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Departments of
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Medicine,
Immunology, and
Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, CO 80262; and Departments of
§
Medicine and
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Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain genes expressed by these CD4+ T cells. In the
lungs of individual patients, as well as among four of five CBD
patients studied, different oligoclonal expansions within the Vß3
subset were found to express homologous or even identical CDR3 amino
acid sequences. These related expansions were specific for CBD
patients, were compartmentalized to lung, and persisted at high
frequency in patients with active disease. Limiting dilution cloning
and analysis of coexpressed TCR -chain genes confirmed that these
TCRs were selectively expanded by a common Ag involving beryllium.
Overall, homologous TCR ß- and -chains showed identical V regions
and invariant charged residues within the CDR3 but considerable
variability in TCRJ usage. Remarkably, CBD patients expressing nearly
identical TCRs did not share common HLA-DRB1 or DQ alleles. These
results implicate particular CD4+ cells in the pathogenesis
of CBD and provide insight into how beryllium is recognized in human
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence suggests that CD4+ T cells are important in the immunopathogenesis of CBD (7, 8, 9, 10). For example, sensitization to beryllium is detected by the ability of CD4+ T cells to proliferate in response to beryllium sulfate (BeSO4) in culture, and the development of granulomatous inflammation in the lung is associated with the accumulation of CD4+ T cells in the bronchoalveolar lavage (BAL) (3, 8, 9, 11, 12). The in vitro T cell response to BeSO4 requires presenting cells expressing class II MHC molecules and has been shown to be MHC restricted (9). How the TCR interacts with beryllium and the MHC remains unknown. Recent studies of BAL CD4+ T cells from CBD patients showed alterations of TCR ß-chain V region (Vß) expression compared with peripheral blood (10). In particular, 11 of 28 CBD patients demonstrated 16 different T cell subset expansions in the BAL, and approximately one-third of these expansions expressed Vß3. We hypothesized that these increased percentages of Vß3+ T cells in CBD patients represent clonal or oligoclonal expansions, consistent with conventional Ag stimulation.
In the present study, we analyzed the expressed TCR ß-chain (TCRB)
and -chain (TCRA) genes and specificity of expanded
CD4+ T cells in the lungs of CBD patients. We
identified oligoclonal expansions within the Vß3 subset that
persisted in BAL over long periods of time but were rarely present in
blood of the same patients. In addition, expressed TCRBV3 genes with
identical or homologous CDR3 amino acid sequences were apparent within
individual patients as well as among four of five CBD patients studied.
Limiting dilution cloning and analysis of BAL
CD4+ T cells expressing homologous TCRB genes
confirmed the coexpression of highly homologous TCRA genes and strongly
suggest that these clones were selected in the lung by a common Ag
involving beryllium. Together, the results implicate these
CD4+ T cells in the pathogenesis of CBD and
provide new insight into T cell recognition of beryllium and possibly
other metals in human disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diagnosis of CBD was established using previously defined
criteria (8, 13), including the presence of granulomatous
inflammation on lung biopsy, a history of exposure to beryllium, and a
positive proliferative response of BAL T cells to
BeSO4 in vitro. Certain relevant characteristics
of the patients studied are shown in Table I
. A group of three healthy individuals
and three sarcoidosis patients served as control subjects for the BAL
studies. Informed consent was obtained from each CBD patient and
control subject, and the protocol was approved by the Human Subject
Institutional Review Board at the National Jewish Medical and Research
Center.
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Analysis of TCRBV3 gene expression in peripheral blood and BAL CD4+ T cells
Mononuclear cells were isolated from heparinized blood by Ficoll-Hypaque density gradient separation and from BAL as previously described (14). Peripheral blood cells from CBD patients and BAL cells from healthy control subjects were stained with FITC-labeled CD4 (Becton Dickinson, San Jose, CA), and CD4+ T cells were sorted using an Epics 751 cell sorter (Coulter, Hialeah, FL). Sorting of BAL cells from CBD and sarcoidosis control subjects for CD4+ T cells was not performed due to the predominance of CD4+ T cells in this compartment (>80% of CD3+ cells expressed CD4). However, as indicated below, all repeated and homologous TCRB sequences in the CBD samples were subsequently shown to be derived from the CD4+ population because subsequent studies were performed on cells sorted for expression of both CD4 and Vß3.
