Recombinant HLA-DP2 Binds Beryllium and Tolerizes Beryllium-Specific Pathogenic CD4+ T Cells

Andrew P. Fontenot, Timothy S. Keizer, Mark McCleskey, Douglas G. Mack, Roberto Meza-Romero, Jianya Huan, David M. Edwards, Yuan K. Chou, Arthur A. Vandenbark, Brian Scott and Gregory G. Burrows
J Immunol September 15, 2006, 177 (6) 3874-3883; DOI: https://doi.org/10.4049/jimmunol.177.6.3874

Abstract

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the α-1 and β-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4-pulsed, plate-bound rHLA-DP2 molecules induced IFN-γ secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.

The majority of studies characterizing T cell epitopes have focused on peptide Ags. However, αβ T cell receptors can recognize ligands other than peptides (1). For example, CD1, an MHC class Ib molecule, presents lipids and glycolipids to T cells (2). Metal ions complexed with the MHC/peptide surface can also stimulate T cells, and the structural basis for MHC-restricted metal recognition remains one of the unanswered questions in the field of T cell activation. Four broad mechanisms contribute to the potency of metal responses by T cells, and data supporting all of these mechanisms have been reported (3, 4, 5, 6, 7, 8, 9, 10). Metal binding-induced conformational changes in the MHC may account for cobalt-induced hard metal disease (3, 4). Metals may affect the processing of Ags, resulting in alternatively processed peptides (5, 6, 7). TCR could recognize metal-modified amino acid residues of the MHC molecule or of the processed peptide itself, such as a beryllium trifluoride (BeFl3) modified glutamic acid residue, as the result of enzymatic activity. This type of adduct has been routinely taken advantage of for stabilizing transition states of ATP binding sites in crystallographic studies using BeFl3 as an ATP analog (MgADP × BeFx) (8). Finally, T cell activation by nickel appears to involve a mechanism similar to haptens such as trinitrophenol or penicillin, with determinants formed by a complex of metal ions with MHC-embedded self-peptides and TCR determinants (1, 9, 10). None of these models is mutually exclusive, and different metals could potentially use distinct or several mechanisms to activate T cells.

The development of chronic beryllium disease (CBD)3 is associated with the accumulation of activated CD4+ T cells in the bronchoalveolar lavage (BAL) (11, 12). Characterization of TCR V region gene usage suggests that T cells are selected in the lung by a common Ag involving beryllium (13, 14). BAL T cells proliferate and secrete effector cytokines in vitro in response to beryllium sulfate (BeSO4) in the presence of APCs that express HLA-DP2 (11, 15, 16, 17). How the TCR interacts with HLA-DP2 and beryllium and the role of peptide as part of the beryllium Ag that gives rise to the pathogenesis of CBD remain important unanswered questions.

To further characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we have constructed rHLA-DP molecules consisting of the α-1 and β-1 domains of HLA-DP genetically linked into a single polypeptide chain. These single chain rTCR ligands (RTLs) (18, 19, 20) refold in a manner that allows binding of allele-specific epitopes (21). In this study, we present data characterizing HLA-DP-derived RTLs, including direct measurement of beryllium binding to rHLA-DP2. Furthermore, we present in vitro data demonstrating the ability of rHLA-DP2 loaded with beryllium to tolerize pathogenic BAL-derived CD4+ T cells.

Materials and Methods

Homology modeling

Sequence alignment of MHC class II (MHC-II) molecules from human, rat, and mouse species provided a starting point for our studies (19). Graphic images were generated with the program Sybyl (version 7.0; Tripos Associates) and an Octane2 workstation (IRIX 6.5; Silicon Graphics) using coordinates deposited in the Protein Data Bank (22). Homology modeling was based on the refined crystallographic coordinates of human HLA-DR2 (23, 24), as well as HLA-DR1 (25, 26), and murine I-Ek molecules (27). The peptide backbones of HLA-DP2-derived RTL600 and HLA-DP4-derived RTL700 were modeled as rigid bodies during structural refinement using local energy minimization.

