells. Thus, these results demonstrate that a single amino acid residue of the MHC class II β-chain dictates beryllium presentation and potentially, disease susceptibility.

Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.

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.

Defective leukocyte GM-CSF receptor (CD116) expression and function in inflammatory bowel disease.

BACKGROUND & AIMS Inflammatory bowel disease (IBD) refers to 2 chronic inflammatory diseases of the intestine, ie, ulcerative colitis and Crohns disease. IBD results from environmental factors (eg, bacterial antigens) triggering a dysregulated immune response in genetically predisposed hosts. Although the basis of IBD is incompletely understood, a number of recent studies have implicated defective innate immune responses in the pathogenesis of IBD. In this regard, there is much interest in therapies that activate innate immunity (eg, recombinant granulocyte-macrophage colony-stimulating factor). METHODS In this study, we screened expression and function of circulating leukocyte granulocyte-macrophage colony-stimulating factor receptor (CD116) messenger RNA and surface protein in 52 IBD patients and 52 healthy controls. RESULTS Our results show that both granulocyte and monocyte CD116 levels, but not CD114 or interleukin-3Rα, were significantly decreased in IBD compared to control (P<.001) and disease controls (irritable bowel syndrome; P<.001; rheumatoid arthritis; P<.025). IBD-associated CD116 repression was more prominent in patients with ulcerative colitis compared to Crohns disease (P<.05), was independent of disease activity (P>.05), and was not influenced by current medications (P>.05). Receiver operating characteristic curve analysis revealed that leukocyte CD116 expression is a sensitive (85%) and specific (92%) biomarker for IBD. Moreover, granulocyte CD116-mediated function (phosphorylation of signal transducers and activators of transcription 3) paralleled decreased expression of CD116 in IBD granulocytes compared to control (P<.001). CONCLUSIONS These studies identify defective expression and function of CD116 as a distinguishing feature of IBD and implicate an associated defect in innate immune responses toward granulocyte-macrophage colony-stimulating factor.

Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease.

Identification of peptides that form complexes with beryllium and class II HLA molecules and are recognized by CD4+ T cells from patients with chronic beryllium disease.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

Significance The essence of the adaptive immune response depends on the specificity of antigen receptors. This report identifies shared alpha–beta T-cell receptor (TCR) pairs from the tissues of HLA-A2+ patients with breast cancer and control donors. Using an emulsion-based RT-PCR assay, we analyzed TCR sequences from tissues ex vivo. We identified multiple TCR pairs shared between tumors, but not control samples. Although recent reports have concluded that anticancer T-cell responses depend on patient-specific mutation-associated neoantigens, this study provides evidence that T cells also recognize shared antigens. This approach has broad application to a variety of research questions where the end goal is to examine T-cell repertoires and/or identify T-cell antigens. Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

Regulatory T cells modulate granulomatous inflammation in an HLA-DP2 transgenic murine model of beryllium-induced disease

Significance Genetic linkage to major histocompatibility complex class II proteins has been observed in many immunological disorders; yet, little is known about the underlying mechanisms of these associations. For chronic beryllium disease (CBD), the linkage to HLA-DPB1 alleles encoding a glutamic acid at position 69 of the β-chain is well established. We tested whether the presence of HLA-DP2 is sufficient for the development of a disease-specific model of CBD. HLA-DP2 transgenic mice developed a beryllium-specific adaptive immune response composed of CD4+ T cells that secrete Th1-type cytokines and are HLA-DP2-restricted, thus replicating the major features of the human disease. In addition, regulatory T cells modulate granuloma formation in the lungs of beryllium oxide-exposed HLA-DP2 transgenic mice. 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-γ 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.

Mutagenesis of Beryllium-Specific TCRs Suggests an Unusual Binding Topology for Antigen Recognition

Unconventional Ags, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of Be-specific TCRs derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2–restricted and express nearly identical TCR Vβ5.1 chains coupled with different TCR α-chains. Site-specific mutagenesis of all amino acids comprising the CDRs of the TCRA and TCRB genes showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the TCR α-chain. Solvent-exposed residues along the α-helices of the HLA-DP2 α- and β-chains were also mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 α-chain. These findings suggest that Be-specific T cells recognize Ag using an unconventional binding topology, with the majority of interactions contributed by TCR Vβ5.1 residues and the HLA-DP2 β1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal Ags, such as Be.

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.

Beryllium-Induced Hypersensitivity: Genetic Susceptibility and Neoantigen Generation

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 β-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 β-chain and peptide and showed that the TCR does not directly interact with the Be2+ 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.