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Beryllium Presentation to CD4⁺ T Cells Is Dependent on a Single Amino Acid Residue of the MHC Class II -Chain¹

Jerome R. Bill^*,, Douglas G. Mack^*, Michael T. Falta^*,, Lisa A. Maier^*,,, Andrew K. Sullivan^*, Fenneke G. Joslin^*, Allison K. Martin^*, Brian M. Freed^*,, Brian L. Kotzin^*, and Andrew P. Fontenot^2,*,

^* Department of Medicine, Department of Immunology, and Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, CO 80262; and Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206

	Abstract

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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 -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 -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- 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 -chain dictates beryllium presentation and potentially, disease susceptibility.

	Introduction

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Chronic beryllium disease (CBD)³ results from beryllium exposure in the workplace and is characterized by a CD4⁺ T cell alveolitis and granulomatous inflammation in the lung (1). Depending on the degree of beryllium exposure and the genetic susceptibility of the individual, this disorder develops in 2–16% of exposed workers (2, 3, 4, 5, 6, 7), with an average latency period of 10 years between exposure and disease development (2, 3, 4, 5, 6, 7). Because of its lightweight, high melting point, and tensile strength, beryllium continues to be used in a variety of high technology industries, with as many as 1 million U.S. workers having been exposed (8, 9). Thus, beryllium exposure in the workplace and CBD remain important public health concerns.

Strong evidence supports a critical role for CD4⁺ T cells in the immunopathogenesis of CBD. Previous studies from our laboratory have shown that large numbers of beryllium-specific, effector memory CD4⁺ T cells accumulate in the lungs of CBD patients, poised to proliferate and secrete Th1-type cytokines following beryllium recognition (10). These CD4⁺ T cells are composed of oligoclonal T cell populations, which persist in the lungs of subjects with active disease (11). Using beryllium salt patch testing as an in vivo model of granuloma formation, disease-relevant CD4⁺ T cells infiltrate the skin before the development of granulomatous inflammation (12). In addition, the current disease definition of CBD requires that blood and/or bronchoalveolar lavage (BAL) CD4⁺ T cells proliferate in the presence of beryllium salts in vitro. Thus, the presence of a known and persistent antigenic stimulus and high frequencies of Ag-specific CD4⁺ T cells in an accessible target organ distinguish CBD from other human conditions, making this disease a model of organ-specific, immune-mediated destruction.

Genetic susceptibility to both beryllium sensitization and CBD has been linked to particular alleles of the MHC class II molecule, HLA-DP (13, 14, 15, 16, 17). In particular, susceptibility has been most strongly associated with HLA-DPB1 alleles that encode a negatively charged glutamic acid (E) at position 69 (Glu⁶⁹) of the -chain. These susceptibility alleles were shown to be functional in that they allowed beryllium presentation to Ag-specific CD4⁺ T cells (18, 19), and an anti-HLA-DP-specific mAb blocked both beryllium-induced proliferation and Th1-type cytokine expression (10, 18, 19, 20). Two polymorphic regions with major charge differences at positions 55–56 and 69 on the DP -chain were apparent in the susceptibility alleles. Because the Glu⁶⁹ and the negatively charged aspartic acid and glutamic acid at positions 55 and 56 frequently coexist in disease-relevant HLA-DP alleles, it is possible that both polymorphisms contribute to disease development after beryllium exposure in the workplace. In addition, 15% of CBD patients do not possess a Glu⁶⁹-containing HLA-DP allele, suggesting the importance of other MHC class II molecules in the genetic susceptibility to beryllium-induced disease (13, 14). In this population of CBD patients, an increased frequency of HLA-DR13 alleles, which possess a glutamic acid at position 71 of the DR -chain (which corresponds to position 69 of HLA-DP) was seen (13).

We hypothesized that beryllium presentation to pathogenic CD4⁺ T cells was solely dependent on a negatively charged glutamic acid at a single position of the MHC class II -chain, irrespective of isotype. In this study, using ex vivo BAL cells and beryllium-specific CD4⁺ T cell lines, we show that beryllium presentation and T cell activation indeed require a glutamic acid at position 69 of the DP -chain and that mutation of the aspartic acid and glutamic acid at positions 55 and 56 to neutral alanines has no effect on beryllium-induced proliferation or IFN- expression. In addition, site-directed mutagenesis of the glutamic acid at position 71 of the DR -chain abolished IFN- secretion from DR13-restricted CD4⁺ T cells. Thus, the results presented herein pinpoint a single amino acid residue in the MHC class II -chain that dictates beryllium presentation and genetic susceptibility to disease.