Total RNA was isolated using an acid guanidinium-phenol chloroform method, and cDNA was synthesized using 2 µg RNA/20 µl reaction. cDNA synthesis reagents included the following: reverse transcriptase (SuperScript RT; Life Technologies, Grand Island, NY), ribonuclease inhibitor (Promega, Madison, WI), and dNTPs and random hexamers (both from Pharmacia Biotech, Piscataway, NJ). One microliter of the cDNA reaction was added per each 50-µl PCR reaction mixture. PCR amplification (AmpliTaq; Perkin-Elmer, Branchburg, NJ) was performed for 35 cycles, and both 5' and 3' oligonucleotide primers were present at a concentration of 0.3 µM. Sequences of the 5' TCRBV3 primer and the 3' CB primer are 5'-GTCTCTAGAGAGAAGAAGGAGCGC-3' and 5'-TTCTGATGGCTCAAACAC-3', respectively. The PCR products were ligated into the pCR II TA cloning vector (Invitrogen, San Diego, CA), and the ligation products were transformed into Epicurian Coli XL-1 Blue supercompetent Escherichia coli cells (Stratagene, La Jolla, CA). Colonies containing inserts were randomly selected for nucleotide sequencing. Cycle sequencing was performed using M13 reverse (5'-CAGGAAACAGCTATGAC-3') and/or M13 forward (5'-CCCAGTCACGACGTTGTAAAACG-3') sequencing primers and an automated ABI 377 sequencer (Applied Biosystems, Perkin-Elmer, Foster City, CA).
Generation of T cell clones from the BAL of CBD patients
To further analyze the TCRB gene repertoire and to identify the TCRA genes coexpressed by particular T cell clones, BAL mononuclear cells were stained for CD4 and Vß3 (clone 8F10; T Cell Sciences, Cambridge, MA) (15, 16), and double-positive cells were sorted and cloned by limiting dilution. Variable numbers (1, 3, or 10 cells per well) of sorted T cells and 1 x 104 irradiated (9000 rad) TK6 feeder cells per well were cultured in 96-well microtiter plates (Falcon, Becton Dickinson Labware, Bedford, MA) in RPMI 1640 media (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 20% HL-1 culture media (Biowhittaker), 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Life Technologies), 0.25 µg/ml PHA (Murex Diagnostics, Dartford, England), and 10% T cell growth factor supernatant (a gift from R. J. Albertini, University of Vermont, Burlington, VT) (17). After 12–14 days of culture, T cell colonies were transferred to 1-ml cultures. Cells from confluent cultures were harvested, and total cellular RNA was isolated using a commercially available kit (RNaid PLUS; BIO 101, La Jolla, CA). cDNA was prepared, and the TCRB gene fragments were amplified using the TCRBV3 primer and TCRBC primer as described above. Each PCR product was purified using a DNA binding membrane spin column (QIAquick PCR Purification Kit, Qiagen, Chatsworth, CA) and sequenced using a TCRBC sequencing primer (5'-CGACCTCGGGTGGGAACA-3').
T cell clones were also derived by limiting dilution from peripheral blood CD4+ T cells of a normal individual after PHA stimulation using methods similar to those described above. After 14 days of culture, Vß3+ clones were selected and transferred to 1-ml cultures in the absence of PHA. One week later, proliferation assays were performed using T cell clones (1 x 105 cells) cultured in 96-well flat-bottom microtiter plates with 1 x 105 autologous, irradiated (3000 rad) PBMCs in the presence of 1 x 10-5 M BeSO4 or 1 µg/ml PHA for 3 days. The wells were then pulsed with 1 µCi of [3H]thymidine for 16 h, and incorporation of radioactivity was determined by ß-emission spectroscopy. Proliferation assays were performed in triplicate.
Beryllium-specific clones were derived after stimulation of BAL cells in 24-well plates (Costar, Cambridge, MA) in 2 ml of culture media in the presence of 1 x 10-5 M BeSO4 for 5 days. Subsequently, Ag was removed, and the lymphoblasts were expanded further in culture media containing 10% T cell growth factor as described above. After 5 days, the lymphoblasts were stained with mAbs to Vß3 and CD4 as described above, and double-positive cells were sorted and cloned by limiting dilution with 1 x 10-5 M BeSO4 in the presence of autologous, irradiated (3000 rad) PBMCs for 12 days. Subsequently, T cell clones were further expanded in culture media containing 10% T cell growth factor supernatant (described above). T cell clones were maintained in culture by cycles of restimulation (every 2–3 wk) with 1 x 10-5 M BeSO4 in the presence of autologous, irradiated PBMCs and further expansion in culture with growth factors. Proliferation assays were performed as described above in the presence or absence of BeSO4 (10-5 M or 10-6 M). Proliferation assays were performed in triplicate.