RTLs

In brief, de novo synthesis of the human DP2-derived RTL600 gene involved a four-step PCR-based gene synthesis protocol, using 75- to 125-bp synthetic oligonucleotide primers with a melting temperature of each overlap equal to 68°C based on G + C = 4°C, A + T = 2°C. Cloned PFU DNA polymerase was purchased from Stratagene, TaqDNA polymerase was purchased from Promega, and the TA cloning kit was purchased from Invitrogen Life Technologies. The amplification reactions resulted in production of dsDNA fragments of the appropriate length, which were gel purified, and the desired bands were then isolated (QIAquick Gel Extraction Kit; Qiagen). TaqDNA polymerase was used to add 3′-A overhangs to all dsDNA fragments. The final 100-μl reaction was gel purified, and the insert was ligated with the PCR2.1 plasmid vector (TA Cloning Kit; Invitrogen Life Technologies). Aliquots of the ligation reaction were transformed into the INVa′F bacterial cloning host. PCR colony screening was used to select a single positive colony, from which plasmid DNA was isolated (QIAprep Spin Miniprep Kit; Qiagen). Plasmid was cut with NcoI and XhoI restriction enzymes (New England Biolabs), gel purified, and the DNA insert containing an exon encoding the HLA-DP2-derived RTL600 was subsequently ligated with NcoI/XhoI digested pET-21d+ plasmid expression vector (Novagen), and transformed into BL21(DE3) expression host (Novagen). Bacterial colonies were selected based on PCR colony and protein expression screening. Plasmid DNA was isolated from positive colonies (Qiagen) and sequenced with (T7) 5′-TAATACGACTCACTATAGGG-3′ and (T7 terminator) 5′-GCTAGTTATTGCTCAGCGG-3′ primers. Of the 12 colonies sequenced, ∼30% contained the desired human DP2 construct insert with single-base insertion or deletion mutations, whereas the remainder showed gross sequence inaccuracies related to nonspecific DNA hybridization during the PCR-based gene synthesis procedure. Plasmid DNA was isolated from one of these single point mutants, and site-directed mutagenesis was used to generate the desired human DP2-derived RTL600 construct. Using the purified pET-21d+/RTL600 plasmid, site-directed mutagenesis was used to mutate four specific residues (V36A, D55A, E56A, and E69K), DPB1 numbering within the RTL600 insert, thus creating RTL700, the DP4-derived equivalent of DP2. After sequence verification, single clones were selected for expression of RTL600 or RTL700.

Expression and in vitro folding of the RTL constructs

Expression, purification, and refolding of HLA-DP-derived RTLs were performed as previously described (20). The final yield of purified refolded RTL600 and RTL700 protein varied between 20 and 30 mg/L bacterial culture. Complete methods for the SDS-PAGE gel shift assay, dynamic light-scattering measurements, circular dichroism (CD) measurements, and the peptide-binding ELISA experiments have been previously described (21).

9Be nuclear magnetic resonance (NMR) spectroscopy

Solutions of 200 mM of RTL600 and RTL700 were prepared in a 20 mM Tris buffer (pH 8.1). Fifteen equivalents of BeSO4 (3 mM) were added. 9Be NMR data were obtained on a Bunker 400-MHz instrument. Chemical shifts are reported relative to external standard Be(H2O)42+ (0.0 parts per million (ppm)) and are in ppm. D2O (10%) was added to the NMR solution to provide a lock signal. A 30° pulse was used with a 1.5 delay and collection of 100,000 scans. Inductively coupled plasma-atomic emission spectrometer (ICP-AES) data were obtained on a Varian Liberty 2020 using the SW846 EPA method 6010. UV/Vis data were obtained on a Hewlett-Packard 8453 spectrometer. 9Be NMR (56.2 MHz, 25°C, Be(H2O)42+): g = 1.12 (w1/2 = 63 Hz). Initial solution concentration of RTL600 was determined to be 200 μM based on an e(280 nm) = 23,045 M−1 cm−1 (28). Inductively coupled plasma results on the NMR solution following purification by ultrafiltration indicated a 6.5:1 ratio of beryllium:RTL600.

Generation and maintenance of beryllium-specific BAL CD4+ T cell lines (TCLs)

The beryllium-specific CD4+ TCLs were developed as previously described (15). Autologous EBV-transformed lymphoblastoid cell lines (LCLs) used as APCs were generated as previously reported (15). Initial and periodic (every 4–6 wk) stimulations of the BAL CD4+ TCLs were conducted with 100 μM BeSO4 (Sigma-Aldrich) in the presence of irradiated (9000 rad) autologous EBV-transformed LCLs (1:1, T cell:APCs) in a final volume of 500 μl/well in complete culture medium (RPMI 1640 culture medium containing 10% FBS supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, and 0.2 mM nonessential amino acids (Invitrogen Life Technologies), 100 μg/ml penicillin G, and 100 μg/ml streptomycin (Invitrogen Life Technologies)) in a 12-well culture plate (BD Biosciences). rIL-2 (R&D Systems) was added to a final concentration of 10 ng/ml twice per week.

Lymphocyte proliferation assay

Beryllium-specific TCLs (5 × 104 cells) were cultured in 96-well flat-bottom microtiter plates with either medium alone or 5 × 104 BeSO4-pulsed, fixed autologous EBV-transformed LCLs. In brief, autologous LCLs were fixed by resuspending pelleted cells in HBSS with 2% paraformaldehyde for 10 min as previously described (15). The cells were pelleted and resuspended in lysine wash solution for 20 min at room temperature (RT) and washed three times before use in culture. For the LCLs that were not pulsed with 1 × 10−4 M BeSO4 before fixation, 1 × 10−4 M BeSO4 was added to the culture. After 72 h of culture, the wells were then pulsed with 1 μCi of [3H]thymidine for an additional 16 h, and incorporation of radioactivity was determined by beta emission spectroscopy. Proliferation assays were performed in triplicate.

Determination of the reversibility of beryllium binding to fixed LCLs

Fixed autologous EBV-transformed LCLs were incubated overnight at 37°C in a humidified 5% CO2 atmosphere in medium alone or medium containing 100 μM BeSO4. Unbound BeSO4 was removed by washing three times with HBSS. The cell pellet was resuspended and distributed into tubes containing HBSS or a 100 mM sodium citrate buffer solution with a pH of either 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0. The cells were washed once in the respective buffers, incubated for 40 min at 37°C, then washed twice in RPMI 1640, and resuspended culture medium.

