Tcf7ΔGzmb CD8+ Tcm Cell Transcriptional Analysis

Wild-type or Tcf7^ΔGzmb CD62L⁺CD8⁺ T_cm cells were sort-purified from the primary recipients at 30–35 dpi and seeded onto flat bottom 96-well plate at 1×10⁵ cells/well and incubated with GP33 peptide (200 nM) for 24 hrs, with dead cells removed using a Dead Cell Removal Kit (Miltenyi Biotec). The cells were used as GP33-stimulated CD8⁺ T_cm cells for RNA-seq and ATAC-seq analyses. For detection of IFN-γ production, Brefeldin A was added during the last 5 hrs of incubation. For mapping pathways leading to Tcf1 downregulation, Wild-type CD8⁺ T_cm cells were enriched with EasySep PE Positive Selection Kit (StemCell Technologies) after staining with CD62L-PE, and the cells were pre-incubated with pharmacological inhibitors or DMSO carrier for 30 min followed by 24-hr coculture with GP33. Benzoxathiole (Abcam, Cat. No. ab145954), Cyclosporin A, LY294002, and Selumetinib (Millipore-Sigma, Cat. No. 30024, 440202, and ADV465749271, respectively) were used at the final concentrations of 10 μM, 140 nM, 14 μM and 70 nM, which were equivalent to 10× their respective reported IC50 (Ref.^{29 (link), 30 (link)}).