	Materials and Methods

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Study population and HLA typing

The diagnosis of CBD was established using previously defined criteria (21, 22), including a history of beryllium exposure, the presence of granulomatous inflammation on lung biopsy, and a positive proliferative response of BAL T cells to BeSO₄ in vitro. HLA typing was performed by standard molecular techniques at the University of Colorado Health Sciences Center ClinImmune Labs. The class II HLA haplotypes of the CBD patients used in this study are shown in Table I. Informed consent was obtained from each CBD patient, and the protocol was approved by the Human Subject Institutional Review Board at the University of Colorado Health Sciences Center and National Jewish Medical and Research Center.

Site-directed mutagenesis of the HLA-DP and HLA-DR -chains and transfection

Mutagenesis of DPB1*0201 and DRB1*1302 cDNA was performed as described previously (23). HindIII-XhoI full-length DPB1*0201 cDNA was mutated at amino acid positions 55, 56, or 69 using overlapping primers. The forward and reverse primers for generating the DP2-K69 mutant were 5'-CTGGAGGAGAAGCGGGCAGTGCCGGACAGGATGTGC-3' and 5'-CGGCACTGCCCGCTTCTCCTCCAGGATGTCCTTCTG-3', respectively. The forward and reverse primers for generating the DP2-A55 mutant were 5'-CTGGGGCGGCCTGCTGAGGAGTACTGGAACAGCCAG-3' and 5'-CCAGTACTCCTCAGCAGGCCGCCCCAGCTCCGTCACCGC-3', respectively. The forward and reverse primers for generating the DP2-A56 mutant were 5'-CTGGGGCGGCCTGATGCGGAGTACTGGAACAGCCAG-3' and 5'-CCAGTACTCCGCATCAGGCCGCCCCAGCTCCGTCACCGC-3', respectively. HindIII-XhoI full-length DRB1*1302 cDNA was mutated at amino acid position 71 using overlapping primers. The forward and reverse primers for generating the DR13-R71 mutant were 5'-CTGGAAGACAGGCGGGCCGCGGTGGACACCTACTGC-3' and 5'-CACCGCGGCCCGCCTGTCTTCCAGGATGTCCTTCTG-3', respectively. PCR fragments containing the mutated DP2 and DR13 DNA were ligated into pcDNA3.1 (Invitrogen Life Technologies), and the sequence was confirmed by DNA sequencing.

Transfection into mouse DAP.3 L cells expressing DPA1*0103 or DRA1*01 was performed using Lipofectamine 2000 (Invitrogen Life Technologies), and selection in the presence of 0.5 mg/ml G418 was performed 48 h after the addition of DNA. After cloning via limiting dilution, the expression of HLA-DP was assessed using the HLA-DP-specific mAb, B7.21, and HLA-DR expression was confirmed using the HLA-DR-specific mAb, LB3.1, as described previously (19). For all experiments, surface expression of mutant HLA-DP and -DR molecules as measured by flow cytometry was equivalent to native DP2 and DR13 expression on the fibroblast cell lines, DP8302 (a gift from W. Marshall, Memorial University of Newfoundland, Newfoundland, Canada) and DAP3-DR1302 (a gift from C. Hurley, Georgetown University, Washington, D.C.), respectively.

Preparation of cells and generation of beryllium-specific T cell lines

BAL was performed as previously described (11, 19), and the generation of the EBV-transformed lymphoblastoid B cell line (LCL) from patient 2 has been described previously (19). EBV-transformed LCLs (SAVC, AMALA, HHKB, and PITOUT) were generous gifts from Dr. G. Nepom (Benaroya Research Institute at Virginia Mason, Seattle, WA). Beryllium-specific T cell lines were derived after initial stimulation of BAL cells in 12-well plates (Costar) with RPMI 1640 supplemented with 10% heat-inactivated human serum (Gemini Bio-Products), 20 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen Life Technologies) and the addition of 100 µM BeSO₄, as described previously (19). After 5 days of culture, cells were washed free of BeSO₄, and the lymphoblasts were expanded further in culture medium supplemented with 20 U/ml human rIL-2 (R & D Systems). The T cell lines were maintained in culture by cycles of restimulation (every 2–3 wk) with 100 µM BeSO₄-pulsed autologous, mitomycin C-treated LCLs and further expanded in culture with rIL-2.