Analysis of TCRB and TCRA gene segments expressed by the T cell clones
T cells were harvested, and total cellular RNA was extracted as described above. cDNA was synthesized, and TCRBV3 gene segments were amplified via PCR as described above. Each PCR product was purified using a DNA binding spin column (QIAquick PCR Purification Kit; Qiagen), and cycle sequencing was performed using a TCRBC sequencing primer (5'-CGACCTCGGGTGGGAACA-3').
RNA from T cell clones expressing the TCRB of interest was used for anchored PCR amplification of TCRA cDNA, utilizing the 5' RACE system (Life Technologies). In brief, TCRA first strand cDNA was synthesized using a TCRAC-specific primer, designated GSP1 (5'-TTCAGTCTAAACAACGAGGTC-3') and SuperScript II reverse transcriptase (Life Technologies). RNA was digested with an RNase mixture, and the cDNA was purified using a silica-based membrane column. A homopolymeric dC tail was added to the cDNA using dCTP and TdT. The dC-tailed cDNA was amplified for 35 cycles of PCR using the Abridged Anchor Primer and a nested TCRAC primer (designated GSP2, 5'-GAACTTCAGGTATCTGGAGTA-3'). A second round of 35 cycles of nested PCR was performed using the Abridged Universal Amplification Primer, GSP3 (5'-TCAAGAGAGTCACCATGTGCC-3'), and 5 µl of the first round PCR product. The anchored PCR product was ligated and cloned as described above. Cycle sequencing was performed using a TCRAC sequencing primer (5'-TGGTACACGGCAGGGTCAGG-3'). The CDR3 of each T cell clone TCRA was verified by a TCRAV-specific PCR utilizing GSP2 and a TCRAV-specific primer (TCRAV22S1, 5'-CCTCCTGAAAGCCACGAAGGCTGA-3').
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously identified a subset of CBD patients with increased
percentages of Vß3+ T cells in BAL compared
with blood (10). We focused the current analysis on five
of these patients who were available for additional studies. Certain
characteristics of these patients are shown in Table I
. Four of the CBD
patients were exposed to beryllium oxide in the ceramics industry,
whereas patient 5 was exposed to beryllium metal in the nuclear defense
industry. The duration of beryllium exposure varied from 9 to 282 mo.
The diagnosis of CBD was established subsequent to the development of
symptoms in patients 1, 2, and 3 while patients 4 and 5 were diagnosed
via a screening program. The presence of symptoms correlated with more
severe functional and radiographic abnormalities (data not shown).
Although three of the five patients were being treated with
corticosteroids at the time of study, all of the patients had active
disease based on worsening symptoms and progressive physiologic and
radiographic abnormalities. We have previously shown (10)
that alterations of TCR V region expression and T cell subset
expansions persist in CBD patients with active disease, despite
treatment with corticosteroids. In that study, we noted that
longitudinal changes in TCR V region expression did not appear to be
different in patients treated with corticosteroids, compared with
untreated patients.
Table I also shows the percentages of Vß3+
CD4+ cells in the BAL and blood of these
patients, as well as their HLA-DRB1, -DQB1, and -DPB1 alleles,
determined by molecular typing. The low percentage of
Vß3+ cells in the peripheral blood of patient 4
is likely to be genetically determined and related to a polymorphism in
the spacer region of the recombination signal sequence of the BV3.1
gene (16, 18, 19). The relatively small
Vß3+ expansion in the lung of this individual
may therefore be secondary to this baseline inefficient generation of
Vß3+ T cells. All of the CBD patients in this
study possessed DPB1*0201, which has a glutamic acid (E) at position 69
and has been associated with an increased risk of developing CBD
(20, 21, 22). In contrast, no common HLA-DR or -DQ alleles
were shared among all individuals. Of note, CBD patient 5 shared
DRB1*13 with patient 2, DRB1*0701 with patient 3, and DQB1*0303 with
patient 1.
