Recombinant mouse il 2

Anti-mouse CD3e (#553057) and anti-mouse CD28 (#553295), recombinant mouse IL-2 (#575404) and IL-12 (#577004), and FITC-conjugated anti-mouse CD4 (#100406), PerCP-conjugated anti-mouse CD8a (#100732), PE-conjugated anti-mouse/human CD44 (#103008), APC-conjugated anti-mouse CD62L (#104412), PE/Cyanine7-conjugated anti-mouse IFN-γ (#505826), APC-conjugated anti-mouse IL-4 (#504106), APC-conjugated anti-mouse IL-17A (#506916), PE-conjugated anti-mouse IL-17A (#506904), and Alexa Fluor 647-conjugated anti-mouse Foxp3 (#126408) antibodies were purchased from the BD Biosciences and BioLegend (both San Diego, CA, USA). Anti-insulin (#4590s), anti-STAT1 (#14994S), and anti-Phospho-STAT1 (Tyr701) (#9167S) antibodies were obtained from the Cell Signaling Technology (Danvers, MA, USA). Anti-MBD2 (#ab188474) antibody was got from Abcam (Cambridge, MA, USA). The anti-Lamin B1 (#12987-1-AP) antibody was obtained from Proteintech (Wuhan, China).

Naïve OT-I CD8⁺ T cells were sorted and labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular probes) at 0.4 μM in pre-warmed PBS (0.1% FCS) for 10 minutes at 37°C, then washed twice with RP10 advanced media (RPMI advanced media, 10% FCS, Hepes, Pen-Strep, L-glutamine, BME, gentamicin). Cells were stimulated in vitro for two days with 100pM OVA₂₅₇ peptide-pulsed, CD45.1 splenocytes that were irradiated at 3000 rads.
Alternatively, 2 x 10⁶ FACS-sorted, naïve OT-I CD8⁺ T cells were stimulated with 4.5 x 10⁶ 200 nM OVA₂₅₇ peptide-pulsed, CD45.1 splenocytes at 37°C for two days in RP10 advanced media. The cells were harvested, washed, and replated with naïve splenocytes in the presence of 5ng/ml TGF-β (R&D Systems) with or without 20 ng/ml recombinant mouse IL-2 (BD Biosciences), IL-7 (Invitrogen Life Technologies), or IL-15 (R&D Systems). Two days later, fresh media and cytokines were added, and two days later, cells were harvested, stained and analyzed by flow cytometry.

Naïve CD8⁺ T cells were isolated from splenocytes using a FlowComp Dynabeads Mouse CD8⁺ positive isolation kit (ThermoFisher Scientific, Karlsruhe, Germany) as described previously [13 (link)]. The cells were cultured at a concentration of 1 × 10⁶ cells per mL in AIMV Medium (ThermoFisher Scientific) supplemented with 10% FCS, 0.5% Pen/Strep and 40 µM BME (Carl Roth, Karlsruhe, Germany). Then, 2 × 10⁶ cells were seeded per well of a 24-well plate and the cells were activated with anti-CD3/anti-CD28 mouse activator beads at a ratio of 1:0.8. Cells were incubated at 37 °C with 5% CO₂ for 7 or 8 days to generate effector CD8⁺ T cells or CTLs. Cells were counted every day after day 2 of activation and split to 1 million/mL as the cells doubled or tripled. Furthermore, 10 U/mL of recombinant mouse IL2 (BD Biosciences, San Jose, CA, USA) was added as supplement to each well during the splitting. Cells activated for 7 or 8 days as described above were used for all experiments.