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J. Exp. Med. 2013 Vol. 210 No. 7 1403-1418
CD4+ T cells play a critical role in the develop-
ment of chronic beryllium disease (CBD), a
fibrotic lung disease characterized by mononu-
clear cell interstitial infiltrates and granuloma-
tous inflammation (Fontenot and Maier, 2005).
Proliferation of blood CD4+ T cells in response
to beryllium (Be) salts in vitro defines sensitiza-
tion (Rossman et al., 1988; Mroz et al., 1991),
and progression to CBD is heralded by the
accumulation of Be-specific, Th1 cytokine–
secreting CD4+ T cells in the lung (Tinkle et al.,
1997; Fontenot et al., 2002). These Be-responsive
cells are characterized by oligoclonally ex-
panded T cell subsets that share a CDR3 motif
among multiple patients with active disease
(Fontenot et al., 1999), and the vast majority
of these T cells recognize antigen in an HLA-
DP–restricted manner (Fontenot et al., 2000;
Lombardi et al., 2001). Importantly, genetic
susceptibility to CBD is strongly linked to
HLA-DP alleles that contain a glutamic acid
at the 69th position of the -chain (Glu69;
Richeldi et al., 1993). Depending on suscepti-
bility and exposure, CBD develops in up to 18%
of Be-exposed workers (Kreiss et al., 1993a,b,
1996; Richeldi et al., 1993). Thus, CBD is a clas-
sical example of a human disease resulting from
The peptide-binding groove of HLA-DP2,
the most prevalent Glu69-containing HLA-
DP molecule, is wider than that of other MHC
class II (MHCII) proteins (Dai et al., 2010). The
gap between the peptide backbone and the
Andrew P. Fontenot:
Abbreviations used: BAL,
bronchoalveolar lavage; Be,
beryllium; CBD, chronic
Identification of beryllium-dependent
peptides recognized by CD4+ T cells
in chronic beryllium disease
Michael T. Falta,1 Clemencia Pinilla,2 Douglas G. Mack,1 Alex N. Tinega,1
Frances Crawford,3,4 Marc Giulianotti,6 Radleigh Santos,6
Gina M. Clayton,3,4 Yuxiao Wang,7 Xuewu Zhang,7 Lisa A. Maier,1,5
Philippa Marrack,3,4 John W. Kappler,3,4 and Andrew P. Fontenot1,3
1Department of Medicine, University of Colorado, Denver, Aurora, CO 80045
2Torrey Pines Institute for Molecular Studies, San Diego, CA 92121
3Integrated Department of Immunology, 4Howard Hughes Medical Institute, and 5Department of Medicine,
National Jewish Health, Denver, CO 80206
6Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987
7Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390
Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of
beryllium (Be)-specific CD4+ T cells into the lung. The vast majority of these T cells recog-
nize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition.
However, the peptides that stimulate Be-specific T cells are unknown. Using positional
scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP mol-
ecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to
MHCII and Be, forming a complex recognized by pathogenic CD4+ T cells in CBD. These
peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that sur-
round the putative Be-binding site and cooperate with HLA-DP2 in Be coordination.
Endogenous plexin A peptides and proteins, which share the core motif and are expressed
in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4
tetramers detected high frequencies of CD4+ T cells specific for these ligands in all HLA-
DP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4+ T cell
involved in metal-induced hypersensitivity and suggest a unique role of these peptides in
metal ion coordination and the generation of a common antigen specificity in CBD.
© 2013 Falta et al. This article is distributed under the terms of an Attribution–
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The Journal of Experimental Medicine
Beryllium-dependent peptides in CBD | Falta et al.
HLA-DP2 -chain -helix opens a solvent-exposed acidic
pocket composed of three HLA-DP2 -chain glutamic acid
residues (including Glu69) that could easily accommodate
a Be-containing compound. Mutation of Glu69 or either
of the other two glutamic acid residues present in this pocket,
Glu26 and Glu68, eliminates Be presentation (Dai et al.,
2010), suggesting that this acidic gap represents the putative
Be-binding site within the footprint of the TCR. How-
ever, the role of peptide in coordinating a Be moiety and
which peptides are recognized by Be-specific CD4+ T cells
To investigate the spectrum of peptides that permit Be
recognition by particular TCRs, we used positional scanning
libraries (Pinilla et al., 1992, 1994; Hemmer et al., 1998) to
screen hybridomas expressing Be-specific TCRs derived from
the lung of an HLA-DP2–expressing CBD patient. We iden-
tified multiple mimotopes and endogenous self-peptides,
including those derived from plexin A proteins, that are rec-
ognized by the T cell hybridomas only in the presence of
Be. These peptides share a core motif of acidic amino acids
adjacent to the putative Be-binding site in HLA-DP2 and
participate in metal ion capture. Be-loaded HLA-DP2–
mimotope and HLA-DP2–plexin A4 tetramers detected
CD4+ T cells that recognize these complexes in the lungs of
many CBD patients, strongly suggesting that these related
ligands play a key role in disease. Thus, the current study is the
first to identify a complete MHCII–peptide–metal ion com-
plex recognized by pathogenic CD4+ T cells in CBD and
provides insight into the role of MHC-bound peptide in
Specific peptide requirement for T cell recognition of Be
Using T cell hybridomas AV22 and AV9 that express related
Be-specific TCRs derived from the lung of a CBD patient
(Fig. 1 A; Bowerman et al., 2011), we sought to identify
Be-dependent peptides capable of stimulating these TCRs.
AV22 and AV9 respond to BeSO4 presented by mouse DAP3.
L cells transfected with HLA-DP2 (designated DP8302) inde-
pendently of the addition of exogenous protein or peptide
(Fig. 1 B). To investigate the role of peptide in the recognition
of Be, we created HLA-DP2–expressing antigen-presenting
cell lines that are incapable of peptide exchange and present
only a single peptide. An HLA-DP2 -chain gene was con-
structed that utilizes a genetically encoded linker to covalently
attach individual HLA-DP2–binding peptides (Okamoto
et al., 1998; Díaz et al., 2005) to the N terminus of the
HLA-DPB1*0201 gene. The DPA1*0103 and DPB1*0201
peptide gene constructs were transfected into a kidney fibro-
blast line derived from C57BL/6 mice that are genetically
Figure 1. Be-specific CD4+ T cells require Be and a specific pep-
tide for antigen recognition. (A) TCR gene segment usage and junc-
tional region amino acid sequence of the Be-specific T cell hybridomas
AV22 and AV9. (B) Be-specific (AV22 and AV9) and dengue virus–specific
(DV-13) T cell hybridomas were stimulated with the following antigen-
presenting cell lines: HLA-DP2–transfected mouse DAP3.L cells (DP8302),
fibroblasts (B6 DK10) derived from MHCII- and invariant chain-deficient
mice and transfected with native HLA-DP2 (DP2.21), and B6 DK10 cells
transfected with HLA-DP2 with a transferrin receptor peptide (DP2-pTf,
EPLSYTRFSLAR) or a dengue virus NS3–derived peptide (DP2-pDV,
REIVDLMCHATF) covalently attached to the N terminus of the DP2
-chain. 200 µM BeSO4 was added to all antigen-presenting cells except
DP2-pDV–expressing fibroblasts, and the IL-2 response (mean ± SEM
pg/ml) by the hybridomas was measured by ELISA. (C) AV22 and AV9
T cell hybridomas were stimulated with 200 µM BeSO4 presented by
HLA-DP2–transfected fibroblasts grown in either FBS or protein-free
(PF) medium. IL-2 secretion (mean ± SEM pg/ml) was measured by
ELISA. (B and C) Data shown are representative of three experiments
performed in triplicate. (D) AV22 hybridoma cells were stimulated over a
range of BeSO4 concentrations (0.3–1,000 µM) using DP8302 cells grown
in FBS-containing medium compared with DP2.21 cells grown in FBS and
protein-free conditions. Data shown (mean IL-2 ± SD pg/ml) are repre-
sentative of three experiments performed in triplicate.