We sorted CD4+ T cells from the blood and BAL of
individual patients from which RNA and cDNA were prepared. TCRBV3
fragments were amplified, cloned in bacteria, and sequenced. Fig. 1A shows CDR3 sequences that
were found at least three times or that appeared to have an identical
or homologous CDR3 amino acid sequence, compared with other cDNA clones
in the same sample. In Fig. 1A, identical amino acid
sequences are shown more than once if they were encoded by a different
nucleotide sequence. Oligoclonal expansions were apparent in the BAL
CD4+ TCRBV3 subset in all five individuals. Each
individual had several clones that occupied over 10% of the
Vß3+ subset, and a few clonal expansions
approached 20–30% of this subset. The probability for one set of
three repeated sequences to be found by chance alone (assuming >5000
cells within a particular TCRBV subset from >100,000
CD4+ T cells sorted) was calculated to be
p = 1.6 x 10-7. Clonal
expansions were much less frequent and smaller in blood. For example,
no repeated sequences were found in the blood
Vß3+ CD4+ subset of
patient 2, and the others demonstrated only a few repeated sequences.
Furthermore, although BAL samples from patients 1–4 were enriched for
sets of clones with homologous TCRs, only patient 1 had matched
isolates in blood (see below). Conversely, of five clonal expansions
(
3 repeated sequences) found in the peripheral blood of these
individuals, none were found in the BAL. These results clearly
demonstrate oligoclonality that predominates in the BAL of these
patients and shows the independent repertoires of BAL vs blood T cells
in CBD patients.
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A). In particular, an aspartic acid (D) was present at
position 96 of the ß-chain in 54%, 53%, 30%, and 68% of the
sequences from patients 1, 2, 3, and 4, respectively. These ß-chain
sequences had CDR3 lengths of 7 or 8 aa. The NDßN regions of these
related CDR3s are shown in bold type in Fig. 1A and usually
had a glycine (G) or lysine (K) at position 95 and an arginine (R) or
glutamine (Q) at position 97. The relatedness of some of these
sequences is also shown in Fig. 2. For
example, sets of TCRs had the sequence CASSFGD, CASSLGD, or CASSSGD,
usually with an arginine (R) or glutamine (Q) at the next position.
Although the Vß and NDßN regions of these clones were similar, the
Jß segments varied considerably. Fig. 2 also shows examples of
identical ß-chain amino acid sequences in the same patient and in
different patients that were encoded by different nucleotide sequences,
i.e., expressed by different Vß3+ T cell
clones. One Vß3-CASSFGD-Jß2.7 sequence was expressed in three
different CBD patients. Nucleotide sequence differences among these
clones precluded the possibility of a PCR contamination or
artifact.
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B). Furthermore, none of
the 108 and 137 sequences from normal subjects or sarcoidosis patients,
respectively, showed an aspartic acid (D) at position 96 of the TCRB
sequence (p < 10-10, compared with
the frequency in CBD patients). Persistence of related Vß3+ clones in BAL
If CD4+ T cells expressing this CDR3 motif
are important in the disease process, we predicted that they should
persist at sites of organ involvement in patients with continued
disease activity. We therefore studied BAL samples, which were
available from prior time points in two patients. Table II shows serial sequence analyses in two
patients studied over 3 to 5 years. All clones were detected in at
least two different serial samples, and the majority of clones were
detected at every time point. Some clones varied considerably in
frequency. For example, in patient 1, one clonal sequence
(Vß3-CASSLKD-Jß2.7) decreased from over 50% of the total sequences
to 1 in 44 sequences 3 yr later. An explanation for the dramatic
decrease in the frequency of this one T cell clone is unclear since
patient 1 continued to require corticosteroid therapy for progressive
lung dysfunction. However, over this same time period, other clones
with the shared CDR3 motif increased in frequency such that the total
frequency of clones with homologous TCRs at the last time point was
about 50%. In patient 2, the frequency of clones with the shared CDR3
motif persisted at 
40–50% of the Vß3+
subset over the 4-yr study period.
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Since recognition of Ag by the TCR involves both TCR ß- and
-chains, TCRs having the same Ag specificity and related ß-chain
sequences should express homologous -chain sequences. To test this
prediction, Vß3+ CD4+ T
cells from the BAL of two CBD patients were cloned by limiting
dilution, and TCR genes expressed by the different clones were
sequenced. T cells expressing the ß-chain CDR3 motif described above
were found among the cloned cells at about the frequency predicted
(data not shown). The TCRA expressed by these clones was determined by
anchored PCR. As shown in Fig. 3, TCRA
genes expressed by these T cell clones showed remarkable similarity.