The BeSO4 loaded, citrate buffer-treated LCLs were incubated alone or with the addition of 100 μM BeSO4. Some of the citrate buffer-treated LCLs were incubated in the presence of 10 ng/ml staphylococcal enterotoxin B (SEB) as a positive control. Following fixation and citrate buffer treatment, LCLs (2.5 × 105) were cocultured with the beryllium-specific CD4+ TCLs (2.5 × 105) for 6 h at 37°C in a humidified 5% CO2 atmosphere with 10 μg/ml brefeldin A added after the first hour of stimulation as previously described (29, 30, 31). After stimulation, cells were washed and stained with mAbs directed against CD3 and CD4 (all from BD Biosciences). Cells were washed with PBS containing 1% BSA and placed in fixation medium (Caltag Laboratories) for 15 min at room temperature. Following washing with PBS containing 1% BSA, cells were added to permeabilization medium (Caltag Laboratories), and stained with mAbs directed against IFN-γ (Caltag Laboratories) for 30 min at 4°C. Fluorescence intensity was analyzed using a FACSCalibur cytometer (BD Biosciences) as previously described (29, 30, 31).

Tolerization assay

Twenty-four-well flat-bottom culture plates (Multiwell 3533047; BD Biosciences) were left untreated (Tris buffer alone) or coated with 40 μM rHLA-DP2 (RTL600) or HLA-DP4 (RTL700) in 1 ml of Tris buffer, overnight at 4°C. Wells were washed three times with RPMI 1640, and 1 ml of complete culture medium containing either no additional components or 20 μM myelin oligodendrocyte glycoprotein (MOG) peptide was added overnight at 4°C. After washing wells three times with RPMI 1640, wells were treated with 1 ml of complete culture medium or medium containing 1 μg/ml BeSO4, overnight at 4°C. After washing wells three times with RPMI 1640, beryllium-specific T cells (1 ml; 1 × 106 cells per well) in complete culture medium were added. Cells were incubated for 3 days at 37°C, followed by washing three times with RPMI 1640, and redistributed into six wells of a 96-well culture plate (200,000 cells per well; 100 μl of complete culture medium) and cocultured in triplicate with untreated LCLs or LCLs pulsed with beryllium, at a 2:1 (LCL:T) ratio for 3 days. At 48 h, 100 μl of supernatant was collected from the top of each well for use in determining IL-2 production using a cytotoxic TCL (CTLL)-2 IL-2-dependent T cell bioassay (see below). After 72 h of culture, 1 μCi of [3H]thymidine was added to wells and the cells were cultured for an additional 16 h, and incorporation of radioactivity was determined by beta emission spectroscopy. Proliferation assays were performed in triplicate.

Quantification of changes in IL-2 production was performed using a CTLL IL-2-dependent T cell bioassay previously described (59). Briefly, supernatants collected from the top of the culture were centrifuged for 1 min at 1000 rpm and then added (in triplicate) into wells containing 5000 CTLL-2 cells in 100 μl of 1% FBS culture medium. After 24 h of culture, 0.5 μCi of [3H]thymidine was added and the cells cultured an additional 6 h, with the incorporation of radioactivity determined by beta emission spectroscopy. Stimulation index was calculated as the cpm of Ag-stimulated cultures divided by the cpm of the cells cultured in medium alone.

Detection and quantification of cytokine production

ELISAs were performed as previously described (18). Ninety-six-well plates (Nunc) were coated with 100 μl/well of capture Ab (purified anti-human IFN-γ mAb, clone 1D1K; Mabtech) diluted to 3 μg/ml in coating buffer (0.1 M carbonate, pH 9.5), incubated overnight at 4°C. After washing (PBS with 0.05% Tween 20), plates were blocked (PBS with 10% FBS, pH 7.0) at RT for 1 h, washed, and then 100 μl of standards, experimental samples, or controls were added into each well and incubated in the dark for 2 h at RT. The plates were then incubated with biotinylated goat anti-human IFN-γ (clone 7B6-1; Mabtech) at RT for 3 h, followed by incubation with peroxidase-labeled Extravidin (Sigma-Aldrich) for 1 h. After washing, 100 μl of tetramethylbenzidine substrate in hydrogen peroxide was added to each well, incubated for 30 min at RT in the dark, followed by addition of 50 μl of tetramethylbenzidine stop solution. Absorbance at 450 nm was read using a Molecular Probes Kinetic Microplate Reader within 30 min of stopping the reaction.

Statistical analysis

The Mann-Whitney U test and the Kruskal-Wallis test with pairwise comparison (Dunn multiple comparison test) were used to determine significance of differences between subject groups (Prism 4; GraphPad Software). A p value of <0.05 was considered statistically significant.

