Clone hit3a

CD14⁺ cells isolated from PBMC using EasySep Human Monocyte Isolation Kit (STEMCELL) were cultured in CellGenix GMP DC serum-free media (CellGenix, #20801-0500) supplemented with 1% Human Serum AB (Millipore Sigma, #H3667) and 1% penicillin–streptomycin-L-glutamine (ThermoFisher Scientific, #10378-016). GM-CSF (1000 U/mL, CellGenix #1412-050) and IL-4 (500 U/mL, CellGenix #1403-050) were added to the medium to generate Mo-DC. On day 5, DC were pulsed with peptide pools or individual peptides at 10 μg/mL, in the presence of IFNα (500U/mL), GM-CSF (1000 U/mL) and IL-4 (500 U/mL). 12,500 to 100,000 Jurkat TCRab dKO/AP1.Luc/hCD8 cells expressing an TCR of interest (Jurkat-TCR) were cocultured with 30,000 DC pulsed with antigen peptides for 5 h before lysed to assess luciferase activity using One-Glo Luciferase Assay (Promega) as per manufacturer’s instructions. Co-culture of 100,000 Jurkat-TCR cells with 30,000 DC pulsed with irrelevant peptides or with DC treated with DMSO was used as negative controls. When a titration of Jurkat-TCR cells were used, Jurkat/NFAT-Luc cells were used as filler cells. Nonspecific activation of TCRs was achieved by soluble anti-CD3 (Clone HIT3a, BD Biosciences) and anti-CD28 (Clone CD28.2, BioLegend). Luminescence signal was measured using the EnVision plate reader. RLU values were plotted after background signals were subtracted.

Cells were suspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a 5% CO₂ atmosphere. The PBMCs (1×10⁶ cells/ml) were cultured for 72 h in 24-well plates and subsequently stimulated with anti-CD3 (3 μg/ml, clone HIT3a; BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD28 (3 μg/ml, clone CD28.2; BD Biosciences) antibodies in the presence or absence of different concentrations (0.01, 0.1 or 1 μmol/l) of GTS-21 (Abcam, Cambridge, MA, USA). Purified CD4⁺ T cells (1×10⁶ cells/ml) were stimulated using anti-CD3-coated 96-well plates (BioCoat™ anti-human CD3 T-cell activation plates; BD Biosciences) plus anti-CD28 antibodies (3 μg/ml), in the presence of IL-12 (15 ng/ml, Peprotech, Inc., Rocky Hill, NJ, USA) and anti-IL-4 antibodies (4 μg/ml, Peprotech, Inc.) for Th1 differentiation for 72 h with GTS-21 (1 μmol/l) alone or combined with α-bungarotoxin (αBgt, 1 μmol/l; Sigma, St. Louis, MO, USA).

Jurkat cells were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI-1640 medium containing 10% FBS, streptomycin sulphate, penicillin, HEPES, and sodium bicarbonate in a 5% CO₂ atmosphere at 37 °C. Jurkat cells were stimulated with 1 μg/ml of plate-bound anti-CD3 mAb (clone HIT3a, BD Bioscience) and then incubated with KITS 1–001 (10, 50, or 100 μM) for 30 min. KIST 1–001 were dissolved in DMSO and added to the culture media in serial dilution (the final concentration of DMSO in all experiments did not exceed 0.1%).