JEM Vol. 210, No. 7
DP2.21), fibroblasts expressing covalently attached HLA-
DP2–binding peptides derived from transferrin receptor
(p6-17) or a dengue virus NS3 peptide (p254-265; Díaz et al.,
2005) failed to stimulate Be-specific T cell hybridomas in
the presence of BeSO4 (Fig. 1 B). Conversely, a dengue virus
deficient for MHCII and invariant chain (B6 DK10; Huseby
et al., 2005). Equivalent cell surface expression of the HLA-
DP2/peptide molecules was confirmed by staining with an
anti–HLA-DP mAb (not depicted). In contrast to B6 DK10
fibroblasts transfected with native HLA-DP2 (designated
Figure 2. Deconvolution strategy to de-
fine Be-dependent peptides that stimulate
T cell hybridomas AV22 and AV9. (A) AV22
cells were stimulated with selected decapep-
tide mixtures with three positions fixed in the
presence of 75 µM BeSO4. Two concentrations
of mixtures were used, and IL-2 response
(mean ± SEM pg/ml) was measured by ELISA.
Mixtures with two fixed positions (D4L5 and
D5E8) were included as positive controls. Data
are representative of three separate experi-
ments performed in triplicate. (B) IL-2 response
of three separate experiments (mean ± SEM)
of AV22 and AV9 to a biased decapeptide
positional scanning library (W2D4L5) is
shown. Peptide mixtures were fixed at W2,
D4, and L5, and one additional peptide position
(p1, p3, p6, p7, p8, p9, and p10) was scanned
with each of 20 amino acids (140 mixtures).
Each panel shows the response of hybridomas
to 20 µg/ml mixtures and 75 µM BeSO4 com-
pared with the W2D4L5 mixture with no
additional fixed positions (Ct). For A and B,
the x axis denotes the amino acid (single letter
code) fixed at each defined position, and mix-
tures did not stimulate hybridomas in the
absence of BeSO4. (C) Selection of amino acids
for each peptide position based on the most
active mixtures in the presence of BeSO4.
Beryllium-dependent peptides in CBD | Falta et al.
Identification of Be-dependent mimotopes
using a decapeptide positional scanning library
Having observed that common HLA-DP2–binding peptides
do not stimulate T cell hybridomas expressing Be-specific
TCRs, we used a decapeptide positional scanning library,
which makes no assumptions regarding peptide composition
and allows a systematic assessment of all peptides of a given
length in a standard T cell activation assay. The initial library
screen and a subsequent screen using less diverse mixtures
containing fixed amino acids at two positions showed that
the most potent mixtures contained an aspartic acid (D) at p4
and a leucine (L) at p5 or a D at p5 and a glutamic acid (E) at
p8 (not depicted). We next synthesized select decapeptide
mixtures with three positions fixed. As shown in Fig. 2 A,
the only mixture that enhanced IL-2 secretion in the pres-
ence of BeSO4 above the control D4L5 and D5E8 mixtures
contained a tryptophan (W) at the p2 position, D at p4, and
L at p5. Importantly, none of these mixtures stimulated
an HLA-DP2–restricted, dengue virus–specific hybridoma
(DV-13) or Be-specific hybridomas in the absence of BeSO4
With preferred amino acids defined at three positions
of the decapeptide, we performed a positional scan of the re-
maining seven positions. All mixtures included three fixed
positions (W2D4L5) and a fourth position fixed with 1 of 20
amino acids at each remaining position of the peptide.
As shown in Fig. 2 B, most positions showed a distinct profile
of allowed amino acids with identical preferences for AV22
and AV9. For example, known anchor residues for HLA-
DP2–binding peptides (e.g., phenylalanine [F] and L) were
preferred at p1 and p6. At positions p3 and p9, related,
nonpolar amino acids (isoleucine [I], valine [V], and L) were
selected. The only amino acid at p7 that enhanced IL-2 secre-
tion by AV22 and AV9 above the control W2D4L5 mixture
was E. Nonrelated amino acids, including threonine (T),
L, and F, were selected at p8. In contrast, no definitive selec-
tion of amino acids was seen at p10.
Based on the defined amino acids at each position of the
peptide (Fig. 2 C), we synthesized a set of 24 mimotopes and
tested their ability to stimulate Be-specific T cell hybridomas. All
of the mimotopes stimulated IL-2 secretion by AV22 in the pres-
ence of BeSO4. In Table 1, mimotopes were ranked by EC50
values (peptide concentration inducing half-maximal IL-2
response), and the ranking of the mimotopes was similar for
AV9 (not depicted). Of note, the three highest ranking mi-
motopes only differed at the p8 position and had similar EC50
values; thus, we focused on mimotopes 2 and 4.
Alanine (A) and analogue scans of mimotope-4 were
performed to determine the critical amino acids for anchor-
ing to HLA-DP2, coordinating Be, or interacting with
TCR. An F to A substitution at either p1 or p6 of the peptide
shifted the dose–response curve to the right, resulting in a
16- and 26-fold reduction in the EC50, respectively (Fig. 3 A).
The preferred anchor residue at p1 and p6 was an F com-
pared with L (Fig. 3 B). Confirming the positional scanning
library data, an A at p2, p4, p5, or p7 abrogated T cell recognition
NS3–specific hybridoma responded to HLA-DP2 with the
covalently linked dengue peptide (Fig. 1 B).
In assessing the role of peptide in mediating Be recogni-
tion, the AV22 and AV9 hybridomas consistently secreted
75% less IL-2 when using DP2.21 fibroblasts as antigen-
presenting cells compared with DP8302 as a result of restric-
tion of the peptide repertoire (Fig. 1, B and C). Adapting
DP2.21 to protein-free medium completely abrogated the
Be-specific hybridoma response, whereas this medium had no
effect on cell viability or growth (not depicted) or the ability
of DP8302 cells to present BeSO4 to the Be-specific T cell
hybridomas (Fig. 1, C and D). Because this cell line does not
naturally present Be to the Be-specific hybridomas, all anti-
gen presentation assays using peptides were conducted using
DP2.21 grown in protein-free medium.
Next, we tested a larger set of 25 known HLA-DP2–binding
peptides (Díaz et al., 2005) in the presence of BeSO4 using
DP2.21 as antigen-presenting cells. None of these peptides
induced IL-2 secretion by the AV22 or AV9 hybridomas
(not depicted), demonstrating that HLA-DP2–restricted
T cell recognition of Be depends on a limited number of
Table 1. Mimotopes that stimulate the Be-specific T cell
1 2 3 4 5 6 7 8 9 10 EC50
F W I D L F E L I
F W I D L F E T I
F W I D L F E F I
F W I D L F E L I
F W I D L L E T I
L W I D L F E F I
F W I D L L E L I
F W I D L L E F I
L W I D L F E L I
L W I D L F E T I
L W I D L F E L I
L W I D L L E T I
L W I D L L E L I
F W I D L L E L I
F W I D L L E F I
F W I D L L E T I
F W I D L F E F I
L W I D L L E F I
L W I D L L E T I
F W I D L F E T I
L W I D L F E F I
L W I D L F E T I
L W I D L L E F I
L W I D L L E L I
Mimotopes are ordered by most to least active (i.e., lowest to highest EC50 values).
Bolded amino acids denote peptide positions that vary in the mimotopes.
JEM Vol. 210, No. 7
with high affinity (Kd = 4.6 µM; Fig. 4 A), confirming that Be
is a necessary component for T cell recognition.