Results

Construction and characterization of DP2-derived RTL600 and DP4-derived RTL700

We have constructed recombinant single-chain TCR ligands from HLA-DP2 (RTL600) and HLA-DP4 (RTL700) using structure-based design (19). There are four amino acid residues (V36A, D55A, E56A, and E69K) that differ between the peptide-binding/TCR recognition domains of HLA-DP2 and HLA-DP4 (shown in bold in Fig. 1⇓). Size-exclusion fast protein liquid chromatography (Fig. 2⇓A) and dynamic light scattering (data not shown) shows that refolding of the molecules resulted in >90% yield of monodisperse monomeric molecules. The presence of the native conserved disulfide bond between cysteines 16 and 78 (RTL600 amino acid numbering) was demonstrated by a gel shift assay (Fig. 2⇓B), and authenticity of the purified proteins was further confirmed by N-terminal amino acid analysis (data not shown). CD confirmed that the RTLs had highly ordered secondary structures (Fig. 2⇓C). RTL600 and RTL700 shared essentially identical secondary structural features (Table I⇓), and comparison with the secondary structures of DR2-derived RTL302 (determined by CD (20)) as well as with MHC-II molecules determined by x-ray crystallography (Table I⇓) provided strong evidence that the RTLs shared the β-sheet platform/anti-parallel α helix secondary structure common to all MHC-II Ag-binding domains (23, 24, 25, 26, 27). Whereas DR2-derived RTL302 required substantial modification to obtain monodispersity (20), the native sequence-derived RTL600 and RTL700 showed extraordinarily well-behaved solution phase characteristics in the absence of the Ig-fold domains that are part of the full-length progenitor MHC-II molecules.

FIGURE 1.
FIGURE 1.

Primary amino acid sequence alignment of human RTL600, RTL700, and RTL302. RTL600 was derived from HLA-DP2 (DPA1*0103/DPB1*0201), RTL700 from HLA-DP4 (DPA1*0103/DPB1*0401), and RTL302 from HLA-DR2 (DRB1*1501/DRA*0101) (20 ). Italics indicate non-native amino acid residues. Gaps in the sequences for optimal alignment (∗) and the β1//α1 junction (← →) are also shown. Note that only four amino acids differ between the DP2- and DP4-derived RTLs (shown in bold). The conserved cysteines that form a disulfide bond are underlined.

FIGURE 2.
FIGURE 2.

Purified human HLA-DP2- and HLA-DP4-derived RTL600 and RTL700 are monodisperse monomers in aqueous buffers, retain the highly conserved disulfide bond of MHC-II molecules, and have highly ordered structures as quantified by CD. A, Size exclusion chromatography of purified, refolded RTL600 and RTL700. Aliquots of RTL600 or RTL700 were loaded on a Superose 75 16/60 and separated according to size. Molecular mass standards: ferritin, 440 kDa; BSA, 67 kDa; OVA, 43 kDa; carbonic anhydrase, 29 kDa; and myoglobin, 17.3 kDa. The RTLs eluted between 62 and 70 ml (>95%) as monodisperse monomers. B, Samples of RTLs were boiled for 5 min in Laemmli sample buffer with or without the reducing agent 2-ME, and then analyzed by SDS-PAGE (12%). Nonreduced RTLs (− lane) have a smaller apparent molecular mass than reduced RTLs (+ lane), indicating the presence of a disulfide bond. First and last lanes, Molecular mass standards carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and bovine α-lactalbumin (14.4 kDa). RTLs (+/− β-ME), as indicated. C, CD measurements were performed at 25°C on an Aviv model 215 CD spectrometer using 0.1-mm cells at 0.5-nm intervals from 260 to 180 nm. The concentration values for each protein solution were determined by amino acid analysis. The buffer was 50 mM Tris (pH 8.5). Analysis of the secondary structure (Table I⇓) was performed using the variable selection method (65 ).

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Table I.

Structural analysis of DP-derived RTLs:comparison with MHC-II β-1/α-1 domains

Saturation-binding kinetics of RTL600, RTL700, and RTL302

A peptide capture ELISA was used to characterize saturation-binding kinetics of antigenic peptides to RTL600, RTL700, and RTL302 (32). Saturation-binding kinetics demonstrated the retention of peptide binding, a necessary prerequisite for using these molecules as Ag-specific attenuators of T cell behavior (33). Saturation binding of MOG35–55, MOG35–55 (W39A), and class II-associated invariant chain peptide (CLIP) 98–117, peptides that showed high, moderate, and low affinity, respectively, are representative of the useful range of information that can be obtained using this method (Fig. 3⇓). Whereas MOG is not found in BAL, this promiscuous MHC-II-binding peptide has proven to be a useful reference peptide because it binds to both the HLA-DP- and HLA-DR-derived RTLs. Competitive binding of peptides to RTL600, RTL700, and RTL302 at pH 5.4 and pH 7.4 was also carefully documented, and binding at equilibrium was plotted by nonlinear regression analysis, assuming a single binding site to quantify an EC50 for peptide binding (Table II⇓). Peptide binding to RTLs derived from HLA-DP2, HLA-DP4, and HLA-DR2 was consistent with previously published (34, 35, 36, 37, 38, 39) peptide-binding motifs for these MHC-II molecules. There was substantial overlap between the published motifs derived by sequencing natural epitopes isolated from HLA-DP2, HLA-DP4, and HLA-DR2, and this was reflected in our data for recombinant versions of these molecules lacking the α-2 and β-2 Ig-fold domains (Table II⇓). Peptide affinities appeared to be dominated by aromatic P1 residues. If this residue is substituted by an alanine (compare MOG35–55 vs MOG35–55, W39A; Fig. 3⇓ and Table II⇓), there was an almost 50-fold decrease in affinity of the peptide at pH 5.4, and a 1500- to 2000-fold decrease in affinity at pH 7.4. As stated above, CLIP98–117 had a low affinity for both RTL600 and RTL 700.

