Soluble anti cd28

Naïve CD4⁺CD25^loCD62^hiCD44^lo cells were isolated by using EasySep Mouse Naïve CD4⁺ T Cell Isolation Kit (Stemcell Technologies) from spleens and LN of Maf^f/f and Maf^ΔTcells mice, and activated with plate-bound anti-CD3 (Biolegend, 5 μg/ml) and soluble anti-CD28 (Biolegend, 1 μg/ml) supplemented with mIL12 (10 ng/ml) and neutralizing antibody anti-IL-4 (clone 11B11, 10 μg/ml) (T_H1), mIL-4 (10 ng/ml) and anti-IFN-γ (clone: XMG-121,10 μg/ml) (T_H2), hIL-2 (50U/ml) and TGF-β (10 ng/ml) (T_reg), TGF-β (5 ng/ml) and IL-6 (40 ng/ml) (FOXP3⁺ and FOXP3⁻ T_H17). Cells were incubated 5 days at 37 °C, 5% CO₂.

Naïve CD4⁺CD62L⁺ T cells were isolated from the spleens of naïve mice by magnetic cell sorting using the CD4⁺CD62L⁺ T Cell Isolation Kit II (Miltenyi Biotec). The purity of the cell isolation was confirmed by FACS analysis.
For T cell differentiation, CD4⁺CD62L⁺ T cell were cultured in RPMI medium supplemented with 10% FCS, 1% Pen/Strep and 1% L-Glu, at 37 °C and 5 % CO₂ in the presence of plate-bound anti-CD3 (5 µg/ml, BD Biosciences) and soluble anti-CD28 (1 µg/ml, Biolegend) antibodies.
For Th1 differentiation, cells were additionally treated with 5 µg/ml anti-IL-4 antibody (BioLegend) and 12 ng/ml recombinant IL-12 (PeproTech).
Afterwards ACK lysis buffer was applied as previously described (27 (link)).

For the proliferation assay, 1 × 10⁶ purified CD4+CD25– T cells were labeled with the carboxyfluorescein succinimidyl ester (CFSE) (1 μM; Invitrogen, Carlsbad, CA, USA) in 1 mL pre-warmed phosphate-buffered saline, followed by incubation for 10 min at room temperature and neutralization with pre-chilled complete medium containing 10% fetal bovine serum. CFSE-labeled cells were re-suspended and cultured in complete medium in the presence of plate-bound anti-CD3 (coating concentration was 2.5 μg/mL, Biolegend, San Diego, CA, USA) and soluble anti-CD28 (2.5 μg/mL, Biolegend, San Diego, CA, USA) in 24-well flat-bottom plates for 72 h. After culture, the cells were collected for anti-TIGIT APC and anti-CD226 PerCP-CY5.5 staining and then analyzed to determine the CFSE intensities. Each experiment was performed and analyzed using a FACS JAZZ instrument.

CD8⁺ T cells were isolated from naive spleens using a negative magnetic bead selection kit (Biolegend). MDSCs were isolated from the spleens of mice bearing 4T1 tumours that had either been treated with vehicle or PI-3065 using a negative magnetic bead selection kit (Stemcell). CD8⁺ T cells were labelled with Tag-It-Violet (Biolegend) prior to stimulation with plate-bound anti-CD3 10 μg/ml (Biolegend, 145–2C11), 1 μg/ml soluble anti-CD28 (Biolegend, 37.51) and 30 IU/ml IL-2. MDSCs were added to the CD8⁺ T cells at a ratio of either 1:1 (MDSCs:CD8) or 4:1. Cocultures were left for 3 days prior to flow cytometric analysis.

Cryopreserved PBMCs were rapidly thawed, washed twice in pre-warmed RPMI-1640 medium, and rested overnight in complete RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES and 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA). The next day, rested PBMCS were resuspended in fresh media and 1x10⁶ cells/well was seeded into a 96 well plate pre-coated with anti-CD3 (2 µg/mL; BD Biosciences, Catalog No. 550368). Soluble anti-CD28 (1 µg/mL; Biolegend, Catalog No. 302934) and anti-CD49d (1 µg/mL; Biolegend, Catalog No. 304340) was added for co-stimulation and the cells stimulated for 2 days at 37⁰ C in 5% CO₂. In select experiments, cells were stimulated with 50 ng/mL of Phorbol-12-myristate 13-acetate (PMA) (Sigma Aldrich, Catalog No. P1585) and 1 µM of Ionomycin (Sigma Aldrich, Catalog No. 19657), and 0.07% Golgi-plug (BD Biosciences, Catalog No. 555029) for 6 hours in fresh complete RPMI media.

For antigen-specific recall assays, 9 d after immunization of the mice, 2.5 × 10⁵ draining inguinal lymph node cells were prepared and cultured in 96-well round-bottom plates for 72 h with the indicated concentrations of MOG_35–55 peptide, a vehicle control, or plate-coated anti-CD3ε (1 µg/ml; BioLegend; catalog no. 100340) together with soluble anti-CD28 (1 µg/ml; BioLegend; catalog no. 102116) as a positive control. During the last 16 h of culture, cells were pulsed with 1 µg/ml BrdU (catalog no. 423401). Single-cell suspensions were stained for surface antigens in the presence of TruStain Fc receptor block (BioLegend), and Fixable Viability Stain 780 (BD Biosciences; catalog no. 565388) was used to discriminate dead cells. Cells were fixed (fixation buffer; BioLegend; catalog no. 420801) and permeabilized using 0.5% Triton X-100, followed by incubation with 40 KU/ml DNase I (Merck; catalog no. 260913-10MU) in PBS with Ca²⁺ and Mg²⁺ for 1 h at 37°C. After DNA digestion, incorporated BrdU was detected by incubation with an anti-BrdU AF647-coupled antibody. The antibodies and the respective antigen, host species, supplier, catalog number, clone, and dilution are listed in Table S8. Data were acquired on an LSR II FACS analyzer (BD Biosciences). Representative gating strategies will be provided upon request.

CD8⁺ T cells were isolated from spleen and lymph nodes from A20^fl/fl and CD4-Cre A20^fl/fl mice with Mouse CD8⁺ T cell Isolation Kit (Stemcell Technologies) according to manufacturer’s instruction. Cells were stimulated in RPMI 1640 medium supplemented with L-glutamine, 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 1% non-essential amino acids, 50 μM β-mercaptoethanol (all from Life Technologies) and 5 mM HEPES (Biochrom) in the presence of 1 μg/ml plate-bound anti-CD3 and 2 μg/ml soluble anti-CD28 (both from BioLegend) for 3 days.
Proliferation was analyzed by staining cells with carboxyfluorescein succinimidyl ester (CFSE, BioLegend) before stimulation.
For active caspase-3/7 measurement, CD8⁺ T cells were stimulated for the indicated time points with the addition of 20 ng/ml TNF or recombinant CD95L (50 ng/ml).