Having identified an MHCII/peptide/Be ligand for
TCRs from a single CBD patient, we tested whether this
ligand could engage TCRs on T cells from other HLA-
DP2–expressing CBD patients. We constructed a tetramer
consisting of HLA-DP2 with mimotope-2 covalently at-
tached to the N terminus of the HLA-DP2 -chain with
and without saturating BeSO4. This reagent specifically
stained the AV22 hybridoma only when Be was included in
the complex and did not bind to either an HLA-DP2–
restricted, dengue virus–specific hybridoma DV-13 or a T cell
hybridoma specific for insulin B:9-23 in the context of IAg7
(8-1.1; Fig. 4 B). In contrast to 8-1.1, which bound to the
IAg7-insulin B:9-23 tetramer (Crawford et al., 2011), AV22
did not stain with this reagent (Fig. 4 B). Identical findings
were seen with AV9 (not depicted). The bronchoalveolar
lavage (BAL)–derived CD4+ T cell line from which AV22
and AV9 were cloned also brightly stained with this tetra-
mer. After one and two cycles of Be stimulation, 1.8 and
25% of the CD4+ T cells specifically recognized this TCR
ligand and not the IAg7-insulin tetramer (Fig. 4 C). After cell
sorting for TCR V5.1 expression, the vast majority of T cells
of the HLA-DP2–peptide/Be complex, whereas alanines
at p8, p9, or p10 had minimal effects on activation of the
Be-specific AV22 hybridoma (Fig. 3 A). In this regard, dele-
tion of the p9 and p10 amino acids had minimal effects on
EC50 values (not depicted), demonstrating that these positions
are much less important for recognition of this ligand. In
addition to W at p2, other amino acids, such as Y, V, and R,
were also allowed at this position with no effect on IL-2
secretion (Fig. 3 C). EC50 values for AV22 and AV9 were
strongly correlated (Fig. 3 D), confirming that these TCRs
recognize the same ligand despite using different TCR V
chains (Bowerman et al., 2011).
HLA-DP2–mimotope-2/Be tetramer staining
of CD4+ T cells from CBD patients
Next, we purified soluble recombinant HLA-DP2–mimotope-2
and AV22 TCR from baculovirus-infected insect cells to de-
termine the affinity of the TCR for the HLA-DP2–mimotope-2/
Be complex, using surface plasmon resonance. The AV22
TCR did not bind to either HLA-DP2–mimotope-2 or
HLA-DR52c–WIRVNIPKRI (denoted HLA-DR52c–WIR;
Yin et al., 2012) in the absence of BeSO4 (Fig. 4 A). However,
after exposing the flow cells to 200 µM BeSO4, the AV22 TCR
bound to HLA-DP2–mimotope-2, but not HLA-DR52c–WIR,
Figure 3. Be-specific T cell hybridoma response to alanine substitutions and analogues of mimotope-4. Peptide dose–response curves were
completed for T cell hybridoma AV22. (A–C) Equal numbers of AV22 cells and DP2.21 antigen-presenting cells were mixed with 75 µM BeSO4 and the
following highly purified peptides: peptides with single alanine substitutions at each position of mimotope-4 (A), analogues of mimotope-4, selected
based on the demonstrated potency of other mimotopes (B), and mimotope-4 variants at the p2 position of the peptide (C). IL-2 secretion was measured
by ELISA after 22 h of culture, and data are plotted as the percentage of maximum IL-2 secretion against peptide concentration in the presence of BeSO4.
EC50 values (mean ± SEM nM) for mimotope-4 and each variant peptide from four independent experiments are shown. Note that error bars have been
left out for viewing clarity. (D) Correlation of EC50 values of all variants of mimotope-4 between hybridomas AV22 and AV9.
Beryllium-dependent peptides in CBD | Falta et al.
Figure 4. HLA-DP2–mimotope-2/Be tetramer staining of Be-specific T cell lines and ex vivo BAL cells from CBD patients. (A) Approxi-
mately 2,000 resonance units (RU) of biotinylated HLA-DP2–mimotope-2 and control HLA-DR52c–WIR complex were immobilized in flow cells of a
BIAcore streptavidin biosensor chip. Various concentrations of soluble AV22 TCR were injected through the flow cells before and after loading with
200 µM BeSO4, and the surface plasmon resonance signal was obtained. Data shown are representative of three independent experiments. (B) Stain-
ing of T cell hybridomas (Be-specific AV22, insulin B:9-23–specific 8-1.1, and dengue virus–specific DV-13) with HLA-DP2–mimotope-2 (DP2-Mim2)
and IAg7-insulin (IAg7-Ins) tetramers prepared into complexes either in the presence or absence of BeSO4. Cells were stained with 20 µg/ml of tetra-
mers, and fluorescence intensity was evaluated by flow cytometry. Representative results from three independent experiments are shown. All hybrid-
omas expressed high levels of TCR (not depicted). (C) Detection of HLA-DP2–mimotope-2/Be tetramer–binding T cells from parental T cell line derived
JEM Vol. 210, No. 7
concentrations, 2 were derived from spectrin and 4 were
derived from plexins. The most potent peptides (i.e., lowest
EC50 values) were derived from plexin A2 (HU25), plexin
A4 (HU31), and erythrocytic spectrin (HU1), although none
were as good as mimotope-4 (Fig. 5 A).
We next expressed HLA-DP2 with plexin A2 (PLXNA2),
plexin A4 (PLXNA4), erythrocytic spectrin (SPTB), or spec-
trin -chain (SPTBN1) peptides covalently attached to the
N terminus of the HLA-DPB1*0201 gene on mouse fibro-
blasts. Titering the number of fibroblasts as antigen-presenting
cells in the presence of BeSO4, both AV22 and AV9 showed
equivalent responses to plexin A2, plexin A4, and erythrocytic
spectrin peptides, as well as mimotopes 2 and 4 (Fig. 5 B). The
peptide derived from spectrin -chain (SPTBN1; HU3)
induced a significantly lower IL-2 response with both puri-
fied peptide and the covalently linked version, likely because
of poor HLA-DP2 binding as a result of an A at the p1 posi-
tion (Fig. 5, A and B). Based on these data, we focused on the
plexin family of proteins to ascertain whether these molecules
might be relevant in vivo in CBD.
Plexins are transmembrane proteins encoded by nine
genes (PLXNA1-4, B1-3, C1, and D1). Only the plexin A
family contains the stimulatory epitope for our Be-responsive
TCRs. However, the presence of this epitope in multiple
plexin A proteins meant that an shRNA knockdown ap-
proach was not feasible. Therefore, we first determined
whether plexin proteins can be processed by antigen-presenting
cells to yield the stimulatory, Be-dependent HLA-DP2–
binding epitope. Purified recombinant proteins encoding the
cytoplasmic domains of several mouse plexin molecules were
generated (He et al., 2009; Wang et al., 2012). All of these
proteins are >600 aa in length and have >98% identity with
human plexins. Each of the plexin A proteins (PLXNA1,
PLXNA2, and PLXNA4) was processed by HLA-DP2+
EBV-transformed B cells and stimulated IL-2 secretion from
AV22 only in the presence of Be (Fig. 5 C). As expected,
PLXNC1 protein, which does not contain the stimulatory
epitope, did not induce IL-2 secretion by AV22, and none of
the plexin proteins stimulated the dengue virus–specific
hybridoma (Fig. 5 C). Furthermore, the natural Be-specific
response observed using HLA-DP2–transfected fibroblasts
(DP8302) as antigen-presenting cells for AV22 was signifi-
cantly enhanced with the addition of all of the plexin A proteins
Next, additional Be-specific T cell lines and ex vivo BAL
cells from CBD patients were stained. Remarkably, all HLA-
DP2–expressing patients (n = 12) who had evidence of a
Be-specific adaptive immune response in the lung had CD4+
T cells in BAL that bound the HLA-DP2–mimotope-2/Be
tetramer (Fig. 4, D and E). For the ex vivo BAL samples (n = 8),
the range was 0.1–4.8% of CD4+ T cells, with an overall fre-
quency of 1.9 ± 0.7% (mean ± SEM; Fig. 4 E). Similar find-
ings were seen with the T cell lines (Fig. 4 E). None of the
HLA-DP2 CBD patients had detectable T cell staining with
tetramer (Fig. 4 E), nor did four HLA-DP2+ Be-sensitized
subjects who had no evidence of a Be-specific immune
response in BAL (not depicted), further validating the speci-
ficity of the reagent.