Immunofluoresence staining and analysis for intracellular cytokine expression

For stimulation of cytokine secretion, ex vivo BAL cells (5 x 10⁵ cells) were placed in 12 x 75-mm polypropylene tubes (Fisher Scientific) containing 1 ml of RPMI 1640 supplemented with 10% heat-inactivated human serum (Gemini Bio-Products), 20 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen Life Technologies) and one of the following experimental conditions: medium alone, 10 ng/ml staphylococcal enterotoxin B (SEB), or 100 µM BeSO₄. Cells were incubated for a total of 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 described previously (10, 24). In some experiments, the presenting MHC class II molecules were determined by the addition of blocking mAbs to HLA-DR (hybridoma LB3.1; American Type Tissue Collection), HLA-DP (hybridoma B7.21; a gift from I. Trowbridge, Salk Institute, La Jolla, CA), and HLA-DQ (hybridoma SVPL3; a gift from Dr. G. Nepom) at the beginning of culture as described previously (10, 19). Control experiments using these Abs have been presented previously (19). IFN- expression with and without the indicated mAb was determined and the extent of inhibition was calculated.

In some experiments, LCLs (1 x 10⁷ cells/ml) were treated with 50 µg/ml mitomycin C for 1 h at 37°C in a humidified 5% CO₂ atmosphere followed by three washes with HBSS plus 5% FCS. The mitomycin C-treated LCLs were pulsed with either medium alone or 100 µM BeSO₄ overnight at 37°C. The cells were washed three times with HBSS and combined in a 1:1 ratio with the beryllium-specific T cell lines (1 x 10⁵ cells of each) in polypropylene tubes and cultured for 6 h as described above. Beryllium pulsing and washing of mitomycin C-treated APCs was necessary to prevent beryllium self-presentation by endogenous MHC class II molecules expressed on activated responder T cells.

After stimulation, cells were washed and stained with mAbs directed against CD3, CD4, and CD8 (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- (BD Biosciences) for 30 min at 4°C. The lymphocyte population was identified using forward and 90° light scatter patterns, and fluorescence intensity was analyzed using an FACSCalibur cytometer (BD Biosciences) as described previously (10, 24).

Lymphocyte proliferation assay

Ex vivo BAL cells (1 x 10⁵ cells) were cultured in 96-well flat-bottom microtiter plates in the presence of medium alone, 10 µM BeSO₄, or 2.5 µg/ml PHA. After 96 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. Inhibition of proliferation was attempted with addition of indicated amounts of mAbs directed to class II HLA molecules at the beginning of culture as described above. Proliferation with and without the indicated mAb was then determined, and the extent of inhibition was calculated. In some experiments, beryllium-specific T cell lines (5 x 10⁴ cells) were cultured in 96-well flat-bottom microtiter plates with 5 x 10⁴ medium or BeSO₄-pulsed, mitomycin C-treated, autologous or allogeneic LCL cells. The transfected fibroblast cells were mitomycin C treated, and the cells were washed three times with HBSS before use as APCs to ensure the absence of free BeSO₄ in culture.

