once to remove excess peptides. MAGE-A3–
specific TCR mutants-transduced SKW3 cells
were resuspended at 5 × 10^5 cells/mL and 200mL
T cells were added to each well with peptide-
pulsed 293T-A1 cells. The stimulation was
performed at 37°C, 5% CO 2 for 14 hours. After
the stimulation, the cells were stained with
anti-CD69-APC and anti-Vb5.1-BV421 (clone
LC4, ThermoFisher Scientific) on ice for 30 min
and analyzed by CytoFLEX flow cytometer
(Beckman).
Transduction of human primary T cells with TCR
Human whole blood from healthy anonymous
volunteer donors was purchased from Stan-
ford Blook Bank under the approved protocol
of APB-2749-KG1018. 6-well plate was coated
with 2 mL of 2.5mg/mL anti-CD3 (OKT3 clone)
overnight. The next day, human peripheral
blood mononuclear cells (PBMCs) were added
to the plate with 5mg/mL anti-CD28 and cul-
tured at 37°C, 5% CO 2 for 3 days. 4 million
LentiX cells were seed in 10-cm dish and
transfected with lentiviral vector of MAGE-
A3–specific TCRachain orbchain. The len-
tivirus was made as described above. In total,
40 mL of TCR virus were concentrated to 500mL
using 100-kDa cutoff filter. 5 million preactivated
human PBMCs were resuspended in 500mL
media and mixed with 500mL concentrated
TCR virus and 5mg/mL Polybrene and 100 U/mL
human IL-2. The virus-cells mixture was pro-
cessed with spin infection under 2800 rpm,
32°C for 2 hours.
Killing assay of tumor cells
Twenty thousand A375 or HCT-116 cells were
seed in each well of 96-well plate. 60,000 MAGE-
A3–specific TCR-transduced human primary
cells were added to each well with tumor cells
and cocultured for 24 hours. The plate was
washed in EDTA-free buffer and stained with
7-AAD (ThermoFisher Scientific) and Annexin
V-APC (BioLegend) for 10 min. The plate was
analyzed by CytoFLEX.
Cytotoxicity, cytokine, and granule
release assays
Two hundred thousand tumor cells or peptide-
pulsed 293T-A1 cells were seeded in each
well of 96-well plate overnight. The next day,
200,000 MAGE-A3–specific TCR-transduced
human primary cells were mixed with 1:100
anti-CD107a-PE (clone H4A3, BioLegend) and
1:1000 brefeldin A, and then added to each
well. Coculture was done for 6 hours at 37°C,
5% CO 2. After 6 hours, the plate was stained
with anti-CD8-BV421 (clone RPA-T8, BD Bio-
sciences), anti-Vb5.1-APC. Then the plate
was fixed with IC fixation and permeabilized
by permeabilization buffer. The plate was
further stained with anti–IFN-g–BV605
(clone B27, BioLegend) and anti-TNF-PE-
Cy7 clone MAb11, BioLegend) on ice for 30 min.
The plate was then washed and analyzed by
CytoFLEX.Production of MHC andb-2-microglobulin
inclusion body
The protein of B35 MHC heavy chain and
humanb-2-microglobulin were made inE. coli
as inclusion body. Specifically, B35 MHC heavy
chain or humanb-2-microglobulin was cloned
into pET28a vector and transformed into BL21
(DE3)E. colistrain. Single colony was picked
and resuspended in 10 mL LB media con-
taining 50mg/mL kanamycin and shake at
250 rpm, 37°C for 12 to 16 hours. Then the 10 mL
culture was added into 1 L LB media containing
50 mg/mL kanamycin and shake at 250 rpm, 37°C
for ~3 hours until the optical density (OD) =
0.5 to 0.6. Isopropyl-b-D-thiogalactopyrano-
side (IPTG) was added into the culture at final
concentration of 1 mM and continued to shake
for another 3 hours. The bacteria culture was
spin down at 6000 rpm for 20 min. The bac-
teria pellet was resuspended in 50 mL buf-
fer1[50mMTris-HCl,pH8.0,100mMNaCl,
1 mM dithiothreitol (DTT), 5% Triton X-100,
1 mM EDTA, 0.2 mM phenylmethylsulfonyl
fluoride (PMSF)]. Then the bacteria were
sonicated under the program of 2 min soni-
cation plus 2 min of rest. The sonication program
was repeated four times continuously. After that,
bacteria were spin 7500 rpm for 15 min. It was
repeated two more times to resuspend the
bacteria pellet in buffer 1 and do the sonica-
tion. The bacteria pellet was then resuspended
in50mLbuffer2(50mMTris-HCl,pH8.0,
100 mM NaCl, 1 mM EDTA). Then the bacteria
were sonicated under the program of 2 min
sonication plus 2 min rest. The sonication
program was repeated four times continuously.