FIGURE 3.
FIGURE 3.

Saturation-binding kinetics of RTL600, RTL700, and RTL302. Saturation-binding data were plotted at equilibrium by nonlinear regression analysis using a sigmoidal dose-response curve with the data fit to the equation Y = Y0 + (Ymax − Y0)/1 + 10(LogEC50 − X)S. X is the logarithm of biotin-peptide concentration (μM) added, Y is the signal (OD405 − background), Y0 is the (OD405 − background) when biotin-peptide = 0, Ymax corresponds to the maximal signal obtained (OD405 − background), S is the hill slope, and the EC50 is the concentration of biotin-peptide at half-maximal saturation (Table II⇓; EC50). These data are a representative example of at least three independent experiments.

View this table:
Table II.

EC50 values derived from competitive binding analysisa

Beryllium binding to RTL600 directly detected by NMR

A previous study (40) suggested that beryllium could displace bound biotinylated CLIP peptide from HLA-DP2. The presence of 1 mM beryllium altered peptide binding to the rHLA-DP2, HLA-DP4, and HLA-DR2 molecules, with the greatest effect seen on RTL600. The absolute amount of 5 μM biotin-MOG peptide bound to RTL600 decreased by 28% at pH 7.4, compared with 6 and 8% decreases for RTL700 and RTL302 under the same experimental conditions (data not shown). Whereas these data demonstrated an effect of beryllium on peptide binding to RTL600, they also suggested a salt effect, with BeSO4 having a subtle but significant effect on all of the molecules tested. A far more rigorous and conclusive confirmation of beryllium binding to rHLA-DP2-derived RTL600 was obtained using 9Be NMR spectroscopy (Fig. 4⇓). The NMR spectrum of a 200 mM solution of RTL600 with 3 mM BeSO4 yields a well-resolved peak at 1.12 ppm. Control solutions of BeSO4 in buffer show no peak in the 9Be NMR, due to precipitation of the beryllium at pH 8.1. The presence of a single peak demonstrates a unique and well-defined binding site for beryllium on the HLA-DP2-derived RTL600, and the intensity indicates a stoichiometry of at least five beryllium atoms per protein (Fig. 4⇓A). Further characterization of the stoichiometry of beryllium binding was done by purifying the NMR sample via ultrafiltration and then determining protein concentration by UV/Vis and the beryllium concentration by ICP-AES. The results based on concentration ratios gave a stoichiometry of 6.5 beryllium atoms per RTL600. Under the same conditions, there was no signal in the 9Be NMR spectrum with RTL700 (Fig. 4⇓B). The peak position of 1.12 ppm observed with RTL600 is similar to that of (Be4O)acetate4 (41) and recently synthesized complexes of beryllium bound to multicarboxylate polymers (42). Although this peak position suggests that multiple carboxylate residues could be involved in the interaction, the exact location of beryllium binding to HLA-DP2 remains unknown. Three of the differences between RTL600 and RTL700 involve substitutions of carboxylate residues (e.g., RTL600, DEE, 55–57; EEE, 67–69; RTL700, AAE, 55–57; EEK, 67–69; see Fig. 1⇑). However, using fibroblasts expressing mutant HLA-DP2 molecules, we recently showed (43) that the critical amino acid in the HLA-DP β-chain for beryllium-induced T cell activation is the glutamate at position 69 (Glu69), with the negatively charged polymorphism at either position 55 or 56 having no effect on beryllium presentation to CD4+ T cells. The absence of Glu69 and possibly the triad of glutamate residues at positions 67–69 explain why RTL700 is unable to bind Be2+. In addition, BeSO4 binding to RTL600, but not RTL700, significantly altered the binding of a mAb (NFLD.M73) specific for an epitope located in the peptide-binding region of HLA-DP2 that is not present in DP4 (Fig. 4⇓C).

FIGURE 4.
FIGURE 4.

Beryllium binds to HLA-DP2, but not HLA-DP4. Beryllium binding to RTL600 (A) and RLT700 (B), as detected directly by NMR, is shown. 9Be spectra showed specific binding of beryllium to RTL600 in the form of a readily detectable bound carboxylate species at ∼2 ppm. No binding to RTL700 was observed under the same conditions. C, Beryllium inhibits binding of mAb, NFLD.M73, to DP2-derived RTL600, but not DP4-derived RTL700.