	Results

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Inhibition of beryllium-induced responses with anti-MHC class II Abs

To determine the importance of amino acid positions 55, 56, and 69 of the HLA-DP -chain in the presentation of beryllium to CD4⁺ T cells, we identified a CBD subject expressing the HLA-DP allele, HLA-DPB1*0402, using MHC class II molecular typing. This allele is identical to the CBD susceptibility allele, HLA-DPB1*0201, except for a lysine (K) instead of a glutamic acid (E) at amino acid position 69 of the -chain. As shown in Table I, patient 2 expressed HLA-DRB1*0701 and *1301 while expressing HLA-DPB1*1001 and *0402. Using intracellular IFN- staining as a measure of T cell response, ex vivo BAL cells were stimulated with either medium alone, 10 µM BeSO₄, or 10 ng/ml SEB for a total of 6 h. Minimal background IFN- expression was seen with medium alone, whereas SEB exposure induced IFN- expression in 45 and 33% of CD4⁺ and CD4^– T cells, respectively (Fig. 1A). Following short-term culture with BeSO₄, 9.2% of this patient’s BAL CD4⁺ T cells were beryllium-specific, as detected by BeSO₄-induced IFN- expression. In contrast, few, if any, CD4^– T cells stained positively for IFN- after BeSO₄ exposure. To determine the restricting MHC class II molecule used to stimulate beryllium-specific CD4⁺ T cells, we used mAbs against HLA-DR, -DP, and -DQ to block beryllium presentation. The addition of the anti-DP mAb inhibited beryllium-stimulated IFN- expression in CD4⁺ T cells by 88% (9.2–1.1%) while the anti-DR mAb resulted in a 17% (9.2–7.6%) reduction in IFN- production, suggesting a dominant role for HLA-DP and a minor role for HLA-DR in the presentation of beryllium to CD4⁺ T cells in this patient (Fig. 1A). The anti-DQ mAb had a minimal effect on beryllium-stimulated IFN- expression. Fig. 1B shows the inhibition of IFN- expression in BAL CD4⁺ T cells from patient 2 when various concentrations of anti-MHC class II mAbs were added. The anti-DP mAb, B7.21, at concentrations of 30, 3, and 0.3 µg/ml resulted in an 88, 86, and 5% inhibition of IFN- expression, respectively. In contrast, the anti-DR mAb, LB3.1, at the same concentrations, resulted in a 17, 17, and 4.3% inhibition of IFN- expression in beryllium-stimulated BAL CD4⁺ T cells. At all concentrations used, the anti-DQ mAb had a minimal effect.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Intracellular IFN- expression and proliferation by ex vivo BAL CD4+ T cells from CBD patient 2 after stimulation. A, Staining patterns are shown for BAL cells after short-term culture with medium alone, 10 µM BeSO4, or 10 ng/ml SEB. The percentage of T cells expressing intracellular IFN- is shown in the upper quadrants of the density plots. Inhibition of beryllium-induced IFN- expression in BAL cells with 30 µg/ml anti-MHC class II mAbs is shown in the lower panels, with the percentage of CD4+ T cells expressing intracellular IFN- shown in the upper right quadrants. B, Inhibition of beryllium-induced intracellular IFN- expression in BAL cells from CBD patient 2 using various concentrations (30, 3, and 0.3 µg/ml) of mAbs directed against MHC class II molecules is shown. C, Inhibition of beryllium-induced T cell proliferation of BAL cells from CBD patient 2. Ex vivo BAL cells (1 x 105 cells) were stimulated with 10 µM BeSO4 in 96-well flat-bottom plates for 96 h. To inhibit proliferation, various concentrations (30, 3, and 0.3 µg/ml) of mAbs directed against MHC class II molecules were added at the beginning of culture. Data are expressed as mean cpm ± SEM.

The restricting HLA-DPB1 and -DRB1 alleles possibly involved in beryllium presentation for patient 2 were determined using a BAL-derived, beryllium-specific T cell line from this patient and a panel of allogeneic EBV-transformed LCLs. Seventy-four percent of the CD4⁺ T cells from this T cell line expressed IFN- in the presence of beryllium presented by the autologous LCL (Fig. 2A). Similarly, allogeneic cells that expressed either DPB1*1001 or DRB1*1301 also resulted in beryllium-induced IFN- expression (Fig. 2A). For example, beryllium presentation by the allogeneic LCLs, SAVC and HHKB, resulted in IFN- expression by 71 and 3.9% of the CD4⁺ T cells, respectively. Allogeneic cells matched only for DPB1*0402 (Fig. 2A) or DRB1*0701 (data not shown) were unable to induce the Th1-type cytokine response. In contrast, only presenting cells expressing DPB1*1001 were able to induce beryllium-specific proliferation of the T cell line (Fig. 2B).

