Soluble anti cd3 and anti cd28 antibodies

Purified monocytes and CD4 memory T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (penicillin 100units/ml and streptomycin 100 ug/ml) and 1% L-glutamine (2 mM). For T cell-monocyte coculture, purified monocytes were seeded at 5×10³ into each well of U-bottomed 96-well plate and incubated with soluble anti-CD3 and anti-CD28 antibodies (1 µg/ml of each; BD bioscience) in the presence of 100ng/ml LPS (Sigma-Aldrich Inc, St. Louis, MO). After incubation for 1 hour, 2.5×10⁴ CD4⁺ memory T cells were added into each well and co-cultured with LPS-activated monocytes for 7 days. In some experiments, purified monocytes were treated for 18 hours with TGF-β (10 ng/ml; R&D systems) in the presence or absence of the indicated recombinant human cytokines such as IL-1β, IL-6 (25 ng/ml of each; both from R&D system), TNF-α (25 ng/ml; ProSpec, East Brunswick, NJ) and IFN-γ (25 ng/ml; eBioscience) and SB431542 (TGF-β signaling inhibitor; Calbiochem, La Jolla, CA), After 18 hour-treatment, the monocytes were stained with the following antibodies: APC-Cy7-CD14, PE-CD16, PE-CD80, and FITC-HLA-DR. The stained cells were acquired by a BD LSRII (BD bioscience).

RPMI 1640 medium and fetal bovine serum for culturing memory CD4⁺ T cells and preparing polarized Th17 cells were purchased from Hyclone. Human T-cell activator anti-CD3 and anti-CD28 antibody cocktail covalently bound to magnetic beads (Dynabeads) was obtained from ThermoFisher Scientific. Soluble anti-CD3 and anti-CD28 antibodies were from BD Biosciences. Ingenol-3-angelate and SAHA were purchased from Cayman Chemical. Recombinant TNF-α was from R&D Systems. Prostratin, PMA and ionomycin were from Millipore Sigma. The sources and catalog information of all commercially obtained inhibitors and antibodies used in the current study are shown in S1 Table. HEXIM1 antibody was custom-synthesized and affinity purified by Covance Research Products. Generation, purification and validation of the polyclonal antibody towards the phospho-Ser-175 CDK9 epitope has been described previously [39 (link)].

Human PBMCs were isolated from the peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque (GE Healthcare-Life Sciences, USA). Regulatory T cells (Tregs) were isolated from PBMCs using magnetic beads technology (CD4⁺ CD25⁺ CD127^dim/- Regulatory T Cell Isolation Kit II; Miltenyi Biotec, USA) according to the manufacturer’s recommendations. Autologous PBMCs were stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 and 0.5 µg/mL respectively; BD Biosciences, USA) in the presence of Tregs (1:1 PBMC to Treg ratio). The therapeutic antibodies were added, and after 3 days of culture, cytokine levels were measured in the culture supernatant using ProcartaPlex immunoassay according to the manufacturer’s recommendations (Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex™ Panel; Thermo Fisher Scientific). Results were expressed as a percentage of inhibition of cytokine secretion relative to the level secreted in the absence of Tregs.