Interleukin 4 (il 4)

RPMI-1640, FBS, penicillin-streptomycin, 1× PBS, TRIzol™, and primers were ordered from Life Technologies (Carlsbad, CA). Norepinephrine with bitartrate salt, epinephrine with bitartrate salt, phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharide from E. coli, were ordered from Sigma-Aldrich (St. Louis, MO). IL-4 from Promega (Madison, WI), Sybr green, Superscript III cDNA kit from Invitrogen (Carlsbad, CA) were purchased and used.

HEK293T cells were cultured to confluence in a 96-well plate and transfected with 40 ng human STAT6, 80 ng luciferase reporter, 3.2 ng ß-galactosidase, and either the FLM-PC1, PC1-p30 or PC1-p15 plasmid. Four h after transfection, 1 ng/ml human IL-4 (R&D Systems) and culture media without penicillin and streptomycin were added to the cells. EGFP in pCDNA4/TO was used as a negative control, and the backbone vector was used for balancing the plasmid amounts in all transfections. Luciferase assays were carried out after 20 h of treatment with IL-4 with luciferase substrate (Promega), and ß-gal activity was detected using 2-nitrophenyl β-D-galactopyranoside in sodium phosphate buffer.

Two female volunteers, aged 27 and 35, were recruited and they provided the informed consents. Peripheral blood was stained with PerCP HLA-A2 (307627, Biolegend, San Diego, CA, USA) and human leukocyte antigen (HLA) subtype was detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from HLA-A2-type volunteer and cultured in 75 cm² flask for 1 h. Suspended cells were removed, and the adherent cells were cultured with fresh X-VIVO 15 medium (Lonza, Alpharetta, GA, USA) containing 5% plasma supplemented with 500 U/ml of IL-4 and 1000 U/ml of GM-CSF (Promega, Madison, WI, USA). After 5 days of culture, DCs were collected for following experiments. CD8⁺ T cells were isolated from PBMCs using the CD8⁺ T Cell Isolation Kit according to manufacturer’s instructions (Miltenyi Biotec, CA, USA).