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Determination of the MHC class II allele restriction of a BeSO4-specific T cell line from CBD patient 2. A, Intracellular IFN- expression of the T cell line in response to either medium alone, 100 µM BeSO4, or 10 ng/ml SEB presented by either autologous or allogeneic LCLs used as APCs is depicted. The percentage of T cells expressing intracellular IFN- is shown in the upper quadrants. The molecular typing of the matching DRB1 or DPB1 allele for each APC is shown on the right. B, Proliferative response of the beryllium-specific T cell line using the same APCs as in Fig. 3A is shown. Data are expressed as mean cpm ± SEM. The molecular typing of the matching DRB1 or DPB1 allele for each APC is shown on the right. The scintillation index for each of the APCs is only shown for beryllium-induced proliferation.

To definitively prove which polymorphic amino acid residues of the HLA-DP2 -chain are critical for beryllium presentation to CD4⁺ T cells, site-directed mutagenesis of the HLA-DPB1*0201 allele was performed. Mutations were targeted at positions 55, 56, and 69 of the DP2 -chain, and the change of sequence was based on the amino acids present in the nonpresenting DPB1*0101 allele (Table II). Mutagenesis was not performed on invariant amino acid residues (i.e., those present in presenting and nonpresenting HLA-DPB1 alleles) such as Glu⁶⁷ and Glu⁶⁸. Thus, three HLA-DP2 mutants were generated, changing the aspartic acid and glutamic acid at positions 55 and 56, respectively, to alanines (DP2-A55 and DP2-A56) and changing the glutamic acid at position 69 to lysine (DP2-K69). The mutant HLA-DP2 -chains were transfected into murine DAP.3 L cells, which had been transfected previously with the HLA-DPA1*0103 molecule. After selection in G418 and limiting dilution cloning, equivalent HLA-DP expression on the surface of the various DP2 mutants as detected by B7.21 staining was seen (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Staining of DAP.3 L cells for surface expression of either HLA-DP or HLA-DR. A, DAP.3 L cells transfected to express either native DP2 (DP8302) or a DP2 mutant (DP2-K69, DP2-A55, and DP2-A56) were stained with either an IgG1 isotype control (open histogram) or an anti-HLA-DP-specific mAb, B7.21 (filled histogram). B, DAP.3 L cells transfected to express either native DR13 (DR1302) or a DR13 mutant (DR1302-R71) were stained with either an IgG2a isotype control (open histogram) or an anti-HLA-DR-specific mAb, LB3.1 (filled histogram).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Induction of beryllium-specific T cell activation using various mutant DP2-expressing fibroblasts. A, Intracellular IFN- expression in the T cell line from patient 4 in response to BeSO4 presentation by fibroblasts expressing either native DP2 (DP8302 cell line) or a DP2 mutant is shown. Three DP2 mutants were used: DP2 with an alanine (A) substitution at position 55, DP2 with an alanine (A) substitution at position 56, and DP2 with a lysine (K) substitution at position 69. The percentage of CD4+ T cells expressing intracellular IFN- is shown in the upper right quadrant of each density plot. B, T cell proliferation of the T cell line from patient 4 using BeSO4-pulsed, mitomycin C-treated fibroblasts expressing either the native DP2 molecule or DP2 mutants is shown. The stimulation index for each of the various presenting cells is shown over each bar, and the data are expressed as the mean cpm ± SEM.

Combining our present findings and those previously published by our group (19), Table II summarizes the polymorphic amino acid residues of the -chains from selected HLA-DP molecules based on their ability or inability to stimulate beryllium-specific T cells. All of the presenting HLA-DP molecules possess a Glu⁶⁹ while the nonpresenting molecules express a positively charged lysine (K). The negatively charged polymorphism at positions 55 and 56 is shared between presenting and nonpresenting HLA-DP molecules. Based on these observations, the critical amino acid in the HLA-DP -chain for beryllium-induced T cell activation is the glutamic acid at position 69. The negatively charged polymorphism at either position 55 or 56 is not necessary for beryllium presentation.