Beryllium presentation occurs in the absence of Ag processing

To determine whether beryllium presentation to CD4+ T cells required Ag processing before presentation, paraformaldehyde fixation of autologous EBV-transformed LCLs was performed. Using LCLs that had been pulsed overnight with 100 μM BeSO4 before fixation induced vigorous T cell proliferation in two HLA-DP2-restricted CD4+ TCLs (Fig. 5⇓). For example, TCL 1041 proliferated in the presence of BeSO4-pulsed, fixed LCL with a thymidine incorporation of 77,996 ± 4,410 cpm compared with background proliferation of 2,504 ± 34 cpm (Fig. 5⇓). Similar findings were seen with the second TCL (1332). Both CD4+ TCLs also proliferated in the presence of fixed LCLs following the addition of 100 μM BeSO4 to the culture. As an example, following the addition of fixed LCLs plus BeSO4, TCL 1041 vigorously proliferated with a thymidine incorporation of 67,427 ± 318 cpm (p > 0.05 compared with BeSO4 pulsed before fixation; Fig. 5⇓). Thus, similar to the findings by Marx et al. (44), beryllium processing is not necessary for presentation and resultant activation of beryllium-specific CD4+ T cells.

FIGURE 5.
FIGURE 5.

Beryllium presentation in the absence of Ag processing. Proliferation of two beryllium-specific CD4+ TCLs in the presence of paraformaldehyde-fixed autologous APCs that were either prepulsed or pulsed after fixation with 100 μM BeSO4 is shown. The data are presented as mean ± SEM. This is a representative example of three independent experiments.

It is not known whether Be2+ binding to HLA-DP2 occurs in a reversible, pH-dependent manner. To address this question, fixed, autologous LCLs were preincubated with BeSO4, extensively washed, and exposed to various pHs in an attempt to remove any bound Be2+ from the MHC-II. The treated cells were then tested for their ability to induce intracellular IFN-γ expression in TCL 1332 with and without the addition of BeSO4 to the culture (Fig. 6⇓). BeSO4-pulsed, fixed LCLs exposed to neutral pH presented Be2+ to TCL 1332 equally well, regardless of whether additional Be2+ was added to the culture, suggesting that Be2+ preincubation saturated the presenting ability of the fixed LCLs. In contrast to Ni2+, in which there was a precipitous drop in the presenting ability of the fixed APC below pH 5.5 (1), there was a more gradual loss of presenting ability as the pH approached 4.0 that could be restored by the addition of BeSO4 to the culture medium. Treatment below pH 3.0 resulted in an irreversible loss of the ability of fixed LCLs to present Be2+ to the TCL. Similar to Ni2+, these results suggest that Be2+ is reversibly bound to the fixed LCLs via a pH-sensitive interaction consistent with the coordination of amino acid side chains from negatively charged residues such as aspartic acid (pK 3.9) and/or glutamic acid (pK 4.2) residues. The ability of the fixed LCLs to present SEB was unaltered over the range of pHs tested (pH 3.0–5.5; Fig. 6⇓).

FIGURE 6.
FIGURE 6.

pH-dependent, reversible binding of Be2+ to fixed autologous EBV-transformed LCLs. LCLs were fixed with paraformaldehyde and incubated with 100 μM BeSO4 overnight at 37°C. Cells were washed to remove unbound Be2+ and exposed to various pHs for 40 min at 37°C. Following washing of cells with HBSS, the treated cells were used as APCs for IFN-γ expression by beryllium-specific CD4+ T cells, alone (▪), plus 100 μM BeSO4 (•),or plus 10 ng/ml SEB (▴). This is a representative example of three independent experiments.

Ability of RTL600 to present beryllium to a beryllium-specific CD4+ TCLs

Using a BAL-derived beryllium-specific CD4+ TCLs (1332) (17, 45), we characterized the effects of rHLA-DP2- and HLA-DP4-derived molecules on T cell proliferation and Th1-type cytokine production. We have previously shown (15, 17, 45) the ability of this TCL to proliferate and secreted IFN-γ, TNF-α, and IL-2 in response to beryllium presentation in the context of HLA-DP2. Using IFN-γ secretion as an indicator of T cell activation, we characterized the ability of RTL600 to activate TCL 1332 in the presence of BeSO4. As shown in Fig. 7⇓A, TCL 1332 exposed to RTL600 loaded exogenously with beryllium secreted IFN-γ. An irrelevant RTL (RTL302) in the presence of beryllium was unable to induce IFN-γ secretion under the same conditions (Fig. 7⇓A). If 1 ng/ml IL-2 was present during the assay (preventing activation-induced cell death), IFN-γ secretion at 24 h showed similar levels to that seen when TCL 1332 was exposed to beryllium in the presence of autologous LCL (Fig. 7⇓B and Refs. 17 and 45). Furthermore, anti-DP mAb completely blocked IFN-γ secretion (Fig. 7⇓B).

FIGURE 7.
FIGURE 7.

IFN-γ secretion assay. A, Plates were coated 24 h with DR2-derived RTL302 or DP2-derived RTL600. Plates were washed, loaded with 10 μM beryllium in conditions as indicated for 24 h and washed again, and then T cells were layered into wells at 1 × 106cells/well in RPMI 1640/FBS. Supernatants were collected at 24 h and IFN-γ was quantified by ELISA. RTL600/Be directly induced IFN-γ production in BAL T cells. B, Experiment 2, as in A, with RTL600/Be in RPMI 1640/FBS containing 1 ng/ml IL-2. Anti-DP Ab at 20 μg/ml completely blocked secretion of IFN-γ. Each panel is representative of two individual experiments.