To examine the frequency of tetramer-staining CD4+
T cells relative to the overall frequency of Be-responsive cells
in the BAL of CBD patients, we coupled tetramer staining
with intracellular cytokine staining for Be-induced IFN-
and IL-2 expression (representative example shown in Fig. 4 F).
We focused on these Th1-type cytokines because they are
the most abundantly expressed T cell–derived cytokines in
CBD lung (Tinkle et al., 1997; Fontenot et al., 2002). For
ex vivo BAL cells, 5.1 ± 1.3% of IFN-–expressing and 3.2 ±
0.8% of IL-2–expressing CD4+ T cells bound tetramer
(Fig. 4 G). The majority of tetramer-binding CD4+ T cells did
not express either IFN- or IL-2 (Fig. 4, F and H). Although
other Be-dependent specificities were present, our data show-
ing the presence of CD4+ T cells specific for this ligand in all
HLA-DP2–expressing CBD patients tested suggest that the
HLA-DP2–mimotope-2/Be complex likely represents a major
ligand in the lung for Be-specific CD4+ T cells.
Delineation of endogenous peptides
that stimulate Be-specific TCRs
To discover endogenous peptides capable of stimulating the
Be-specific AV22 and AV9 hybridomas, we performed a bio-
metric analysis (Hemmer et al., 1999; Zhao et al., 2001; Pinilla
et al., 2003) using a matrix derived from the stimulatory po-
tency of each mixture defined with an amino acid at every
peptide position of the W2D4L5 positional scanning library.
As shown in Table 2, we screened 35 human peptides using
IL-2 secretion from AV22 and AV9 as a readout for T cell
response. Of the 11 peptides that gave positive results at low
from CBD patient 1332. BAL T cells after one and two cycles of stimulation with BeSO4 and after V5.1 sorting were stained with the HLA-DP2–
mimotope-2/Be (top) and IAg7-insulin (bottom) tetramers, and the frequency of CD4+ T cells stained with each tetramer is shown in each density plot.
Representative staining results from two independent experiments are shown. (D) HLA-DP2–mimotope-2/Be tetramer staining of ex vivo BAL cells
from CBD patients who are HLA-DP2+ (8722) and HLA-DP2 (1089). Density plots show tetramer staining of CBD patients, excluding cells staining
with CD8, CD14, and CD19, and gating for CD3 and CD4 expression. Results are representative of ex vivo BAL cells from eight HLA-DP2+ and four
HLA-DP2 CBD subjects. (E) Frequency (mean) of CD4+ T cells staining with the HLA-DP2–mimotope-2/Be tetramer is shown for ex vivo BAL cells
(n = 12) and BAL T cell lines (n = 7) derived from DP2+ and DP2 CBD patients. Frequency was determined by subtracting nonspecific staining ob-
served with the control tetramer from staining with the HLA-DP2 tetramer. (F) Flow cytometric analysis of dual intracellular cytokine and tetramer
staining of ex vivo BAL cells from a DP2+ CBD patient is shown. Density plots show IFN- (left) and IL-2 (right) expression relative to HLA-DP2–
mimotope-2/Be tetramer staining. Data are representative of the eight HLA-DP2+ CBD subjects studied. (G and H) For ex vivo BAL cells, the mean fre-
quency of IFN-– and IL-2–expressing CD4+ T cells that bind the HLA-DP2–mimotope-2/Be tetramer (G) and the frequency of tetramer-positive cells
that secrete these cytokines (H) are shown. (E, G, and H) Mean values for each group are indicated with horizontal bars.
Beryllium-dependent peptides in CBD | Falta et al.
affinity of the AV22 TCR for this complex in the presence
of BeSO4. As shown in Fig. 6 A, the AV22 TCR binds to
HLA-DP2–PLXNA4 only in the presence of Be, with identi-
cal binding kinetics for each of the four concentrations of
TCR injected. The measured TCR affinity (4.3 µM) for this
MHCII is very similar to that of the HLA-DP2–mimotope-2/
Be complex (compare with Fig. 4 A). An HLA-DP2–PLXNA4
tetramer, saturated in BeSO4 and labeled with PE, stained the
AV22 hybridoma with slightly lower fluorescence intensity
than the PE-labeled HLA-DP2–mimotope-2/Be tetramer
(Fig. 6 B, top). Using a mimotope-2/Be tetramer labeled with
(not depicted). Titering the concentration of plexin A pro-
teins to as low as 6 µg/ml still resulted in robust stimulation
of the AV22 hybridoma for PLXNA1 and PLXNA4 (Fig. 5 D).
Conversely, PLXNA2 was 15-fold less potent at stimulating
Be-dependent IL-2 secretion and could be detected begin-
ning at 50 µg/ml.
Be-loaded HLA-DP2–plexin A4 tetramer
staining of CD4+ T cells in BAL
Similar to our analysis of mimotope-2, we generated soluble
recombinant HLA-DP2–PLXNA4 and assessed the binding
Table 2. Human peptides screened for the ability to stimulate the AV22 and AV9 Be-specific T cell hybridomas
ID/rankRatio to max Peptide sequence GenBank
Designations in bold indicate peptides that stimulate AV22 and AV9 in the presence of BeSO4. For Ratio to max, the score of each peptide was divided by the matrix maximum
score. Peptide positions are shown in bold if peptides contain W2D4L5E7. Protein designations (if available) are provided only for peptides that gave a positive response.
Hybridomas were stimulated with 0.5 µg/ml of peptide and BeSO4 at 75 µM.
JEM Vol. 210, No. 7
majority of CD4+ T cells that bind the mimotope-2/Be tet-
ramer also bind the PLXNA4/Be tetramer, as indicated
by the diagonal staining distribution (Fig. 6 E). For CBD pa-
tient 6042, the dual staining cells are shifted to the right and
bind to the PLXNA4/Be tetramer more strongly than in
patients 8133 and 8845 (Fig. 6 E). Importantly, a discrete pop-
ulation of BAL T cells that only bind the PLXNA4/Be tetra-
mer was seen in these CBD patients (Fig. 6 E). Thus, these
data highlight the heterogeneity of the BAL CD4+ T cells that
bind these ligands and demonstrate a unique population of cells
that specifically recognizes only the HLA-DP2–PLXNA4/
Be complex, strongly supporting a role of plexin A as an
endogenous antigen for a set of Be-specific TCRs.
Plexin A protein expression in antigen-presenting cells,
BAL fluid, and lung tissue
To address whether plexins are present at the site of inflam-
mation in CBD, we used Western blot analysis to assess ex-
pression of plexin A2 and A4 proteins in the antigen-presenting
brilliant violet 421 (BV421), hybridoma cells stained with
both tetramers showed a uniform pattern of costaining
(Fig. 6 B, bottom). Using ex vivo BAL cells from four of
the HLA-DP2+ CBD patients previously stained with the
HLA-DP2–mimotope-2/Be tetramer in Fig. 4 E, we identi-
fied PLXNA4/Be tetramer–staining CD4+ T cells in all sub-
jects (Fig. 6, C and D). No PLXNA4/Be tetramer–staining
T cells were seen using BAL cells from an HLA-DP2 CBD
patient (not depicted). In three of the HLA-DP2+ CBD
patients, the frequency of PLXNA4/Be tetramer–staining
T cells was higher than that seen with the mimotope-2/Be
tetramer (Fig. 6 D).