Ability of certain HLA-DR molecules to present beryllium to CD4⁺ T cells

Approximately 15% of CBD patients do not possess a HLA-DP allele encoding a Glu⁶⁹, suggesting that other MHC class II alleles may be involved in the activation of CD4⁺ T cells (13, 14). In a recent study, it was shown that subjects who did not possess a Glu⁶⁹-containing HLA-DP allele had an increased frequency of HLA-DR13 alleles (13, 14). In the present study, it appeared that certain HLA-DR alleles, such as DRB1*1301, were capable of binding beryllium and stimulating IFN- expression in CD4⁺ T cells (Figs. 1 and 2). To further investigate this possibility, we identified two HLA-DP Glu⁶⁹-negative CBD subjects expressing the HLA-DR allele, HLA-DRB1*1302. We performed intracellular IFN- staining on ex vivo BAL cells from patient 1 (DRB1*1302 and DPB1*0101/*0402) and patient 6 (DRB1*0301/*1302 and DPB1*0101/*0501) (Fig. 5, A and B). Following short-term culture with BeSO₄, 3.7% of patient 1’s BAL CD4⁺ T cells were beryllium-specific, as detected by BeSO₄-induced IFN- expression. Using blocking mAbs against HLA-DR, -DP, and -DQ, the majority of beryllium-stimulated IFN- expression was inhibited in the presence of 30 µg/ml anti-DR mAb while the anti-DP and -DQ mAbs had no effect on IFN- expression (Fig. 5A). Thus, in patient 1, the beryllium-presenting MHC class II molecule was DRB1*1302. Similar findings were seen using BAL cells from patient 6 where the majority of beryllium-stimulated IFN- expression was blocked by the addition of 30 µg/ml anti-DR mAb with anti-DP and -DQ mAbs having no effect (Fig. 5B). Fig. 5C shows the inhibition of IFN- expression in BAL CD4⁺ T cells from patients 1 and 6 using various concentrations (30, 3, and 0.3 µg/ml) of anti-MHC class II mAbs.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Beryllium-specific intracellular IFN- expression by ex vivo BAL cells from patients 1 (A) and 6 (B). Staining patterns are shown for ex vivo BAL cells after short-term culture with medium alone, 10 µM BeSO4, or 10 ng/ml SEB. The percentage of T cells expressing intracellular IFN- is shown in the upper quadrants. Inhibition of the beryllium-induced IFN- expression in BAL cells with 30 µg/ml anti-MHC class II mAbs is shown in the lower panels. The percentage of CD4+ T cells expressing intracellular IFN- is shown in the upper right quadrants. C, Inhibition of beryllium-stimulated intracellular IFN- expression in BAL cells from CBD patients 1 and 6 using various concentrations (30, 3, and 0.3 µg/ml) of mAbs directed against MHC class II molecules is shown.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Beryllium-specific intracellular IFN- expression (A) and proliferation (B) of the T cell line from patient 6 using allogeneic LCLs as APCs is shown. The percentage of CD4+ T cells expressing intracellular IFN- is shown in the upper right quadrants. The molecular typing of the matching DRB1 or DPB1 allele for each APC is shown on the right. For the proliferation assay, data are expressed as mean cpm ± SEM, and the stimulation index for each of the APCs is only shown for beryllium-induced proliferation.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Intracellular staining for IFN- expression in a T cell line from patient 6. The T cells were stimulated in short-term culture with either medium alone, 100 µM BeSO4, or 10 ng/ml SEB using fibroblasts expressing either native DR1302 or DR1302-R71 mutant as APCs. The number in the upper right quadrant of each density plot indicates the percentage of CD4+ T cells expressing intracellular IFN-.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