Plate-bound RTL600 loaded with beryllium in complete culture medium blocked both proliferation (Fig. 8⇓A) and IL-2 production (Fig. 8⇓B) when cells were later exposed to LCLs in the presence of BeSO4, effectively tolerizing the T cells. RTL700 showed no effect on either proliferation or IL-2 production when used under the same conditions (Fig. 8⇓, A and B). When RTL600 was exposed to saturating concentrations of MOG (20 μM), a peptide with a very high affinity for RTL600 (EC50 = 0.2 μM at pH 7.4; Fig. 3⇑) and then loaded with beryllium, RTL600 (RTL600 plus Be plus MOG35–55) did not block proliferation (Fig. 8⇓A) nor did it inhibit IL-2 production upon re-exposure to LCLs in the presence of beryllium salts (Fig. 8⇓B). Anti-HLA-DP2 (clone B7/21) mAb effectively blocked the ability of DP2-derived RTL600 to tolerize TCL 1332 (Fig. 8⇓C). These data demonstrated that RTL600 in the presence of beryllium could be used to tolerize pathogenic CD4+ T cells, preventing activation of these T cells upon re-exposure to beryllium in culture.

FIGURE 8.
FIGURE 8.

Preculturing BAL-derived TCL 1332 with RTL600 loaded with beryllium blocks T cell proliferation and IL-2 production upon re-exposure of the TCLs to APC/Be. Plates coated with RTL600 loaded with beryllium tolerize TCL 1332 and inhibit proliferation (A) and IL-2 production when exposed to APC/Be (B). The effect is specific, and DP4-derived RTL700/Be does not tolerize the TCLs. C, Anti-DP2 mAb blocks the ability of DP2-derived RTL600 to tolerize TCL 1332. Cells were exposed to RTL/Be for 24 h, washed, and then exposed to APC (1:1 ratio) plus 20 μM BeSO4. Each panel is a representative example of three independent experiments.

Discussion

Despite recent advances in our understanding of the immunopathogenesis of beryllium-induced disease, the mechanism(s) involved in the development of fibrotic lung disease in only a minority of beryllium-exposed workers remains poorly understood. In addition to beryllium exposure in the workplace, genetic susceptibility plays a major role in disease development, with Glu69-containing HLA-DP alleles being strongly associated with the development of beryllium sensitization (46, 47, 48, 49). Functional studies have also shown (43) that the Glu69 residue is of critical importance for beryllium presentation and subsequent CD4+ T cell activation. In addition, all Glu69-containing HLA-DP alleles express two other glutamate (E) residues at position 67 and 68, thus forming a triad of glutamate residues (50). Our results reported herein demonstrate the ability of Be2+ to directly bind to single chain HLA-DP2 constructs, but not similar constructs derived from HLA-DP4. In addition, the HLA-DP2-derived RTLs are functional, being able to stimulate beryllium-specific T cells, resulting in IFN-γ secretion. Pretreatment of CD4+ T cells with plate-bound DP2-beryllium renders them less responsive to subsequent T cell activation, raising the possibility of the use of these molecules as a therapeutic agent. The generation of these DP2 RTLs represent a major advance in the field of beryllium-induced disease and should assist in the delineation of the beryllium binding site on HLA-DP2, as well as the role, if any, of peptides.

Experiments presented in this manuscript describe, for the first time, the ability of recombinant single-chain HLA-DP-derived molecules to modulate the behavior of pathogenic CD4+ T cells isolated from the lungs of CBD patients. Soluble recombinant MHC-II molecules in various forms have been developed, from full-length detergent solubilized MHC-II heterodimers (51, 52) to various forms of the extracellular a1a2/b1b2 heterodimeric domains (53, 54, 55), and recently the a1 and b1 domains genetically linked into a single polypeptide chain (18, 19, 20, 21, 56). The latter appears to be the smallest structure that retains the peptide binding/TCR recognition features of MHC-II molecules (33). Although the ability of HLA-DR2-derived RTLs to modulate T cell behavior in vitro (21, 57) and in vivo (32, 58, 59) has been extensively studied, the nature of the interactions among HLA-DP2, beryllium, and CD4+ T cells has been hampered by the chemistry of the components being studied. Whereas HLA-DP2-derived RTL600 and HLA-DP4-derived RTL700 have good solution-phase properties under physiologically relevant conditions, these molecules aggregate and precipitate below pH 5.4. In addition, beryllium precipitates above pH 6.0. By coating 96-well plates with RTL molecules and then loading them with beryllium, we have identified conditions that have allowed us to study HLA-DP2/beryllium interactions with T cells.