Having tetramers labeled with different fluorophores
enabled us to simultaneously stain ex vivo BAL cells from
CBD patients with the HLA-DP2–mimotope-2/Be and
HLA-DP2–PLXNA4/Be tetramers. As shown in Fig. 6 E,
distinct populations of CD4+ T cells emerged that differen-
tially stain with the two tetramers. Although the relative affin-
ity of the BAL CD4+ T cells for each tetramer varies, the
Figure 5. Characterization of candidate endogenous peptides that stimulate Be-specific T cell hybridomas. (A) Dose–response curves to stimu-
latory peptides identified from the biometric analysis of T cell hybridoma AV22 are shown. Equal numbers of AV22 cells and DP2.21 antigen-presenting
cells were mixed with 75 µM BeSO4 and highly purified peptides. IL-2 secretion was measured by ELISA after 22 h of culture, and data are plotted as the
percentage of maximum IL-2 secretion against concentration of peptide in the presence of BeSO4. EC50 values (mean ± SEM nM) for mimotope-4 and
each human peptide from four independent experiments are shown. Note that error bars have been left out for viewing clarity. (B) AV22 response to
antigen-presenting cells expressing HLA-DP2 with spectrin and plexin peptides covalently attached to the N terminus of the -chain. Percentage of max-
imum IL-2 secretion versus the number of antigen-presenting cells added per well is shown. Results are representative of three independent experiments.
(C) IL-2 response (mean ± SD pg/ml) of AV22 and dengue virus–specific hybridoma DV-13 to plexin proteins presented by an EBV-transformed B cell line
derived from CBD patient 1332 is shown. Plexin proteins (A1, A2, A4, and C1, all at 100 µg/ml) were added in the presence and absence of 75 µM BeSO4.
Control peptides, mimotope-4 and dengue viral peptide, were added at 100 nM and 20 µM, respectively. Representative results from three independent
experiments are shown. (D) Dose–response curves of AV22 to plexin A1, A2, and A4 proteins presented by EBV-transformed B cells in the presence of
75 µM BeSO4 are shown. Representative results from three independent experiments (mean IL-2 ± SD pg/ml) performed in triplicate are shown.
Beryllium-dependent peptides in CBD | Falta et al.
driving T cell activation. In the case of CBD, Glu69-
containing HLA-DP molecules and Be are known require-
ments for activation of Be-specific T cells. Until now, the
identity of the peptides that complete this ligand has remained
unknown. Using positional scanning libraries and biometric
data analysis, we delineated a spectrum of mimotopes and
endogenously derived human peptides that stimulate Be-
specific TCRs isolated from the lung of a patient with active
CBD. Be-loaded HLA-DP2 tetramers with mimotope-2 and
plexin A4 peptides in the binding groove identified antigen-
specific CD4+ T cells in the target organ of all HLA-DP2–
expressing CBD patients evaluated, confirming the importance
of these related ligands in driving recruitment of these patho-
genic T cells to the lung.
Because of the inability of effector memory CD4+ T cells
obtained from a target organ to vigorously undergo repeated
rounds of stimulation (Fontenot et al., 2005), delineating
antigen specificity of human CD4+ T cells has been difficult.
We overcame this obstacle by using the immortalized T cell
hybridoma line 54 expressing the TCRs of interest and an
unbiased positional scanning library. From the deconvolution
cells routinely used in Be-specific T cell hybridoma activation
assays. Using mouse brain as a positive control, plexin A2 was
present in both HLA-DP2–expressing fibroblasts (DP8302
and DP2.21) and EBV-transformed B cells, whereas plexin
A4 was expressed in DP8302 and DP2.21 (Fig. 7 A). Using
concentrated BAL fluid from CBD patients, we documented
expression of plexin A2 in 6/6 and plexin A4 in 5/6 samples
analyzed (Fig. 7 A). Using the same plexin A2 mAb and mouse
cerebellum as a positive control, Fig. 7 B shows plexin A2
staining in Purkinje cells and white matter. Staining of a trans-
bronchial lung biopsy from a CBD patient showed plexin A2
expression predominantly within the bronchial epithelium
(Fig. 7 C). Similar findings were observed in lung tissue ob-
tained from two other CBD patients (not depicted), and isotype
control antibody staining was negative. Thus, we demonstrate
that plexin A proteins are readily available as a source of anti-
gen in lung tissue and BAL fluid of CBD patients.
Understanding the pathogenesis of CD4+ T cell–mediated
diseases requires an elucidation of the ligands responsible for
Figure 6. HLA-DP2–PLXNA4/Be tetramer staining
of ex vivo BAL cells from CBD patients. (A) The
binding kinetics of soluble AV22 TCR to biotinylated
HLA-DP2–PLXNA4 with and without Be are shown.
Four concentrations of soluble AV22 TCR were injected
through the flow cells before and after loading with
200 µM BeSO4, and the surface plasmon resonance
signal was obtained. Data shown are representative of
three independent experiments. (B) Staining of the
AV22 T cell hybridoma with HLA-DP2–mimotope-2,
HLA-DP2–mimotope-2/Be, and HLA-DP2–PLXNA4/Be
tetramers individually (top) and costaining with Be-
saturated mimotope-2 (BV421 labeled) and PLXNA4 (PE
labeled) tetramers (bottom). The fluorescence intensity
of cells was evaluated after staining with 20 µg/ml of
tetramers for 2 h at 37°C. Representative results from
three independent experiments are shown. (C) Density
plots showing control HLA-DP2–mimotope-2 without
Be (left) and HLA-DP2–PLXNA4/Be (right) tetramer
staining of CD4+ T cells from ex vivo BAL cells of a HLA-
DP2+ CBD patient. Results are representative of four
HLA-DP2+ CBD subjects stained. (D) Summary of the
frequency of tetramer staining of ex vivo BAL CD4+
T cells from four HLA-DP2+ CBD patients individually
stained with the HLA-DP2–mimotope-2/Be and HLA-
DP2–PLXNA4/Be tetramers. (E) Density plots of ex vivo
BAL cells from four DP2+ CBD patients costained with
HLA-DP2–PLXNA4/Be and HLA-DP2–mimotope-2/Be
tetramers are shown.
JEM Vol. 210, No. 7
hybridoma to its ligand (NS3, p254-265) is not affected by
the presence of Be (unpublished data). Because the NS3 pep-
tide does not contain acidic amino acids at p4 and p7 (L and
histidine [H], respectively), it is thus incapable of coordinating
Be, providing an explanation of why Be does not inhibit TCR
interaction with the HLA-DP2–dengue viral complex.
The identification of Be-dependent mimotopes enabled
us to answer several critical questions regarding TCR recog-
nition of this unconventional antigen. Using surface plasmon
resonance, the AV22 TCR only interacted with the HLA-
DP2–peptide complex if Be was present, and this interaction
occurred with high affinity (Kd = 4.6 µM), with the majority
of TCR/pMHCII affinity constants (Kd) ranging between 10
and 100 µM (Davis et al., 1998; van der Merwe and Davis,
2003). In addition, we predict that Be binding to HLA-DP2
is nearly irreversible, based on the stability of the complex on
the BIAcore chip as well as the stability of the staining inten-
sity of Be-specific T cells for the BeSO4-soaked HLA-DP2–
mimotope tetramer over an 8-mo period.
The ability to track antigen-specific T cells using tetra-
mers of MHC molecules has been a significant advance over
the last decade (Nepom, 2012). However, the application of
this technology to CD4+ T cells has proven difficult, predom-
inantly because of a low frequency of antigen-specific CD4+
T cells that expand in response to antigen (Homann et al.,
2001). Consequently, in vitro expansion or magnetic bead
capture of antigen-specific CD4+ T cells is typically necessary
before staining (Novak et al., 1999; Day et al., 2003; Moon et al.,
2009; Wambre et al., 2012). Initially, we used an HLA-DP2–
mimotope-2/Be tetramer to stain ex vivo CD4+ T cells from
the BAL of CBD patients and detected large numbers of
CD4+ T cells specific for this ligand in all DP2-expressing
CBD patients tested, with frequencies ranging from 0.1 to 4.8%.
of the positional scanning library data, important characteris-
tics of Be-dependent mimotopes were identified, including
(a) preference for bulky hydrophobic or nonpolar amino
acids at the p1 and p6 anchor positions that match the known
HLA-DP2–binding motif (Díaz et al., 2005; Sidney et al.,
2010), (b) independence of Be-dependent TCR recognition
on the C terminus of the peptide, (c) distinct profile of
allowed amino acids at the TCR contact sites p2, p3, and p5,
and (d) negatively charged aspartic and glutamic acid residues
at p4 and p7 that represent potential Be coordination sites.
