Supplemental Materials
Metal-specific CD4+ T cell responses induced by beryllium exposure in HLA-DP2
transgenic mice
Michael T. Falta, PhD1, Alex N. Tinega1, Douglas G. Mack, PhD1, Natalie A. Bowerman, PhD1,
Frances Crawford2,3, John W. Kappler, PhD2,3, Clemencia Pinilla, PhD4, and Andrew P. Fontenot.
MD1,2
Departments of 1Medicine and 2Immunology, University of Colorado Anschutz Medical Campus,
Aurora, CO 80045 USA
3
Howard Hughes Medical Institute, National Jewish Health, Denver, CO 80206 USA
4
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121 USA
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Supplemental Methods
Flow cytometric analysis of TCR Vβ expression
Prepared splenocytes, lung and BAL cells isolated from unexposed or BeO-exposed HLA-DP2 Tg
FVB/N mice were stained either individually or as pooled samples with fluorescently conjugated
mAb specific to CD4 (PerCP or APC) and murine TCR Vβ (FITC). The panel of anti-TCR Vβ
mAb (BD Pharmingen) included anti-mouse Vβ2 (clone B20.6), Vβ3 (KJ25), Vβ4 (KT4), Vβ6
(RR4-7), Vβ7 (TR310), Vβ14 (14-2), and Vβ17 (KJ23). Cells were run on a FACSCalibur flow
cytometer (BD), and the frequency of CD4+ T cells in the lymphocyte scatter gate that expressed
the different Vβ’s was assessed using FlowJo software (Tree Star).
Transfectomas expressing particular murine Be-specific TCRs
TCRs expressed by select Be-specific hybridomas were cloned into expression vectors and stably
transfected into the mouse hybridoma cell line 54ζ, which expresses human CD4 (1-4). Separate
expression vectors encoding the variable domains of the TCRA and TCRB genes juxtaposed with
either the murine AC or BC gene segments were generated and co-transfected into recipient cells
by electroporation. Transfectants were selected in G418-containing medium (1 mg/ml; Cellgro),
screened for TCR expression using a PE-labeled H57-597 mAb (eBioscience), and cloned by
limiting dilution to identify subclones with the highest TCR expression (data not shown).
HLA-DP2 transfected fibroblast cell lines
Murine fibroblasts transfected with genes encoding wild-type HLA-DP2 or HLA-DP2 with single
amino acid substitutions in the β-chain were used as antigen-presenting cells to present Be and
peptides. DP2.21 cells are kidney fibroblasts derived from IAb-/-, Ii-/- C57BL/6 mice (5) and co-
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transfected to express HLA-DP2 (DPA*0103/DPB1*0201). For T cell transfectoma activation
assays, this cell line was adapted to grow in protein-free tissue culture medium (OptiPro SFM;
Invitrogen), supplemented with 4 mM glutaMAX (Invitrogen). DP8302 is a DAP.3 L cell line that
also expresses wild-type HLA-DP2 (6, 7).
For HLA-DP2 β-chain variants, site-directed
mutagenesis of the DPB1*0201 gene using overlapping PCR primers to encode single amino acid
gene mutations was performed as previously described (6, 8). HLA-DP2 variants were expressed
on the DAP.3 L cell line and stained with an anti-HLA-DP mAb (clone B7.21) to ensure similar
surface expression as wild-type (data not shown). All DAP.3 L cell lines were grown in complete
culture media (C-IMDM) consisting of IMDM/glutamax tissue culture medium (Invitrogen)
supplemented with 10% heat-inactivated FBS (Hyclone), 1 mM sodium pyruvate, 100 units/ml
penicillin, 100 µg/ml streptomycin (all from Invitrogen).