It has become apparent that a significant proportion of CBD patients do not possess a Glu⁶⁹-containing HLA-DP allele, raising the possibility that other MHC class II molecules may be involved in the genetic susceptibility to beryllium-induced disease (13, 14). In this population of patients, Maier et al. (13) recently reported an increased frequency of HLA-DR13 alleles, which possess a glutamic acid at position 71 (Glu⁷¹) of the DR -chain (which corresponds to position 69 of HLA-DP). In addition, increased expression of the HLA-DR13 allele, DRB1*1302, has been seen in beryllium sensitization and CBD (13). Very few HLA-DR molecules express a glutamic acid at this position of the -chain. Besides the majority of DR13 molecules (Table III), several DR11 molecules also possess this amino acid. Interestingly, HLA-DRB1*1101 possesses a glutamic acid and aspartic acid at positions 69 and 70 with an arginine at position 71 but is unable to present beryllium to Ag-specific CD4⁺ T cells (Table III). In the current study, two CBD patients who were HLA-DP Glu⁶⁹-negative expressed HLA-DRB1*1302 (patients 1 and 6). Beryllium presentation to CD4⁺ T cells from these patients was mediated through DRB1*1302. A comparison of the key amino acid sequences of presenting and nonpresenting DRB1 alleles shows significant charge differences with only one polymorphic amino acid residue shared between all presenting alleles, Glu⁷¹ (Table III). Because position 71 of the DR -chain corresponds to position 69 of the DP -chain, we performed site-directed mutagenesis at position 71 of the DR13 -chain to determine whether this amino acid position in HLA-DR was as critical in mediating beryllium presentation as in HLA-DP. As shown in Fig. 7, fibroblasts expressing DR1302 with an arginine (R) at position 71 were no longer able to induce IFN- expression after short-term culture in the presence of beryllium. Comparing the amino acid residues present in DQB1, DRB3, DRB4, and DRB5 alleles, none express a glutamic acid at position 71 of the -chain, and as expected, these alleles have not been linked to genetic susceptibility of beryllium-induced disease (13, 14). Taken together, these results indicate that a single amino acid position determines beryllium presentation and subsequent T cell activation. Analysis of MHC class II molecular typing of 25 CBD patients has shown that all possess a glutamic acid at the critical amino acid position of either HLA-DP or -DR, raising the possibility that this position is solely responsible for genetic susceptibility to disease (our unpublished data). In addition, when both HLA-DP and -DR alleles contain a glutamic acid at this position, a dominant role for HLA-DP and a minor role for HLA-DR in the presentation of beryllium to CD4⁺ T cells were seen as in patient 2.

One fundamental unanswered question is what makes this particular position critical for beryllium recognition. In addition, how does the beryllium moiety interact with this amino acid? To date, no crystal structure for HLA-DP2 or DR13 exists, but HLA-DP2 modeling based on the known crystal structure of HLA-DR1 suggests that the Glu⁶⁹ is located on the -helix of the -chain with side groups pointing into the peptide-binding cleft (19, 27). Based on HLA-DP2 modeling studies, the Glu⁶⁹ appears to be involved in the formation of the P4 pocket (27). Berretta et al. (27) suggested that the altering of the HLA-DP2 molecule with a lysine at position 69 (Lys⁶⁹) as opposed to a glutamic acid induced significant changes in both the shape and charge distribution of the P4 pocket, as well as the neighboring P6 pocket. In particular, HLA-DP2 possessed a deeper P4 pocket compared with HLA-DP2 Lys⁶⁹. In all HLA-DP molecules capable of presenting beryllium to CD4⁺ T cells, a cluster of three negatively charged glutamic acids exist at positions 67–69 of the -chain. Equally interesting is the lack of a role played by the other cluster of negative charges at positions 55–57. It remains possible that the invariant Glu⁶⁷ and Glu⁶⁸ residues (as well as other negatively charged residues) in addition to Glu⁶⁹ may be critical for beryllium binding and presentation by HLA-DP. The same may also be true for HLA-DR.

The role of peptides bound to MHC class II molecules in beryllium presentation remains an important unanswered question. There are undoubtedly differences in the composition of the peptides presented by fibroblasts as opposed to professional APCs. For the current experiments, if particular peptides are required for beryllium presentation, those present in these transfected fibroblasts are sufficient because fibroblasts expressing native HLA-DP2 or -DR1302 stimulate T cell activation of beryllium-specific CD4⁺ T cells.