Modeling of mimotope-2 in the peptide-binding groove of
the solved HLA-DP2 structure reveals the acidic pocket com-
posed of glutamic acid residues at positions 26, 68, and 69 of
the HLA-DP2 -chain with an additional two acidic amino
acids contributed by the peptide in the Be-binding pocket
(Fig. 8). The positions of the p4 and p7 amino acids of the
peptide strongly suggest their role in capturing Be for T cell
recognition. Thus, this acidic cluster provides multiple elec-
tron donors for the coordination of a Be moiety within the
TCR footprint. Our present and previously published data
(Bill et al., 2005; Dai et al., 2010) show that mutagenesis of
any of these five acidic residues abolishes T cell activation,
suggesting that this acidic pocket is the Be-binding site respon-
sible for the induction of an adaptive immune response. In the
absence of Be, this solvent-exposed collection of acidic amino
acids is likely stabilized by other divalent cations (e.g., magne-
sium) or a water molecule. In retrospect, our inability to identify
a Be-dependent peptide from the list of peptides commonly
bound to HLA-DP2 is likely caused by the preference of
those peptides to possess a positively charged arginine and
lysine at the p4 position (Díaz et al., 2005). As further evidence
of the critical contribution of peptide in coordinating Be, the
response of the HLA-DP2–restricted dengue virus–specific
Figure 7. Distribution of plexin A proteins in antigen-presenting cells, BAL fluid, and lung tissue derived from CBD patients. (A) Western blot
analysis of cell extracts from the mouse fibroblast cell lines (DP8302 and DP2.21) and EBV-transformed B cells from CBD patient 1332 and BAL fluid from
two representative CBD patients is shown. Anti-plexin antibodies cross-react to mouse and human plexin A2 and A4. BAL fluid samples were concen-
trated 300-fold and resolved on a 7.5% polyacrylamide gel. Results are representative of a minimum of three independent experiments for cell extracts
and BAL fluids. (B and C) Immunofluorescence staining of plexin A2 (red) and cell nuclei (blue) in mouse cerebellum (B) and transbronchial lung biopsy
tissue from a CBD patient (C) is shown. An enlarged view of the area within the white box in C showing plexin A2 expression in bronchial epithelial cells is
shown in the right panel. Results are representative of the three transbronchial lung biopsy specimens analyzed.
Beryllium-dependent peptides in CBD | Falta et al.
association with Be to our T cell hybridomas. Furthermore,
these proteins are present in lung tissue and BAL fluid from
CBD patients and likely are accessible to antigen-presenting
cells during Be-induced lung inflammation. Using an HLA-
DP2–PLXNA4/Be tetramer to stain ex vivo BAL cells from
CBD patients, CD4+ T cells that bind to this complex were
present in all subjects. Furthermore, simultaneous staining
with mimotope-2/Be and PLXNA4/Be tetramers revealed
distinct populations of T cells that bind these tetramers with
varying affinities. Although the amino acid sequence of mi-
motope-2 was dictated based on the preferences of a single
TCR, the binding patterns of ex vivo BAL CD4+ T cells
reflect a heterogeneous collection of T cells that display a
range of affinities for both ligands. Our findings of a distinct
population of BAL CD4+ T cells that only bind to the
PLXNA4/Be tetramer provide strong support that plexin A
proteins are a source of endogenous self-peptides in vivo that
are required for T cell recognition of Be.
Although we have identified mimotopes and endogenous
plexin A peptides that stimulate Be-specific CD4+ T cells in
the presence of HLA-DP2 and Be, our data indicate that
there are other Be-responsive T cells that do not bind these
tetramers and must recognize other peptides. It is unknown
whether the number of additional peptides is large or small.
Furthermore, although the source of these stimulating pep-
tides is likely from self-proteins, they may also be derived
from exogenous sources such as viruses or bacteria. However,
based on our library screen, we predict that the common
theme of Be-dependent peptides will be the presence of acidic,
Be-coordinating amino acids at p4 and p7, with amino acids
at p2, p3, and p5 being dictated by the TCR contact require-
ments of particular Be-specific T cells.
In addition to the role of conventional MHC-bound
peptides in anchoring to MHC and interacting with TCR,
our data establish a novel function for peptide in metal hyper-
sensitivity, that of metal ion capture. Importantly, this process
can result in the conversion of a self-peptide into a neoanti-
gen. The creation of neoantigens that are absent in the thymus
and arise in target organs plays a key role in the genesis of
autoimmunity (Marrack and Kappler, 2012). For example, in
celiac disease, transglutaminase enzymatically modifies glia-
din, resulting in deamidated gliadin peptides that are recog-
nized as nonself by CD4+ T cells and lead to the generation
of a T cell–mediated response in genetically susceptible hosts
(Molberg et al., 1998). The abacavir hypersensitivity syn-
drome, which occurs in HLA-B*57:01 patients treated with
abacavir, results from binding of the drug to the peptide-
binding groove of the MHCI molecule and generation of
altered peptides that activate antigen-specific CD8+ T cells
(Illing et al., 2012). In the case of CBD, self-peptides bound in
the HLA-DP2–binding groove, such as those derived from
plexin A and spectrin proteins, may be altered in the presence
of Be, resulting in the conversion of endogenous peptides into
neoantigens and culminating in Be-specific adaptive immunity.
Alternatively, Be binding to HLA-DP2 may partially neutral-
ize the acidity of the p4 pocket and subtly alter the peptide
In addition, lungs of HLA-DP2 transgenic mice exposed
to BeO contain CD4+ T cells that share the same TCR speci-
ficity and bind to the DP2-mimotope/Be tetramer (unpub-
lished data), reinforcing the importance of this ligand in
the generation of Be-specific adaptive immunity in humans
Our data also suggest that the frequency of Be-responsive
T cells as determined by intracellular cytokine staining un-
derestimates the number of Be-specific T cells in the lung
because two thirds of Be-specific, tetramer-binding T cells fail
to express IFN- and/or IL-2. We have previously shown that
some Be-specific CD4+ T cells proliferate poorly after anti-
gen exposure, while maintaining the ability to secrete Th1-
type cytokines (Fontenot et al., 2002, 2005). The current study
extends those findings by showing that a portion of Be-
responsive T cells in BAL have lost their Th1 cytokine–secreting
ability, suggesting a terminally differentiated T cell phenotype
and consistent with up-regulated programmed death-1 ex-
pression on Be-specific CD4+ T cells in CBD (Palmer et al.,
2008). Although it is possible that the tetramer-positive cells
may be secreting cytokines other than IFN- and IL-2, we
have never detected IL-4, IL-10, or IL-17 in BAL-derived
CD4+ T cells in response to either BeSO4 or staphylococcal
enterotoxin B (unpublished data). Overall, addition of HLA-
DP2 tetramer staining to Be-induced cytokine staining may
allow a more accurate assessment of the frequency of Be-
specific T cells in the lung and could potentially be developed
into a biomarker of disease development and progression.
To identify endogenous peptides for the Be-specific
TCRs, we used the biased W2D4L5 positional scanning library
data to rank all overlapping peptides in a human protein data-
base for their stimulatory potential. We focused on plexin
A peptides based on screening of a limited number of these
peptides and show that plexin A proteins are efficiently pro-
cessed by HLA-DP2–expressing EBV-transformed B cells
and fibroblasts to present the core TCR recognition motif in
Figure 8. Model of mimotope-2 in the peptide-binding groove of
HLA-DP2. The amino acids of mimotope-2 were introduced into the
peptide of the HLA-DP2–pDRa structure (Protein Data Bank accession no.
3LQZ) using Swiss PDB Viewer 4.0. Rotamers of the amino acids of mimo-
tope-2 that avoided conflict with the structure were selected. Ribbon
representations of DP2 (cyan)- and DP2 (magenta)-chains are shown.
