Journal: Frontiers in Oncology
Article Title: A Potent and Selective Dual Inhibitor of AXL and MERTK Possesses Both Immunomodulatory and Tumor-Targeted Activity
Figure Lengend Snippet: Inhibition of phagocytosis and reversal of macrophage-mediated suppression of T cells by INCB081776 in primary human immune cells. (A) Human whole blood was treated with INCB081776 at 10 nM and 100 nM along with dimethyl sulfoxide control. Fluorescent microspheres were added to peripheral blood mononuclear cells (PBMCs) prelabeled with anti-CD14 and anti-CD16, and acquisition performed at intervals over a course of 4 min. Plot indicating the mean of the uptake of fluorescent microspheres by CD14 ++ CD16 + monocyte population over time is shown. Data are representative of three healthy donors. (B) Primary macrophages differentiated in vitro from human PBMCs were pretreated for 2 h with INCB081776 followed by stimulation with the MERTK-specific agonist antibody MAB8912 for an additional 30 min. Levels of phospho-MERTK (pMERTK) and total MERTK were determined by Western blot. (C) Primary macrophages differentiated in vitro from human PBMCs were pretreated with various concentrations of INCB081776 overnight, followed by incubation with carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells for an additional 5 days. T-cell proliferation was determined by FACS analysis ( N = 2 per group). (D) Following incubation of macrophages and T cells in the presence of increasing concentrations of INCB081776 as in panel (C) , supernatants were collected and interferon (IFN)-γ cytokine production was measured ( N = 2 per group, except for 111 and 333 nM [ N = 1]). SD, standard deviation.
Article Snippet: Human peripheral blood mononuclear cells (PBMCs) were obtained from normal leukapheresis of two healthy donors (Biological Specialties, Colmar, PA, USA).
Techniques: Inhibition, In Vitro, Western Blot, Incubation, Labeling, FACS, Standard Deviation