HLA-DP is the least polymorphic and least abundantly expressed of the human class II Ags. For example, Gorga et al. (28) obtained 60- to 100-fold lower yields of purified DP protein compared with DR. Similar to HLA-DR and -DQ, certain HLA-DP alleles have been implicated in the genetic susceptibility of a variety of diseases, including hard metal lung disease (29), pauciarticular juvenile chronic arthritis (30), and Graves disease (31). In particular, cobalt exposure in the workplace has been associated with hard metal lung disease, and cobalt has been shown to bind to Glu⁶⁹-containing HLA-DP alleles (32). The increased susceptibility to disease associated with MHC class II molecules is most likely due to the ability of those molecules to present critical epitopes to pathogenic T cells. We have previously shown that the HLA-DP susceptibility alleles associated with beryllium-induced disease match those capable of presenting beryllium salts in culture to Ag-specific CD4⁺ T cells, thus providing a mechanism for this association (19). We have further advanced those findings here by showing that the increased frequency of HLA-DRB1*1302 in CBD as reported by Rossman et al. (14) is indeed due to the ability of those Glu⁷¹-containing HLA-DR molecules to bind and present beryllium to CD4⁺ T cells.

The ability of a single amino acid residue to affect disease susceptibility is not unprecedented. In type 1 diabetes, position 57 in DQ has been associated with susceptibility and resistance to disease (33, 34). For example, the presence of an aspartic acid at this position protects against diabetes while a noncharged amino acid residue predisposes to disease development. This amino acid position is involved in the formation of a salt bridge with a conserved arginine (R) residue at position 79 of the class II -chain that is critical in the formation of the Ag-binding cleft (35). In rheumatoid arthritis, the shared epitope among HLA-DR4 alleles includes amino acid position 71 (36). The disease-associated DR4 alleles in rheumatoid arthritis include HLA-DRB1*0401 and *0404, which contain a positively charged lysine (K) or arginine (R), respectively, at position 71. Peptides with a negative charge at the p4 position allow for strong binding to rheumatoid arthritis-associated DR4 alleles but poor binding to nonassociated alleles (37). As we have shown in CBD, these findings suggest that the DR4 molecules expressing the shared epitope are involved in the presentation of arthritogenic peptides to autoreactive T cells, although this has been a difficult hypothesis to prove in the setting of an unknown inciting autoantigen(s).

One interesting and unexpected observation of this study was the ability of TCR engagement by beryllium-specific CD4⁺ T cells to induce IFN- expression in the absence of T cell proliferation. As shown in Fig. 2, 3.9% of CD4⁺ T cells from the T cell line of patient 2 expressed IFN- in response to the beryllium-pulsed LCL, HHKB, while no proliferation above background levels was seen. In addition, 30 µg/ml anti-DR mAb blocked 17% of IFN- expression using ex vivo BAL cells from this same subject (Fig. 1A), while this same concentration of Ab did not affect beryllium-induced T cell proliferation (Fig. 1C). In the lung, it is clear that certain beryllium-specific effector memory CD4⁺ T cells proliferate poorly after in vitro Ag exposure but retain the capacity to secrete Th1-type cytokines (10, 38). These observations raise the possibility that different intracellular signaling mechanisms in BAL T cells govern proliferation vs Th1-type cytokine secretion.

The current studies extend our understanding of the interaction of beryllium with certain MHC class II molecules. We have definitively shown that the glutamic acid at position 69 of HLA-DP and position 71 of HLA-DR dictates the ability of certain class II molecules to bind and present beryllium salts to pathogenic lung CD4⁺ T cells. Whether this interaction occurs through a peptide intermediary or direct interaction of the beryllium moiety with this amino acid remains a fundamental unanswered question. Thus, beryllium presentation and potentially genetic susceptibility to disease have been linked to a single amino acid on the -chain of MHC class II molecules. These findings further our understanding of T cell recognition in beryllium-induced disease and have broad implications for all genetically determined CD4⁺ T cell-dependent disorders.

	Disclosures

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The authors have no financial conflict of interest.

	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 HL62410, ES06358, and ES011810, and General Clinical Research Center Grant M01-RR00051 from the Division of Research Resources.

² Address correspondence and reprint requests to Dr. Andrew P. Fontenot, Division of Clinical Immunology (B164), University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: andrew.fontenot{at}uchsc.edu

³ Abbreviations used in this paper: CBD, chronic beryllium disease; BAL, bronchoalveolar lavage; BeSO₄, beryllium sulfate; LCL, lymphoblastoid B cell line; SEB, staphylococcal enterotoxin B.

Received for publication June 27, 2005. Accepted for publication September 1, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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