Wireframe representations of the side chains of mimotope-2, Glu26,
Glu68, and Glu69 with CPK coloring are shown.
JEM Vol. 210, No. 7
scanning library was synthesized. This library consists of 200 mixtures pre-
pared in an OX9 format, where O represents a specific amino acid at a de-
fined position and X represents an equal molar mixture of 20 natural amino
acids (except cysteine) in each of the remaining 9 positions. Each OX9 mix-
ture consists of 3.2 × 1011 different decapeptides, and the total number of
peptides in the library is 6.4 × 1012. Mixtures containing multiple fixed posi-
tions and a biased W2D4L5 positional scanning library were synthesized as
previously described (Pinilla et al., 1994; Hemmer et al., 1999).
Individual candidate peptides were initially synthesized using the PEP-
Screen 96-well array (Sigma-Aldrich). Peptides chosen for further study
were synthesized on a larger scale and tested at 95% purity (CPC Scientific).
All peptides were first dissolved in DMSO at high concentration before mak-
ing a working stock in PBS. DNA encoding the cytoplasmic domains of
mouse plexins (A1, residues 1269–1894; A2, 1264–1894; A4, 1263–1893; C1,
975–1571) was cloned into a modified pET28 vector (EMD Millipore) and
expressed in ArticExpress bacteria (Agilent Technologies). Proteins were puri-
fied to >95% with a HisTrap column (GE Healthcare) followed by ion ex-
change chromatography (1 ml resource Q; GE Healthcare) and resuspended
in 10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, and 2 mM DTT (He
et al., 2009; Wang et al., 2012).
T cell hybridoma activation assays. T cell hybridomas (2.5 × 104–5 cells
per well) were cultured in protein-free medium in 96-well flat-bottomed
tissue culture plates (Falcon) in the presence of HLA-DP2–transfected mouse
fibroblasts (105 cells) and 75 µM BeSO4. Initial experiments involving the
decapeptide positional scanning library were performed using mixtures at
200 µg/ml. Subsequent mixtures were tested multiple times in duplicate at
concentrations ranging from 20 to 200 µg/ml. Concentrations of crude pep-
tides were 0.5, 5, and 50 µg/ml. For dose–response curves to purified pep-
tides in the presence of BeSO4, peptide was added at concentrations ranging
from 0.1 nM to 10 µM. For testing plexin proteins, assays were completed
in IMDM/GlutaMAX tissue culture medium (Invitrogen) supplemented
with 10% FBS (Hyclone), 1 mM Na pyruvate, 100 U/ml penicillin, and
100 µg/ml streptomycin (all from Invitrogen). 6–400 µg/ml of protein was
added to wells in triplicate containing autologous EBV-transformed B cells
(2 × 105 cells) or DP8302 fibroblasts (105 cells) in either the absence or pres-
ence of 75 µM BeSO4.
After 22–24 h, supernatants were harvested, and mouse IL-2 was mea-
sured by ELISA (eBioscience). In some experiments, the concentration of
peptide that provided 50% of the maximum IL-2 response (EC50) for each
hybridoma was determined using nonlinear regression (sigmoidal-fit; Prism;
Scoring matrix and database searches. A scoring matrix was generated
by assigning numerical values to the stimulatory potency of defined amino
acids at each position in the W2D4L5 decapeptide positional library. The
values were calculated as the logarithm of IL-2 (pg/ml) in the presence of
peptide mixtures/Be. For the three defined positions (W2D4L5), a value of
0 was assigned to all the amino acids except for the amino acid that was fixed
at that position (i.e., W in position 2, D in position 4, and L in position 5).
The value for these amino acids was assigned the maximum stimulatory
potency measured among all the mixtures at all positions (E7). The predicted
stimulatory potential of a peptide, or score, was calculated by summing the
values associated with each amino acid in each position of the peptide. The
sum of the maximum values at each position is defined as the maximum
matrix score. Using a web-based search tool, the scoring matrix was applied
to rank, according to their stimulatory score, all of the overlapping peptides
within each protein sequence of a human GenPept (version 156) protein
database, as previously described (Hemmer et al., 1999; Zhao et al., 2001;
Pinilla et al., 2003).
BIAcore measurement of TCR affinity and kinetics. Approximately
2,000 resonance units of HLA-DP2–mimotope-2, HLA-DP2–PLXNA4, or an
irrelevant MHCII molecule, HLA-DR52c–WIR (Yin et al., 2012), were cap-
tured in separate flow cells of a BIAcore streptavidin biosensor chip. Various
repertoire that binds to this MHCII, contributing to the gen-
eration of de novo antigen-specific responses. Although Be is
an absolute requirement for T cell recognition, it is unknown
whether the CDR loops of the AV22 TCR directly contact
a solvent-exposed Be moiety as suggested by our model of
the HLA-DP2–mimotope-2/Be complex or recognize an
altered self-peptide with no direct contacts between Be and
the TCR. Collectively, our findings further blur the distinc-
tion between hypersensitivity and autoimmunity, showing
how a modified self-peptide can create a neoantigen as the
target in these inflammatory diseases.
In conclusion, our findings identify the first complete
ligand for a metal-specific TCR and demonstrate the require-
ment for additional acidic amino acids contributed by the
peptide for Be capture and presentation. This additional func-
tion of the peptide in metal hypersensitivity provides an ex-
planation for the generation of a human disease in a genetically
susceptible host exposed to an environmental antigen.
MATERIALS AND METHODS
Study population and Be-specific T cell lines. 17 patients with CBD
were enrolled in this study. CBD was diagnosed using previously defined cri-
teria, including a history of Be exposure, the presence of granulomatous in-
flammation on lung biopsy, and a positive proliferative response of blood
and/or BAL T cells to BeSO4 in vitro (Rossman et al., 1988; Newman et al.,
1989). For select subjects, BAL-derived, Be-specific T cell lines were gener-
ated as previously described (Fontenot et al., 2000; Bill et al., 2005). HLA-DP
typing was performed by ClinImmune Labs. Informed consent was ob-
tained from each patient, and the Human Subject Institutional Review
Boards at the University of Colorado Denver and National Jewish Health
approved the protocol.
Generation of mouse hybridomas expressing TCRs from human
T cell clones. TCRs expressed by two Be-specific T cell clones derived from
the lung of CBD patient 1332 (Bowerman et al., 2011) and a dengue virus–
specific T cell clone were introduced into the TCR-negative mouse recipient
hybridoma cell line 54, which expresses human CD4 (Boen et al., 2000). In
brief, PCR fragments encoding the extracellular variable domains of the
TCR - and -chains of each T cell clone were introduced into separate
expression vectors that encode either a mouse C or C domain. The full-
length chimeric TCRA and TCRB gene constructs were cotransfected into
54 recipient cells by electroporation, and transfectants were selected in
1 mg/ml G418-containing medium (Cellgro; Bowerman et al., 2011). Trans-
fectants were screened for TCR expression using a PE-labeled H57-597
mAb (eBioscience). Subcloning by limiting dilution was performed, and the
subclones with the highest TCR expression, as determined by staining with
the H57-597 mAb, were chosen for stimulation experiments.
EBV-transformed B cells and fibroblast cell lines. The EBV-transformed
B lymphoblastoid cell line from CBD patient 1332 was generated from PBMCs
as previously described (Fontenot et al., 2000). Mouse fibroblasts transfected
with genes encoding wild-type HLA-DP2 or DP2 with peptides covalently
attached were used as antigen-presenting cells to present Be and peptides.
DP8302 expresses the DPA*0103 and DPB*0201 (HLA-DP2) genes (Bill
et al., 2005). DP2.21 cells were derived from kidney fibroblasts of IAb/, Ii/
C57BL/6 mice (Huseby et al., 2003) and express DP2 (DPA*0103/
DPB1*0201) on their surface. For T cell hybridoma activation experiments,
DP2.21 cells were adapted to grow in the protein-free medium OptiPro SFM
(Invitrogen) supplemented with 4 mM GlutaMAX (Invitrogen).