Thus, all utilized TCRAV22S1, out of a possible 75–100 TCRAV gene
segments to choose from (25). Most used AJ49, and nearly
all had similar junctional sequences and length. In addition, all of
the coexpressed TCRAs encoded an asparagine (N) at amino acid position
95 and all but one had a leucine (L) at position 92. In addition, two
subsets of TCRAs were apparent: one encoding a serine (S) at position
93 and utilization of AJ8 or AJ11 and another encoding an arginine (R)
at position 93 and utilization of AJ49.
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It was important to determine whether T cells with the related
CDR3 motif could be stimulated by BeSO4 in
culture. BAL and blood T cells from CBD patients were cultured for 5
days with optimal concentrations of BeSO4
(3, 12, 26). Consistent with previous studies (3, 12), cultures of cells from CBD patients showed vigorous
proliferation, whereas cultures containing cells from control subjects
showed no response and were similar to cultures without added
BeSO4 (data not shown). TCR ß-chain sequences
expressed by cells before and after stimulation are shown in Fig. 4. At day 0, the frequency of TCRs with
the shared CDR3 motif was 53% and 30% in patients 2 and 3,
respectively. After 5 days of stimulation with BeSO4, blasting
CD4+ T cells predominated in the cultures and expressed the
shared motif at about the same frequency. The continued presence of
these cells indicates that they did proliferate in culture. Otherwise,
their expressed TCRs would have been diluted out by the surrounding
proliferating cells. The results also suggest that these cells with the
particular ß-chain CDR3 sequence are not the only
Vß3+ beryllium-responsive T cells in the lungs
of these patients and that perhaps most of the BAL
Vß3+ population, as well as selected cells in
other BAL Vß subsets, are capable of responding to
BeSO4 in culture.
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Vß3+ T cell clones with the shared ß-chain CDR3 motif respond to beryllium in vitro
BAL cells from patient 2 were stimulated in culture in the
presence of BeSO4 and then cloned at limiting
dilution in the presence of autologous, irradiated PBMCs in culture
media containing BeSO4 and T cell growth factors.
After several rounds of repeated stimulation, 24 clones were expanded.
Eight clones were randomly chosen for sequence analysis, and four
(50%) expressed the homologous CDR3 motif as defined above. Two
isolates were identical with the ß-chain junctional sequence
Vß3-CASSLGDQPQHFG-Jß1.5 and two others were identical with the
sequence Vß3-CASSWADREAFFG-Jß1.1. In addition, all four expressed
TCRAV22S1 and had the invariant asparagine (N) at position 95 and
leucine (L) at position 92 of the -chain, as shown above for other T
cell clones with the conserved CDR3 motif. Four clones were analyzed
for proliferative response to BeSO4 using
autologous, irradiated PBMCs as APCs, and all four clones proliferated
in an Ag-specific manner (Fig. 5). One of
the four clones studied, RP1, expressed the shared CDR3 motif. The
frequency of this TCR ß-chain sequence ranged from 9 to 17% of
the Vß3+ T cells in this patient’s BAL over 4
yr. The same ß-chain sequence was found in two other T cell clones,
including one from patient 3. Irrelevant Vß3+
CD4+ T cell clones were also derived from
peripheral blood CD4+ T cells of a normal
individual after stimulation with PHA and selection of clones
expressing Vß3. As shown in Fig. 5, these clones proliferated
vigorously to PHA but all failed to respond to
BeSO4 in vitro. In fact, exposure to
BeSO4 appeared to inhibit proliferation in six of
eight T cell clones analyzed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of expanded CD4+ T cell clones in the BAL of patients with CBD was neither surprising nor specific for this disease. Consistent with past studies, patients with sarcoidosis, another granulomatous disease, also harbored oligoclonal expansions in their BAL CD4+ cells (28, 29, 30). In the present work, we also found oligoclonal expansions in the BAL CD4+ TCRBV3 subset of healthy individuals, including clones that occupied 25–30% of this subset. These clones appear to be larger than those previously identified in normal individuals (31). The clonal expansions in normal controls may represent resident lung T cell populations or result from recent exposures to foreign Ags and indicate that the presence of oligoclonal populations in lung is not necessarily evidence of pathology. In regard to the current study, the repertoire of BAL Vß3+ T cells in CBD was found to be distinct from that in sarcoidosis patients and normal controls and, therefore, does not appear to represent the stimulation of a resident lung population. Thus, the conserved TCR ß-chain CDR3 motif found in four of the five CBD patients was not seen once in the BAL of sarcoidosis patients or normal controls. Furthermore, despite a data bank of hundreds of TCRBV3 sequences in our laboratory derived from the synovial fluid and/or peripheral blood CD4+ T cells from rheumatoid arthritis patients, other patient groups, and healthy individuals, we have not previously found this related CDR3 motif.
