Mouse anti human cd28

Peripheral blood mononuclear cells (PBMCs) were isolated from whole human blood using Ficoll (Fisher) gradient centrifugation. Cell suspensions were washed in PBS and stained with 10 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) cell stain (CellTrace CFSE Cell Proliferation Kit, Invitrogen). Cells were incubated for 6 minutes at 37°C protected from light, reaction was stopped by adding an excess of ice‐cold T‐cell media (10% FBS, 2 mM Pen/Strep, 2 mM l‐glutamine, 0.1 M nonessential amino acids, 1 mM sodium pyruvate and 55 μM β‐mercaptoethanol in RPMI 1690 [Sigma Aldrich]). 2 × 10⁵ CFSE‐labelled T cells were added to a 96‐well plate, they were stimulated using mouse anti‐human CD3 0.5 mg/mL (BD Biosciences) and mouse anti‐human CD28 10 mg/mL (BD Biosciences). Stromal cells were added at ratios of 1:10, 1:50 and 1:200. Cells were harvested after 4 days and stained using PE‐Cy7 mouse anti‐human CD4 (BD Biosciences) and analysed using a FACS Canto II (BD Biosciences). All analyses were carried out using FlowJo. ≥3 generations of CD4⁺ T‐cell proliferation was determined in order to quantify the ability of stromal cells to suppress sustained proliferation.

In order to block CD226, PBMCs were washed and resuspended and an anti-CD226-FITC antibody, which can facilitate CD226 blocking, as well as the subsequent flow cytometry analysis, was then added, followed by incubation for 20 min. Next, leukocyte activation cocktail containing GolgiPlug was added for 4 h to activate the cells. CD3, CD4, CD8, CD107a, IFN-γ, and TNF-α were stained as described above to determine the functional and activation changes in T cells due to CD226 blocking.
To assess the proliferation, PBMCs were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) at 37°C for 15 min, and then washed and resuspended in RPMI 1640 medium containing 10% fetal bovine serum. These labeled PBMCs were incubated with mouse anti-human CD3 (5 μg/mL; BD Bioscience) and mouse anti-human CD28 (5 μg/mL; BD Bioscience) antibodies for 72 h at 37°C in a humidified atmosphere containing 5% CO₂, until the surface markers CD3, CD4, and CD8 were stained; then, the cells were analyzed by flow cytometry. In order to avoid a quenching effect, most of the above-mentioned procedures were performed in the dark.

All reagents were endotoxin free. rhGM-CSF and rhTNF-α were from PeproTech (Rocky Hill, NJ, USA); IL-4 and IFN-γ were from R&D Systems Europe (Oxford, United Kingdom); Ultrapure TLR4 agonist (Salmonella Minnesota LPS) was from InvivoGen (San Diego, CA, USA); rhIL-1β and rhIL-6 were from ImmunoTools (Friesoythe, Germany); IL-23 was from eBioscience (San Diego, CA, USA); and PGE₂, indomethacin, and forskolin were from Sigma-Aldrich (Dorset, United Kingdom). Mouse anti-human CD4-PE was from BD Biosciences (Oxford, United Kingdom); Mouse anti-human CD4-PE-Cy7, mouse anti-human CD45RA-FITC, mouse anti-human CD14-PE-Cy5.5, and matching isotype controls were from eBioscience. For CD4 activation, mouse anti-human CD28 was obtained from BD Biosciences, IL-2 from R&D Systems Europe, and CD3 (OKT3) was produced in-house. The annexin V/PI staining kit was obtained from BD Biosciences.