title: GSM6423875: COVID_AIM_AIM_CD8_040_TCR; Homo sapiens; OTHER
library_construction_protocol: PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C. AIM assay was performed on PBMC samples collected at P2, P3, and P8. PBMC were thawed by warming frozen cryovials in a 37°C water bath and resuspended in 10mL of in Culture Medium (CM) [10% fetal bovine serum (MP Bio Japan), 1% HEPES (Nacarai) and 1% penicillin/streptomycin (Nacarai) in RPMI-1640 medium (Nacarai)]. Cells were resuspended in 1mL of CM, then plated in 24-well plates and incubated overnight (about 16 hours) in an incubator at 37°C, with 5% CO2. After incubation, cells were then stimulated for 24 hours in stimulation medium [CM with BD FastImmune CD28/CD49d Costimulatory Reagent (final x100, BD Biosciences) and anti-human CD40 (final concentration of 0.5?g/mL, clone HB14, BioLegend)] with or without PepTivator SARS-CoV-2 Prot_S Complete (final concentration of 1 mg/mL, Miltenyi). 20 hours post-stimulation, an anti-CD107a-FITC antibody (final concentration of 5?g/mL, clone H4A3, BioLegend) was added to the culture. After four hours, cells were washed in PBS supplemented with 10% FBS (10% FBS/PBS). Cells were stained for 20 minutes at 4°C with antibody mix 2 (Supplementary Table S1). Cells were washed once in 10% FBS/PBS. AIM+ non-naïve CD4+, AIM- non-naïve CD4+, AIM+ non-naïve CD8+, and AIM- non-naive CD8+ cells were sorted using FACS Aria II or Aria III (BD Biosciences) and FACSDiva software (BD Biosciences, v8.0.1). AIM+ CD4+ T cells were defined by dual-expression of CD69 and CD40L. AIM+ CD8+ T cells were defined by expression of either CD107a or CD137. Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining (PI, Sigma-Aldrich). Data were analyzed using FlowJo software. TCRseq libraries of AIM+ and AIM- non-naive T cells  were prepared for sample collected at P3. 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 ?L 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 S2 and S3, respectively.