Considering the enormous diversity of the TCR repertoire, related TCRs
are predicted to occur very rarely by chance alone and almost certainly
represent selection by the same or similar Ag. In four of the five CBD
patients investigated, related CDR3 sequences appeared to be present in
the BAL Vß3+ subset. This CDR3 motif was not
stringent in a number of positions but appeared to require an aspartic
acid (D) at the 96th position of the ß-chain, frequently followed by
a glutamine (Q) or arginine (R) at the next position. These CDR3s also
had a similar length of 7 or 8 aa. Interestingly, nearly any TCRBJ
usage was allowed, which raised questions about whether these TCRs were
truly related. The ultimate form of related TCRs is that which
expresses different TCRB genes (i.e., different CDR3 nucleotide
sequences) and therefore is derived from different T cell clones, but
expresses identical TCR ß-chain amino acid sequences. Examples of
such clones were found in each CBD patient as well as between different
patients. Selection by the same conventional Ag should also be
reflected in both the TCRA and TCRB gene sequences coexpressed in
related clones. Therefore, we cloned BAL CD4+ T
cells by limiting dilution, identified those clones that expressed TCRB
sequences predicted to be related, and sequenced the coexpressed TCRA
genes. Despite variation in TCRBJ usage among these T cell clones, the
coexpressed TCRA sequences were remarkably related to each other, with
identical TCRAV usage and invariant amino acids at certain
nongermline-encoded positions of the -chain CDR3. The similarity of
the TCRAs provides unequivocal evidence for the relatedness of these
TCRs and their selection by a common Ag.
Although conserved usage of homologous TCRs among different animals has been frequently seen after immunization with particular proteins or peptides in rodent studies, their demonstration in different patients with the same disease has been rare. In a recent study of patients with rheumatoid arthritis, despite selection of patients for particular HLA-DR4 alleles, we could not demonstrate similar TCRs in the synovial fluid among different patients (32). We have also not been able to demonstrate related TCRs in patients with sarcoidosis (30).
It is of interest that the only patient who did not share the conserved CDR3 motif was exposed to beryllium in a nuclear weapons plant whereas the other four patients were exposed in the ceramics industry. In the ceramics industry, beryllium exposure occurs in the form of beryllium oxide, as opposed to beryllium metal in the nuclear defense industry. Others have suggested that this difference, as well as differences in particle size and solubility, may influence the immune response to beryllium and possibly account for the higher frequency of disease following exposure in the ceramics industry (1). Our present results suggest that qualitative differences in CD4+ T cell recognition may also be involved. Further studies with larger numbers of patients will determine whether different TCR repertoires are related to these different types of beryllium exposures.
We also noted a remarkable separation of lung and peripheral blood CD4+ T cell repertoires in these patients. The compartmentalization in lung of a subset of beryllium-reactive cells occurred despite the presence of beryllium-responsive cells in peripheral blood in most patients with CBD (12), including the two patients tested in our study. It is well-known that beryllium-exposed individuals may demonstrate evidence of sensitization and yet display no evidence of lung pathology or physiologic abnormalities (27). Thus, in a subset of exposed individuals, peripheral blood T cells will proliferate in culture after the addition of BeSO4, but these patients may demonstrate no clinical, physiologic, or lung histopathologic abnormalities even after long-term follow-up (27). Therefore, only a subset of beryllium-reactive CD4+ T cells appears to have pathogenic potential. The present results support the contention that these clones are compartmentalized because of their different beryllium-related specificity.
We also stimulated BAL and blood cells with BeSO4 and analyzed TCR expression after T cell stimulation. Numerous studies have indicated that BeSO4 is not a polyclonal T cell activator (8, 9, 13, 26). In contrast to patients with CBD and those individuals sensitized by a previous exposure to beryllium, cells from normal individuals or patients with other diseases do not proliferate upon BeSO4 exposure. This forms the basis for the diagnostic test used in the evaluation of beryllium-exposed individuals (3, 7, 8, 12, 13). In the present work, T cells expressing the shared CDR3 motif were prevalent in the blast population after in vitro stimulation of BAL cells, indicating their ability to respond to BeSO4 in vitro. This capability to respond to beryllium in vitro was also shown for T cell clones derived from the BAL of a patient but not for irrelevant Vß3+ T cell clones.
