Anti cd28 antibody

Cells from lymph nodes or spleens were cultured in IMDM medium supplemented with heat-inactivated 10% FBS, 2 mM L-glutamine, 100 U/mL Penicillin, 100 mg/mL Streptomycin and 50 μM 2-mercaptoethanol (all Sigma-Aldrich). For in vitro cell proliferation assays cells were labeled with 500 nM CellTracer Violet (Life Technologies). N4, T4, and G4 peptides (Peptide Synthetics) were added to culture medium at indicated concentrations. Recombinant IL-7 (Peprotech) was used at 20 ng/mL, recombinant IL-10 (Peprotech) was used at indicated concentrations. For cytokine recall responses cells were restimulated with 20 ng/mL phorbol 12,13-dibutyrate (PdBU) and 0.5 μg/mL Ionomycin (all Sigma-Aldrich) in presence of 1 μg/ml Brefeldin A for 4 h before intracellular staining (antibodies identified above). Transgenic OT-1 T cells were restimulated with T4 peptide (SIITFEKL) in presence of 1 μg/ml brefeldin A for 4 h. Where stated cells were stimulated in media with 0.5 μg/mL anti CD3ε antibody and 2 μg/mL anti CD28 antibody (both BioLegend) for 4 h.

The anti-CD3 microbeads (Miltenyi Biotec, Germany) was used to sort CD3⁺ T cells in mouse spleen. Then, anti-CD3 antibody (2 μg/mL) and anti-CD28 antibody (1 μg/mL) (BioLegend, San Diego, CA, USA) were used to activate T cells.

T cells were isolated from the spleens of wildtype BALB/c mice, stained with eFluor 450 and activated by culturing in the presence of 3 mg/ml of anti-CD3 antibody (BioLegend, catalogue number 100302) and 1 mg/ml of anti-CD28 antibody (BioLegend, catalogue number 102102) as previously described.^{38 (link)} Cells were then incubated with MDSCs for 4 days at 37 °C. CD8⁺ T-cell proliferation was then analysed.

On the day before transduction, HEK293T cells were seeded at 8 × 10⁶ cells per 100-mm dish. Twenty-four hours later, virus was generated by transfecting HEK293T cells with a plasmid encoding NL4-3 or NL4-3 BaL, using a polyethylenimine transfection system and following the manufacturer’s instructions. Supernatants were harvested after 48 h, centrifuged (10 min, 500 × g, room temperature), and filtered through a 0.45-μm-pore-size membrane to remove the cell debris. Viruses were concentrated by centrifuging with a 25% volume of 50% polyethylene glycol 6000 and a 10% volume of 4 M NaCl. Concentrated virions were resuspended in complete medium and stored at −80°C. The virus concentration was estimated by p24 titration using an enzyme-linked immunosorbent assay (ELISA). Different CD4⁺ T cell subsets cultured in basal medium or stimulated for 3 days with anti-CD3 antibody at 1 μg ml⁻¹ (BioLegend) and anti-CD28 antibody at 1 μg ml⁻¹ (BioLegend) were infected with NL4-3 Bal or NL4-3 virus (p24 titer, 50 ng ml⁻¹). Infected CD4⁺ T cells were further cultured in basal medium and incubated at 37°C in a humidified incubator with 5% CO₂. Then, infected cells were analyzed by flow cytometry at 3 to 7 days postinfection.

CD3⁺ T-cells were purified from the spleen of wildtype and TRAIL^−/− mice using the MACS^® cell-sorting system (Pan T cell isolation kit II, Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions, specifically using an antibody cocktail (provided by the manufacturer) against CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC-class II, and Ter-119 over MACS LD columns. Resulting cells were >95% CD3 positive by FACS analysis. 96-well plates were incubated over at least one hour at 37 °C with an anti-CD3 antibody at a concentration of 1 µg/mL and afterwards washed three times with PBS. CD3⁺ T-cells were stained with carboxyfluorescein succinimidyl ester (CFSE, LifeTechnologies, Invitrogen) at a concentration of 4 µM and 5 × 10⁴ cells per well in the coated 96-well plate and distributed in 200 µL. Furthermore, an anti-CD28 antibody (BioLegend) at a concentration of 1 µg/mL was added. The cells were then treated with recombinant soluble murine TRAIL (Biolmol GmbH, Hamburg, Germany) (purity 95%, <1.0 EU per 1 g) and dissolved in sterile RPMI-1640 medium in different concentrations (0, 100 and 1000 ng/µL final concentration). Proliferation was checked by FACS on day 2 and 3.