Ccl3 mip 1α

CCL3 (MIP-1α) and IL-2 were purchased from Millipore (Billerica, MA, USA). Monoclonal antibodies anti-Stat3 (clone: 79D7), anti-phospho-Stat3 (Tyr705) (clone: D3A7), and anti-GAPDH (clone: D16H11) were purchased from Cell Signaling Technology (Beverly, MA). Ly6G mAb (clone 1A8) was from BioExpress. Mouse IL-18 binding protein (IL-18BP) was from BIOHJ Corporation (USA). Anti-NKG2D (MI-6), anti-NKp46 (29A1.4) and anti-G-CSF antibodies were from R&D Systems. G-CSF, GM-CSF, IL-6, and TNF-α were from PeproTech (Rocky Hill, NJ). STAT3 inhibitor (FLLL32) was from Selleck. Rabbit anti-DX5 (clone EPR5788), anti-PD-L1 (clone EPR20529), and anti-PD-1 (clone J43) antibodies were from Abcam. Rabbit anti-mouse CCR1 (clone PA1–41062) and HRP-conjugated Goat anti-Rabbit IgG were from ThermoFisher Scientific. PE-conjugated anti-mouse IFN-γ (clone XMG1.2), PE-conjugated anti-mouse PD-L1 (clone MIH5), PE-conjugated anti-mouse PD-1 (clone RMP1–30) and PE-conjugated anti-mouse DX5 were from eBioscience (San Diego, CA).

Immune mediators in the supernatants from monocyte cultures were measured using a Milliplex Multiplex assay panel measuring levels of TNFα, IL-6, IL-1β, G-CSF, CCL3(MIP-1α), and IL-2 (Millipore, Burlington, MA). Differences in induction of proteins post stimulation were calculated using log₁₀(pg/mL fold-change +1). Undiluted samples run in duplicates on the Magpix Instrument (Luminex, Austin, TX). Data were fit using a 5P-logistic regression on xPONENT software (version 7.0c).