The 9Be NMR spectroscopy clearly demonstrates that beryllium binds to a unique, well-defined site on the RTL600. This is the first time that beryllium binding to an HLA-DP analog has been directly observed. In the absence of beryllium binding, no NMR signal for the RTL700 was observed. The 63-Hz width at half-height of the peak observed with RTL600 suggests that the beryllium atoms reside in similar chemical environments and are not undergoing rapid exchange between a variety of sites on the protein. The stoichiometry of the binding was determined to be at least five beryllium atoms per RTL600 based on the intensity of the NMR signal and further refined to a value of 6.5 beryllium atoms based on analysis of a purified solution by a combination of UV/Vis for protein concentration and ICP-AES for beryllium concentration. The value of 6.5 may be high due to potential amorphous beryllium atom precipitates in solution that would not be observed in the NMR but would be detected by the ICP-AES. The ppm shift of the 9Be peak is consistent with a carboxylate-binding site that is expected to be present in RTL600 and not in RTL700 (60). In addition, the exposure of BeSO4-pulsed, fixed APCs to various pHs resulted in dramatic reduction in IFN-γ expression that occurred near the pK of glutamic acid residues.

rHLA-DP2-derived RTL600 showed potent bioactivity when loaded with beryllium. Using a beryllium-specific CD4+ TCL, BeSO4-loaded RTL600 clearly induced IFN-γ secretion. T cell activation was occurring through the HLA-DP2 molecule since blocking this molecule with an anti-HLA-DP mAb inhibited IFN-γ secretion. One of the most important observations, presented in this study for the first time, is that rHLA-DP2 loaded with beryllium tolerizes beryllium-specific pathogenic CD4+ T cells. This suggests a straightforward approach toward tolerizing and/or deleting pathogenic T cells from BAL of patients with CBD. The tolerization and/or deletion of pathogenic CD4+ T cells through the use of these RTLs could represent an important therapeutic advance, especially in a disease characterizing by persistent Ag exposure. Studies (61, 62, 63) have identified beryllium in the lungs of CBD patients years after beryllium exposure cessation. Consistent with the persistence of beryllium in the lung, we have also identified persistent oligoclonal T cell populations in the lungs of CBD patients at every time point analyzed over a 5-year period (14). In addition, preliminary experiments have shown that the frequency of beryllium-specific CD4+ T cells in the lung of CBD patients remains stable over time despite corticosteroid treatment. Thus, it is not surprising that the natural history of this disease is characterized by a gradual decline in lung function with one-third of untreated patients historically progressing to end-stage respiratory insufficiency (64).

9Be NMR data and the ability of beryllium to inhibit the binding of a mAb specific for an epitope within the HLA-DP binding groove suggest a unique binding site for beryllium on HLA-DP2-derived RTL600. Modeling studies (60) predict that a high-affinity beryllium binding site exists within the HLA-DP2 β-1 domain. Although there are two carboxylate clusters within the HLA-DP2 molecule, residues DEE (55, 56, 57) and EEE (67–69), a recent study from our group using fibroblasts expressing mutant versions of HLA-DP2 strongly suggested that the critical polymorphic amino acid in the HLA-DP β-chain for beryllium-induced T cell activation was Glu69 (43). The negatively charged polymorphisms at position 55 or 56 were not required for beryllium presentation to lung-derived CD4+ T cells in vitro. In addition, the polymorphism at positions 55 and 56 was shared between both beryllium-presenting and -nonpresenting HLA-DP molecules (43).

In summary, we have directly shown that Be2+ binds to soluble HLA-DP2 molecules and the ability of these DP2 RTLs to stimulate T cell proliferation and IFN-γ secretion. The assays can be used to monitor the bioactivity of rHLA-DP2 molecules as we engineer RTL600 variants that retain the ability to tolerize BAL CD4+ T cells, but do so in the absence of beryllium. Such molecules would potentially provide an immunotherapeutic option for the treatment of subjects with progressive fibrotic lung disease secondary to CBD.

Acknowledgments

We thank Jeffrey Mooney and Justin Chang for expert assistance with protein purification and characterization, as well as Dorian LaTocha for assistance with the IFN-γ ELISA.

Disclosures

G. G. Burrows and A. A. Vanderbark, and the Oregon Health and Science University have a significant financial interest in Virogenomics, Incorporated, a company that may have a commercial interest in the results of this research and technology. This potential conflict was reviewed, and a management plan was implemented after approval by the Oregon Health and Science University Conflict of Interest in Research Committee, Integrity Program Oversight Council, and the Conflict of Interest Committee at the Portland Veterans Affairs Medical Center.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grants ES10554 (to G.G.B.), LANL Directed Research Grant 20051027DR (to M.M.), and HL62410 and ES11810 (to A.P.F.) and by Department of Neurology, Oregon Health and Science University and the Veterans Affairs Medical Center at Portland, Oregon.

  • ↵2 Address correspondence and reprint requests to Dr. Gregory G. Burrows, UHS-46, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239. E-mail address: ggb{at}ohsu.edu

  • ↵3 Abbreviations used in this paper: CBD, chronic beryllium disease; BAL, bronchoalveolar lavage; RTL, rTCR ligand; NMR, nuclear magnetic resonance; ICP-AES, inductively coupled plasma-atomic emission spectrometer; TCL, T cell line; LCL, lymphoblastoid cell line; CTLL, cytotoxic TCL; RT, room temperature; CD, circular dichroism; MOG, myelin oligodendrocyte glycoprotein; CLIP, class II-associated invariant chain peptide; SEB, staphylococcal enterotoxin B.

  • Received April 17, 2006.
  • Accepted July 7, 2006.

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