Generation of IL-10Rα-Deficient Murine P14 CD8+ T Cells

Wild type (WT) P14 CD8⁺ T cells were isolated from splenocytes of Cas9 knock in P14 TCR transgenic mice using CD8 negative selection kit (Miltenyi Biotec) and activated using mouse T-activator CD3/CD28 Dynabeads® (Gibco / Thermo Fisher Scientific) for 24 h followed by 4-day expansion in complete RPMI medium containing IL-2 (10 ng/mL) prior to use. To generate control and IL-10Rα-KO P14 CD8⁺ T cells, WT P14 CD8⁺ T cells were isolated and activated for 24 h similarly, followed by transduction with retroviruses containing scramble control guide RNA (gRNA) or IL-10Rα-targeting gRNA on plates coated with RetroNectin (5 μg/cm², Clonetech / Takara). Transduced CD8⁺ T cells were then expanded similarly for additional 4 days prior to use. The pool of gRNAs targeting IL-10Rα (IL-10Rα1: 5’-TCTGGCTTCAAACCACACAT-3’, IL-10Rα2: 5’-GCAGTGTTTACTTATCACGA-3’, IL-10Rα3: 5’-GTGGGGACAACACGGACAGT-3’) and a scramble control gRNA control (5’-GCGAGGTATTCGGCTCCGCG-3’) were designed using the publicly available online gRNA design tool CRISPick^{54 (link)}.