T cell hybridoma/transfectoma activation assays
T cell hybridomas or transfectomas (1 x 105 cells per well) were cultured in 96-well flat-bottomed
tissue culture plates (Falcon) in the presence of HLA-DP2-transfected murine fibroblasts (1 x 105
cells) and varying amounts of BeSO4 and peptides. For screening of the decapeptide positional
scanning library, mixtures were added (200 µg/ml) in duplicate to DP2.21 cells in protein-free
medium for 3 hours prior to the addition of T cell transfectoma cells and BeSO4 (75 µM). Identical
experimental conditions were used to test crude preparations of mimotopes (0.5, 5.0 and 50 g/ml)
as well as for performing dose response curves to purified peptides (0.1 nM to 10 M) in the
presence of BeSO4. Standard dose response curves to BeSO4 (range concentrations) and analysis
of transfectoma response to HLA-DP2 variants presenting different pure peptides were performed
in C-IMDM.
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Cloning of PLXNA4/Be-specific TCRs from CD4+ T cells isolated by single cell sorting
A single cell suspension was prepared from the lungs of BeO-exposed HLA-DP2 Tg mice as
described previously (9). Cells were stained with mAbs directed against CD4 (AF700), CD3 (PECy7), CD8 (eFluor 450), B220 (eFluor 450), F4/80 (eFluor 450), CD44 (PerCP-Cy5.5), and PElabeled HLA-DP2-PLXNA4/Be tetramer (4). Tetramer-staining CD3+CD4+CD44+ T cells were
single cell sorted into reverse transcription buffer in a 96-well plate using a FACSAria flow
cytometer. TCRAV and TCRBV gene expression was determined using 5’ RACE and a nested
PCR method as previously described (3, 10). In some experiments, tetramer-staining T cells were
sorted directly into complete IMDM media in a 96 well round-bottomed plate and stimulated with
anti-CD3 and anti-CD28 beads (Invitrogen) and recombinant human IL-2 (30 U/mL) for two
weeks. Stimulated cells were transferred into a 24 well plate for further stimulation and expansion.
The TCRAV and BV gene expression in the expanded cells was determined as described above.
T cell transfectomas expressing HLA-DP2-PLXNA4/Be binding TCRs were generated as
described previously (2, 4). Cells expressing the highest levels of TCR as detected by staining with
anti-mouse TCR β-PE (H57-597) mAb (BD Biosciences) were purified by cell sorting and
screened for Be specificity using an IL-2 ELISA.
In all experiments, supernatants were harvested after 22-24 hrs, and murine IL-2 was
measured by ELISA (eBioscience). For dose response calculations, 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, GraphPad Software, Inc.).
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Supplemental Figure Legends
Figure S1. TCRA gene conferring Be-specificity for T cell hybridomas LB9-18 and LB10-17.
Three Be-specific T cell hybridomas derived from the lungs of BeO-exposed HLA-DP2 Tg mice
expressed multiple TCR α-chains paired with a TCR Vβ6-chain (LB9-18/LB10-9 are clonal
replicates and LB10-17; see Figure 1B). To ascertain which TCR α/β pair conferred Be-specificity
for each hybridoma, the TCRA genes from each hybridoma were introduced separately into
transfectomas expressing the appropriate TCRB gene. TCR surface expression on the resultant
transfectomas was confirmed by staining with an anti-mouse TCR β-chain mAb (H57-597) (data
not shown). Transfectomas were tested for their ability to secrete IL-2 (mean ± SEM pg/ml) in
response to BeSO4 (200 µM) presented by HLA-DP2 transfected fibroblasts. Shown are the correct
TCR α/β pairs for each hybridoma (Vβ6/Vα4.2 for LB9-18; Vβ6/Vα1.1 for LB10-17) as indicated
by IL-2 production.
Figure S2. T cell transfectoma response to mimotope 6 presented by transfected DAP3.L cell
lines expressing wild-type or variant HLA-DP2 genes. LB9-18 and LBM10-10 cells (1 x 105 cells
per well) were mixed in complete IMDM with an equal number of DAP3.L fibroblast cells
transfected with either wild-type (8302), or variant HLA-DP2 molecules containing single-site
mutations at critical Be-coordinating residues (amino acid positions 26, 68 and 69) of the HLADP2 β-chain. Cells were stimulated with either Be (200 µM) or mimotope 6 (5 µM) for 22 hours
and IL-2 secretion (mean ± SEM pg/ml) measured by ELISA. Data shown are representative of
three independent experiments done in triplicate.
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