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Hiasa A. et al., Page 1
Supplementary Figure Legends
Supplementary Figure 1. Identification of MAGE-A4143-151-specific TCR and 
chain cDNA.
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Total RNA was extracted from MAGE-A4143-151-specific CTL clone2-2812 by RNeasy
Mini Kit (Qiagen). Single strand cDNA was synthesized with oligo dT primer by RTase
M-MLV (Takara Bio, Shiga, Japan). TCR and  cDNA were synthesized by a RACE
method. 5’ RACE was performed using 5’ and 3’ RACE primer of gene-specific primer
TCRC
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-specific
reverse
TCRC1-specific
primer
reverse
(5’-TCAGCTGGACCACAGCCGCAGCGT-3’),
prime
r(5’-TCAGAAATCCTTTCTCTTGAC-3’),
TCRC2-specific reverse primer (5’-CTAGCCTCTGGAATCCTTTCTCTT-3’) by Cap
Fishing Full-length cDNA premix Kit (SeeGene, Seoul, South Korea). PCR products
were cloned into pT7blue T-vector (Novagen, San Diego, CA). 96 plasmids were
sequenced to choose a correct TCRand  cDNA from MAGE-A4143-151-specific CTL
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clone2-28.
Supplementary Figure 2. The procedure of retroviral TCR transduction.
CH-296 (RetroNectin, Takara Bio)-coated 6-well plates (Nunc, Roskilde, Denmark)
with 3x106 pfu viral supernatant were centrifuged at 32°C. After 2 hour incubation at
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37°C, viral supernatant was removed and 1x106 PBMCs in GT-T503 medium (Takara
Bio) with 10% AB plasma containing 10 ng/ml anti-CD3 antibody (Janssen Pharma,
Basel,
Switzerland)
and
40
IU/ml
human
IL-2
(kindly
provided
Takeda
Pharmaceutical, Osaka, Japan) were applied into the wells and infected twice on day
2 and 3.
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Supplementary Figure 3. MAGE-A4143-151-specific TCR is dependent on CD8
Hiasa A. et al., Page 2
(A). Diagram of retroviral vectors containing CD8  and  genes. RNA was isolated
from PBMCs derived from a healthy donor, and CD8  and  genes were amplified
by RT-PCR. Amplified CD8  and  genes were cloned into retroviral vector
pDON-AI-2 (Takara Bio, Ohtsu, Japan) together with murine PGK promoter to
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construct pDON-AI-2-CD8 
in which M-MLV LTR drives  gene and PGK
promoter drives  gene. (B). PBMCs from healthy donors with no history of
autoimmune disease collected as a part of study approved by the Ethics Committee
of Mie University Graduate School of Medicinewere transduced with retroviral vector.
TCR expression determined by MAGE-A4143-151/HLA-A*2402 tetramers in TCR
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gene-modified PBMCs was analyzed with flow cytometry.
Supplementary Figure 4. Sequence of TCR from CD8+ T cell clones generated
from retrovirally TCR gene-modified PBMCs.
TCR from CD8+ T cell clones generated from retrovirally TCR gene-modified
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PBMCs was sequenced from the R region of 5’LTR to the R region of 3’LTR and
compared to the original sequence used in retroviral vector MS-bPa.