THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets

Abstract

Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA. However, there is currently no widely available monocyte or macrophage cell line expressing all these receptors. Common sources of monocytes for differentiation into macrophages, such as human peripheral blood monocytes and the monocytic leukemia cell line THP-1, generally lack the expression of FcγRIIIA (CD16A). Here, we utilized a lentiviral system to generate THP-1 cells stably expressing human FcγRIIIA (CD16F158). THP-1-CD16A cells treated with phorbol 12-myristate 13-acetate for 24 hours phagocytosed anti-D-opsonized human red blood cells primarily utilizing FcγRI with a lesser but significant contribution of IIIA while phagocytosis of antibody-opsonized human platelets equally utilized FcγRI and Fcγ IIIA. Despite the well-known ability of FcγRIIA to bind IgG in cell free systems, this receptor did not appear to be involved in either RBC or platelet phagocytosis. These transgenic cells may constitute a valuable tool for studying macrophage FcγR utilization and function.

Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP

“…The binding of 17C02-based molecules to FcγRIIIA was evaluated using THP-1 transgenic cells expressing the CD16A molecule (THP-1-CD16A cells) 9 and peripheral blood mononuclear cells (PBMC) from human donors. The 3 molecules 17C02-albumin, 17C02-IgG2a (deglycosylated), and 17C02-IgG1 OA bound to both THP-1-CD16A and NK cells in a dose-dependent manner ( Figure 3 A-B,D-E,G-H).…”

“…THP-1-CD16A cells 9 were maintained in a 37°C, 5% CO 2 environment in complete RPMI medium. The extracellular domain of human FcγRs fused with a polyhistidine tag at the C-terminus were purchased from Sino Biological (Chesterbrook, PA).…”

“…Phagocytosis of antibody-opsonized platelets was performed as previously described. 9 Briefly, THP-1-CD16A cells were seeded on sterile glass coverslips (Thermo Fisher Scientific, Canada) in a 24-well polystyrene plate at 2 × 10 5 cells per well in complete RPMI and differentiated to macrophages by treating them with PMA (BioShop, Canada). Whole blood collected in citrate-dextrose solution (BD) was used to obtain platelet-rich plasma.…”

Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice

“…Furthermore, the ability of THP-1-CD16A monocyte-macrophages to engulf (phagocytose) antibody-opsonized human red blood cells and platelets had previously been demonstrated using ADCP assay. 24 Employing ADCP assay, we explored the potential of BiKE:E5C1 to enhance the anticancer activity of CD16+M1 macrophages (downstream effect) against SKOV-3 cells. To achieve this, THP-1-CD16A monocytes that were engineered to stably express CD16a receptor ( figure 1F ), were treated with LPS and IFN-γ to polarize them into M1 macrophages.…”

“…THP-1-CD16A monocytes stably overexpressing CD16A were generously provided by Dr. Alan H. Lazarus’ research group at the University of Toronto. 19 For cell culture details, please see online supplemental method 1 .…”