Positional scanning libraries, mixtures, peptides, and plexin proteins.
A decapeptide, N-acetylated, C-terminal–amidated, L–amino acid positional
Beryllium-dependent peptides in CBD | Falta et al.
7.5% polyacrylamide gel (Bio-Rad Laboratories) with mouse brain extract
(Santa Cruz Biotechnology, Inc.) as a positive control and transferred to poly-
vinylidene fluoride membranes (EMD Millipore). After blocking with TBS–
5% nonfat powdered milk, membranes were incubated with polyclonal rabbit
anti–human plexin A2 (H-70; Santa Cruz Biotechnology, Inc.) or a rabbit
anti–plexin A4 (C5D1; Cell Signaling Technology) mAb at 4°C overnight.
Membranes were incubated with goat anti–rabbit IgG-HRP secondary anti-
body (Santa Cruz Biotechnology, Inc.), and immunoreactive proteins were
visualized by an ECL system (Thermo Fisher Scientific).
For microscopic demonstration of plexin A2 expression in human lung
tissue, formalin-fixed, paraffin-embedded transbronchial lung biopsies from
three CBD patients and mouse cerebellum (positive control) were deparaf-
finized, followed by epitope retrieval (heat induced, Tris/EDTA, pH 9.0) and
blocking with Fc receptor block (Miltenyi Biotec) in 1% BSA/PBS. Slides
were incubated for 3 h with 2 µg/ml of the anti–plexin A2 mAb or rabbit
polyclonal IgG antibody (negative control), followed by 2 µg/ml of highly
cross-adsorbed Alexa Fluor 555 goat anti–rabbit IgG (Invitrogen) for 1 h.
Hoechst 33342 was used to stain the nuclei of cells. Slides were visualized on
an Axio microscope (Carl Zeiss).
Concentration of BAL fluid. Previously frozen (80°C) BAL fluids from
CBD patients were filtered (0.22-µm filter; Corning), and 15-ml aliquots
were concentrated in an Ultra-15 Centrifugal Filter Unit (50,000 MWCO;
Amicon). Retentates were further concentrated in an Ultra-0.5 Centrifugal
Filter Unit (100,000 MWCO; Amicon) to a final volume of 50 µl. The integ-
rity of BAL fluid proteins was assessed by SDS-PAGE, and equal amounts of
protein were resolved by Western blot.
We thank Tina Gibbins for the synthesis of peptide mixtures.
This work was supported by National Institutes of Health grants HL062410,
ES011810, HL102245 (to A.P. Fontenot), and GM088197 (to X. Zhang) and Clinical
and Translational Sciences Institute grant UL1 TR000154 from the National Center
for Advancing Translational Sciences. The work was also supported by the Multiple
Sclerosis Research Institute (grant to C. Pinilla) and the Welch Foundation (grant
I-1702 to X. Zhang).
The authors declare no competing financial interests.
Submitted: 29 October 2012
Accepted: 31 May 2013
Bill, J.R., D.G. Mack, M.T. Falta, L.A. Maier, A.K. Sullivan, F.G. Joslin, A.K.
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tion of antigen-specific T cells with multivalent soluble class II MHC
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Marrack, G. Eisenbarth, and J.W. Kappler. 2011. Specificity and detec-
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diabetic (NOD) mouse. Proc. Natl. Acad. Sci. USA. 108:16729–16734.
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concentrations of soluble AV22 TCR were injected through the flow cells
before and after loading with 200 µM BeSO4, and the surface plasmon reso-
nance signal was recorded during and after the injections. The resonance
signal in the control flow cell was subtracted from those in the other flow
cells to correct for the fluid phase signal. The kinetics of AV22 binding to
HLA-DP2–mimotope-2 and HLA-DP2–PLXNA4 after loading with Be2+
were calculated using BIAeval software (version 4.1).
Preparation of soluble MHCII–covalent peptide tetramers. Gene
fragments encoding the extracellular domains of the - and -chains of
HLA-DP2 were cloned into separate baculovirus transfer vectors. Sequence
encoding mimotope-2 (FWIDLFETIG) or PLXNA4 peptide (FVDDLFETIF)
and a flexible linker were inserted between the signal peptide and the
N terminus of the mature -chain to tether the peptide to MHCII. The
complete constructions were incorporated into Sapphire baculovirus DNA
(Orbigen) to produce high-titer virus stock. Soluble HLA-DP2–mimotope-2
and HLA-DP2–PLXNA4 proteins were purified from supernatants of
infected High5 insect cells (Invitrogen) by immunoaffinity chromatogra-
phy and size-exclusion chromatography using Superdex200 10/300GL (GE
Healthcare). Both proteins were biotinylated with the BirA enzyme (Avidity)
and incorporated into saturated complexes with PE-streptavidin (Prozyme)
or BV421-streptavidin (BioLegend; Crawford et al., 1998), with and without
200 µM BeSO4. Tetramers were separated from monomeric protein by FPLC
size-exclusion chromatography. An IAg7-insulin B:9-23 tetramer and a T cell
hybridoma specific for this ligand (8-1.1; Crawford et al., 2011) were used as
Tetramer and dual intracellular cytokine/tetramer assay. Hybridoma
cells were incubated in 25 µl of culture medium containing the HLA-DP2–
mimotope-2, HLA-DP2–mimotope-2/Be, DP2-PLXNA4/Be, or IAg7-
insulin tetramer (20 µg/ml) for 2 h at 37°C in a humidified 10% CO2
incubator, with gentle mixing every 20 min. In separate tubes, cells were
stained with PE-labeled H57-597 mAb (eBioscience) at 4°C to confirm
For stimulation of cytokine expression, 5–20 × 106 cells were exposed
to either medium or 100 µM BeSO4 for 6 h at 37°C, with 10 µg/ml
brefeldin A (BD) added after the first hour. For T cell lines, an equal num-
ber of autologous EBV-transformed B cells was added to the assay as
antigen-presenting cells. Cells were washed and stained with 20 µg/ml
PE-labeled DP2–mimotope-2/Be tetramer or IAg7-insulin tetramer. After
2 h, cells were stained for 30 min at 4°C with the following mAbs: anti-
CD3–Texas Red, anti–CD4-PerCP-Cy5.5, anti–CD8-v500, anti–CD14-
FITC, and anti–CD19-FITC (all from eBioscience). Cells were fixed,
permeabilized, and stained with anti–IFN-–PE-Cy7 (BD) and anti–IL-2–
AF700 (BioLegend) mAb for 30 min. The viable mononuclear cell popula-
tion was evaluated for fluorescence intensity on an LSR-II flow cytometer
(BD). Data were analyzed with FlowJo software (Tree Star). Cells express-
ing CD8, CD14, and CD19 were excluded from the analysis. T cells posi-
tively staining for CD3 and CD4 were gated and analyzed for tetramer
binding and cytokine expression.
For tetramer costaining of ex vivo BAL cells from CBD patients, cells
were incubated in 25 µl of staining medium containing the HLA-DP2–
mimotope-2 (control), HLA-DP2–mimotope-2/Be, or HLA-DP2–PLXNA4/
Be tetramer for 2 h at 37°C. Cells were surface stained and analyzed as
Western blot analysis and immunofluorescence microscopy. Cells
from fibroblast lines DP8302 and DP2.21 and EBV-transformed B cells were
lysed in RIPA buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Triton
X-100, 0.1% SDS glycerol, 1 mM PMSF, 0.5% Na deoxycholate, and protease
inhibitor cocktail [Sigma-Aldrich]), incubated for 30 min at 4°C with con-
tinuous agitation, and centrifuged at 16,000 g for 20 min at 4°C. Total protein
was determined using a Bradford protein assay (Bio-Rad Laboratories).
Equivalent amounts of protein (50 µg) were resolved by SDS-PAGE on a
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