Soluble αcd28 37.51

Thy1.1⁺-enriched cells after lentiviral gRNA vector transduction were activated in vitro with 3 µg/ml plate-bound α-CD3ε (145-2C11, Biolegend) and 1 µg/ml of soluble α-CD28 (37.51, Biolegend). Cells were collected after 3–7 d after activation for preparation of whole cell lysates in radioimmunoassay precipitation buffer for subsequent immunoblot analysis to assess the efficiency of gene inactivation. Total cell lysates equivalent to 2–5 × 10⁵ cells were loaded into each lane for SDS-PAGE and subsequent protein transfer to a polyvinylidene difluoride membrane. Antibodies for Western blotting against HDAC3 (2632, 85057), Blimp-1 (9115), Runx3 (9647), and GAPDH (2118) were purchased from Cell Signaling Technology. Membranes were incubated with primary antibodies overnight at 4°C with gentle shaking at a dilution of 1:1,000 in SuperBlock T20 (TBS) (Thermo Fisher Scientific), except for α-GAPDH antibodies, which were diluted 1:5,000. Membranes were then incubated with secondary goat α-rabbit HRP-conjugated antibodies (1:10,000; Cell Signaling Technology) for 1 h at room temperature and then treated with enhanced chemiluminescence reagent (Thermo Fisher Scientific). Protein bands were visualized on autoradiography film (Denville Scientific), and protein KO levels were quantified from scanned film images using FIJI software (National Institutes of Health).

Naïve CD4⁺ T cells were isolated by negative selection with mouse Naïve CD4⁺ T cell isolation kit (Miltenyi). 1 × 10⁵ cells were cultured for 4 days in 96-well plates precoated with 1 μg/ml of α-CD3ε (145-2C11; Biolegend) and 1 μg/ml of soluble α-CD28 (37.51; Biolegend) in RPMI 1640 media supplemented with 10% FBS, 1% penicillin/streptomycin, 1% pyruvate (11360070; Thermo Fisher Scientific), 1% MEM/NEAA (11140050; Thermo Fisher Scientific), 2.5% HEPES (15630080; Thermo Fisher Scientific), and 55 μM β-mercaptoethanol (21985023; Thermo Fisher Scientific) under CD4⁺CD8αα⁺ T cell conditions: RA (10 nM, 0064-1G, Tokyo Chemical Industry), TGFβ (2 ng/ml, 7666-MB-005, R&D systems), IFNγ (20 ng/ml, 315-05, Peprotech). For the treatment with inhibitors, Rapamycin (1 μM, Sigma-Aldrich), Torin-1 (1 μM, Selleckchem) and 2DG (1 mM, Sigma-Aldrich) were added into the cell culture. In some experiments, cells were placed inside a sealed air tight container which contains an anaeropack (Mitsubishi Gas Company, Tokyo, Japan). The anaeropack contains a gas-controlling reagent which absorbs oxygen and generates carbon dioxide resulting in a hypoxic atmosphere (8% O₂).