After that, bacteria were spin 7500 rpm for
15 min. It was repeated one more time to
resuspend the bacteria pellet in buffer 2 and
do the sonication. The inclusion body was
pelleted and solubilized in 25 mL buffer (8 M
urea, 50 mM Tris-HCl pH 8.0, 10 mM EDTA,
10 mM DTT).Refolding of pMHC
Refolding buffer was prepared as 100 mM
Tris-HCl pH 8, 400 mM arginine, 5 M urea,
0.5 mM oxidized glutathione, 5 mM reduced
glutathione, 2 mM EDTA. 30 mg peptide was
dissolved in DMSO and added to each liter of
refolding buffer. For each liter of refolding
buffer, 30 mg MHC heavy chain inclusion
body and 30 mg humanb-2-microglobulin
inclusion body were mixed in a syringe and
added into each liter of refolding buffer drop
by drop. Then, the refold buffer/protein were
poured into dialysis tubing (Spectrum Labs)
and dialyzed into 10 L 10 mM Tris pH 8.0. The
10 L 10 mM Tris pH 8.0 buffer was changed
every 12 hours and repeated four times in total.
Then, the protein was purified by using weakanion exchange resin (DEAE Cellulose, Santa
Cruz Biotechnologies). Specifically, DEAE-
Cellulose was equilibrated with 10 mM Tris-
HCl, pH 8.0 in a column. Then the dialyzed
refolded protein solution flowed through the
cellulose column drop by drop and repeated
the flowing one more time. The refolded
proteinwaselutedin30mL10mMTris-HCl,
pH 8.0 plus 0.5 M NaCl. The protein was buffer
exchanged into 10 mM Tris-HCl, pH 8.0 and
concentrated to 500mL and biotinylated over-
night. Biotinylated refolded protein was
analyzed by size exclusion chromatography
(Superdex 200, GE Healthcare) and ion ex-
change (MonoQ, GE Healthcare) on AKTAPurifier
(GE Healthcare).pMHC tetramer
For staining each 10 million cells, 20mg
biotinylated pMHC protein and 30mg
streptavidin-PE (Thermo Fisher Scientific)
were aliquoted. 20% of total amount of
streptavidin-PE were added into biotinylated
pMHC each time at an interval time of 1 hour
and repeated five times. During the interval
time, the tetramer was incubated on ice. The
pMHC tetramer was stored at 4°C overnight
before using.Production of TCR protein by Expi293
The TCR protein used for SPR was produced
in Expi293 cells (Thermo Fisher Scientific).
Specifically, TCRachain was cloned into pD649
vector with basic zipper, and TCRbchain was
cloned into pD649 vector with acid zipper. 15mg
TCRachain constructs and 15mg TCRbchain
constructs were transfected into 75 million
Expi293 cells according to the manufacturer’s
protocol. Four days after the transfection, the
cell culture was spin down at 400 g for 5 min
and the supernatant was saved. A onefold
volume of PBS was added to the supernatant
and final concentration of 20 mM Tris-HCl
pH 8.0 buffer was added. 2 mL nickel-NTA was
added to the supernatant and the solution was
rotated overnight at 4°C. Then, the solution
was flowed through a column to collect the
Ni-NTA and bounded protein. 1× HBS pH 7.2
containing 10 mM imidazole was used to wash
the Ni-NTA and protein once, and the protein
was eluted by 1× HBS pH 7.2 containing 300 mM
imidazole. The protein was concentrated in a
30-kDa filter (Millipore) and buffer exchanged in
1× HBS pH 7.2. The protein was purified by size-
exclusion chromatography using Superdex200
column on AKTAPurifier (GE Healthcare). The
purified protein was collected from the accord-
ing fraction based on the size and run on SDS–
polyacrylamide gel electrophoresis (SDS-PAGE)
to check the size and 1:1 stoichiometry.Production of TCR protein by insect cells
The TCRachain was cloned into pAcGP67a
vector with basic zipper, and the TCRbchainZhaoet al.,Science 376 , eabl5282 (2022) 8 April 2022 11 of 14
RESEARCH | RESEARCH ARTICLE