GSM6692869: B16_CD8_dLN_CD4PDL1_9; Mus musculus; OTHER
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice. dLN cells were prepared by standard method. Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter). Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi). CD8+, CD8+ PD1-, CD8+ PD1+ Ly108+ Tim3-, CD8+ PD1+ Ly108+ Tim3+, and CD8+ PD1+ Ly108- Tim3+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%. Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2021). In brief, to amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed as follows. Beads containing cDNA was resuspended with the 12.5 mL of first PCR mixture comprised 0.5 mL of 10 mM trP1 primer, and 1 mL of TCR primer mix (Trac_ex, and Trbc_ex), 4.75 mL of water, and 6.25 mL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems). The thermal cycling conditions were programmed as follows: denaturation at 95°C for 3 minutes, 5 cycles of denaturation for 20 seconds at 98°C, annealing for 15 seconds at 65°C and extension for 30 seconds at 72°C, followed by a final extension at 72°C for 2 minutes. Next, 2.5 mL of the first-PCR products were added into the 22.5 mL of second PCR mixture consisted of 1.75 mL of 10 mM primer mix (trP1, Trac_in-Bio, and Trbc_in-Bio), 8.25 mL of water and 12.5 mL of KAPA Hifi Hotstart ReadyMix. The thermal cycling conditions were the same as the first PCR except for the cycle number, which was 25 cycles. Next, 25 mL of the second-PCR products were purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio (beads to sample) and eluted in 20 mL of 10mM of Tris HCl pH 8.0. Fragmentasion and adaptor ligation were performed using 10-20ng of second-PCR product as a template with NEBNext FS DNA Library Prep Kit (New England Biolabs) with some modifications: NEBNext Adaptor for Illumina in “Adaptor Ligation” step was substituted by 5mM Adaptor P1, and the reaction volume was 1/4 of the recommended in all steps. Adaptor-ligated DNA was purified using Agencort AM Pure XP kit (Beckman Coulter) at a 0.8:1 ratio (beads to sample) and eluted in 20 mL of 10mM of Tris HCl pH 8.0. The third PCR was carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The third PCR mixture consisted of 1 mL of 10 mM trP1 primer, 1 mL of 10 mM IonA-BC-Trbc primer, 3 mL of template and 5 mL of NEBNext Ultra II Q5 Master Mix (accessory of NEBNext FS DNA Library Prep Kit). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 seconds, 10 cycles of denaturation for 10 seconds at 98°C, annealing and extension for 1minute 15 seconds at 65°C, followed by a final extension at 65°C for 5 minutes. The PCR product was purified and subjected to size selection using Agencort AM Pure XP kit (Beckman Coulter) at a 1.0:1 ratio (beads to sample) and eluted with 20 mL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu, Kyoto, Japan). Primers used for library preparations were summarized in Supplementary Table 1.  Final TCR libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion 540 Kit Chef, an Ion 540 Chip kit, and an Ion Genestudio S5 Sequencer (Thermo Fisher Scientific) according to the manufacturer's instructions, except the flow number (500).