Previous studies have shown that T cells from patients with CBD respond to BeSO4 in culture in a class II MHC-restricted manner (9). Considering the expression of related TCRs among four different patients, it is surprising that these CBD patients demonstrated such diverse HLA-DR and -DQ types. Thus, a shared HLA-DRB1 or -DQB1 allele was not found in this group of CBD patients. patient 5 did share HLA-DR and -DQ alleles with other CBD patients studied and yet did not demonstrate BAL Vß3+ cells utilizing the related CDR3 motif. In contrast to HLA-DR and -DQ, all of the patients expressed DPB1*0201, which has been associated with an increased risk of developing CBD after exposure to beryllium (20, 21, 22). In preliminary studies, however, the responses to BeSO4 of lung-derived CD4+ T cell clones, including those with the shared CDR3 motif, were blocked by anti-DR mAbs and therefore appear to be restricted to HLA-DR (A. P. Fontenot, L. S. Newman, and B. L. Kotzin, unpublished observations).
The nature of the Ag that interacts with the TCR and MHC is not known. It has been hypothesized that beryllium binds to different self proteins (or peptides), which are then presented as "foreign" to T cells by surface class II MHC molecules. There is no direct evidence to suggest that metals (e.g., beryllium, nickel, and gold) can directly interact with the MHC molecule (33). Studies of nickel-induced contact dermatitis have demonstrated recognition of nickel in association with HLA-DRw11 (34) and suggested that nickel binds directly to the peptide component of the MHC-peptide complex via a histidine residue. The interaction of other transitional metals (Co2+, Cu2+, and Zn2+) with histidine residues has also been demonstrated (35). Whether beryllium might interact with a particular exposed amino acid position of the bound peptide remains unknown. However, our finding of a CDR3 motif with invariant charged residues at particular positions, such as the ß-chain aspartic acid, is consistent with a direct interaction of the TCR with a positively charged beryllium molecule.
In summary, we believe that these studies have identified pathogenic T cells in patients with CBD and have provided insight into an unusual form of T cell recognition. Based on the repertoire of beryllium-reactive peripheral blood cells, the TCRs expressed appear to be diverse. However, only a subset of these clones, including a subset expressing homologous TCRs, appears to have pathogenic potential. Why these particular cells seem to be enriched in the lungs of patients with CBD is of critical importance in our understanding the immunopathogenesis of this disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Brian L. Kotzin, Division of Clinical Immunology (B164), University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address:
3 Abbreviations used in this paper: CBD, chronic beryllium disease; BeSO4, beryllium sulfate; BAL, bronchoalveolar lavage; TCRA, TCR -chain gene; TCRB, TCR ß-chain gene; Vß, TCR ß-chain V region.
Received for publication October 15, 1998. Accepted for publication April 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. G. Mack, A. K. Lanham, B. E. Palmer, L. A. Maier, T. H. Watts, and A. P. Fontenot 4-1BB Enhances Proliferation of Beryllium-Specific T Cells in the Lung of Subjects with Chronic Beryllium Disease J. Immunol., September 15, 2008; 181(6): 4381 - 4388. [Abstract] [Full Text] [PDF]
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B. E. Palmer, D. G. Mack, A. K. Martin, M. Gillespie, M. M. Mroz, L. A. Maier, and A. P. Fontenot Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease J. Immunol., February 15, 2008; 180(4): 2704 - 2712. [Abstract] [Full Text] [PDF]
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 I. G. Luzina, N. W. Todd, A. T. Iacono, and S. P. Atamas Roles of T lymphocytes in pulmonary fibrosis J. Leukoc. Biol., February 1, 2008; 83(2): 237 - 244. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, T. S. Keizer, M. McCleskey, D. G. Mack, R. Meza-Romero, J. Huan, D. M. Edwards, Y. K. Chou, A. A. Vandenbark, B. Scott, et al. Recombinant HLA-DP2 Binds Beryllium and Tolerizes Beryllium-Specific Pathogenic CD4+ T Cells J. Immunol., September 15, 2006; 177(6): 3874 - 3883. [Abstract] [Full Text] [PDF]
