GSM7998094
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C. For preparing Peptides-HLA class I tetramers for SARS-CoV-2 Spike epitopes, S448 and S1208 peptides were purchased from GenScript. S269 and S919 peptides were purchased from GL Biochem Ltd. (Shanghai, China). Tetramers for S448, S1208, and S269 were generated by QuickSwitchTM Quant HLA-A*24:02 Tetramer Kit-BV421, HLA-A*24:02 Tetramer Kit -PE, and HLA-A*02:01 Tetramer Kit-PE (MBL), respectively, according to the manufacturer's protocol. Tetramer for S919 was generated using Flex-T™ HLA-B*15:01 Monomer UVX (BioLegend) with D-Biotin (Nacalai) and BV421 Streptavidin (BD) according to the manufacturer's protocol. The rate of peptide exchange was reported to be more than 95% in our previous report32. Thawed PBMCs were treated with a protein kinase inhibitor, Dasatinib (50 nM, AdooQ), for 30 min at 37 °C, and Human TruStain FcX for 5 minutes at room temperature. Then, PBMCs were stained with tetramers for 30 minutes at room temperature. After tetramer staining, cells were stained for antibody mix 3 (anti-CD4 (PerCP/Cyanine5.5), anti-CD14 (PE/Dazzle™ 594), anti-CD19 (PE/Dazzle™ 594), anti-CD45RA (APC/Cyanine7), anti-CD27 (APC), anti-CD8 (FITC, clone HIT8a)) for 20 minutes at room temperature. After washing twice with FACS buffer, CD8+ tetramer+ cells were sorted using FACS Aria II or Aria III and FACSDiva software (v8.0.1). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. Data were analyzed using FlowJo software (v10.8.1). TCR-seq libraries of tetramer+ T cells were prepared on sample with (i) more than 100 tetramer+ cells and (ii) the proportion of tetramer+ cells was more than 0.1% in CD8+ T cells. S1208-tetramer+ sample with high background (ID22) was excluded from the analysis. Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2023).  In brief, mRNA in T-cell lysates were captured by incubating with 10 μL of Dynabeads M270 streptavidin (Thermo Fisher Scientific) bound to 2.5 pmol of BioEcoP-dT25-adapter primers for 30 minutes at room temperature under gentle rotation. mRNA-trapped beads were suspended in 10 µL of RT mix and incubated for 60 min at 42ºC to perform reverse transcription (RT) and template-switching. The composition of the RT mix was as follows; 1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed using KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), followed by the purification of PCR product using an AM Pure XP kit (Beckman Coulter) at a 0.7:1 beads-to-sample ratio. The third PCR was performed to amplify TCR libraries and add adaptor sequences for the next-generation sequencer. The third-PCR products were purified as second PCR. Final TCR libraries, whose lengths were about 600 base pairs, were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). TCR libraries were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina) using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles, Illumina). Only read2 contained the sequence regarding the definition of T-cell clones. Oligonucleotides and reaction conditions for library preparation were summarized in Table S1 and S2, respectively. Bulk TCR-seq