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J. R. Bill, D. G. Mack, M. T. Falta, L. A. Maier, A. K. Sullivan, F. G. Joslin, A. K. Martin, B. M. Freed, B. L. Kotzin, and A. P. Fontenot Beryllium Presentation to CD4+ T Cells Is Dependent on a Single Amino Acid Residue of the MHC Class II {beta}-Chain J. Immunol., November 15, 2005; 175(10): 7029 - 7037. [Abstract] [Full Text] [PDF]
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 A. K. Sullivan, P. L. Simonian, M. T. Falta, J. D. Mitchell, G. P. Cosgrove, K. K. Brown, B. L. Kotzin, N. F. Voelkel, and A. P. Fontenot Oligoclonal CD4+ T Cells in the Lungs of Patients with Severe Emphysema Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 590 - 596. [Abstract] [Full Text] [PDF]
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Y. K. Chou, D. M. Edwards, A. D. Weinberg, A. A. Vandenbark, B. L. Kotzin, A. P. Fontenot, and G. G. Burrows Activation Pathways Implicate Anti-HLA-DP and Anti-LFA-1 Antibodies as Lead Candidates for Intervention in Chronic Berylliosis J. Immunol., April 1, 2005; 174(7): 4316 - 4324. [Abstract] [Full Text] [PDF]
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M. Facco, L. Trentin, L. Nicolardi, M. Miorin, E. Scquizzato, D. Carollo, I. Baesso, M. Bortoli, R. Zambello, G. Marcer, et al. T cells in the lung of patients with hypersensitivity pneumonitis accumulate in a clonal manner J. Leukoc. Biol., May 1, 2004; 75(5): 798 - 804. [Abstract] [Full Text] [PDF]
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 M. Amicosante, F. Berretta, A. Franchi, P. Rogliani, C. Dotti, M. Losi, R. Dweik, and C. Saltini HLA-DP-unrestricted TNF-{alpha} release in beryllium-stimulated peripheral blood mononuclear cells Eur. Respir. J., November 1, 2002; 20(5): 1174 - 1178. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, L. A. Maier, S. J. Canavera, T. B. Hendry-Hofer, M. Boguniewicz, E. A. Barker, L. S. Newman, and B. L. Kotzin Beryllium Skin Patch Testing to Analyze T Cell Stimulation and Granulomatous Inflammation in the Lung J. Immunol., April 1, 2002; 168(7): 3627 - 3634. [Abstract] [Full Text] [PDF]
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M. D. ROSSMAN, J. STUBBS, C. W. LEE, E. ARGYRIS, E. MAGIRA, and D. MONOS Human Leukocyte Antigen Class II Amino Acid Epitopes . Susceptibility and Progression Markers for Beryllium Hypersensitivity Am. J. Respir. Crit. Care Med., March 15, 2002; 165(6): 788 - 794. [Abstract] [Full Text] [PDF]
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C. Saltini, L. Richeldi, M. Losi, M. Amicosante, C. Voorter, E. van den Berg-Loonen, R.A. Dweik, H.P. Wiedemann, D.C. Deubner, and C. Tinelli Major histocompatibility locus genetic markers of beryllium sensitization and disease Eur. Respir. J., October 1, 2001; 18(4): 677 - 684. [Abstract] [Full Text] [PDF]
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L.A. Maier, R.T. Sawyer, S.S. Tinkle, L.A. Kittle, E.A. Barker, R. Balkissoon, C. Rose, and L.S. Newman IL-4 fails to regulate in vitro beryllium-induced cytokines in berylliosis Eur. Respir. J., March 1, 2001; 17(3): 403 - 415. [Abstract] [Full Text] [PDF]
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G. Lombardi, C. Germain, J. Uren, M. T. Fiorillo, R. M. du Bois, W. Jones-Williams, C. Saltini, R. Sorrentino, and R. Lechler HLA-DP Allele-Specific T Cell Responses to Beryllium Account for DP-Associated Susceptibility to Chronic Beryllium Disease J. Immunol., March 1, 2001; 166(5): 3549 - 3555. [Abstract] [Full Text] [PDF]
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 A. P. Fontenot, M. Torres, W. H. Marshall, L. S. Newman, and B. L. Kotzin Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease PNAS, October 23, 2000; (2000) 220430797. [Abstract] [Full Text]
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A. P. Fontenot, M. Torres, W. H. Marshall, L. S. Newman, and B. L. Kotzin Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease PNAS, November 7, 2000; 97(23): 12717 - 12722. [Abstract] [Full Text] [PDF]
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