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

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

Analysis of FcγRIIIA transfection in HEK293T cells.

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THP-1-CD16A macrophages with phagocytosed IgG-opsonized human platelets.

Three different images are shown of IgG-opsonized platelets incubated with THP-1-CD16A macrophages. Platelets were labelled with 5- chloromethylfluorescein diacetate (CMFDA) (green) before phagocytosis. Non-phagocytosed platelets were differentiated after phagocytosis using an AF647-conjugated anti-human CD42a antibody (red). Platelets were additionally defined by size (roughly 1.5 to 3.5 μm) to distinguish them from internalized microparticles or platelet aggregates. THP-1-CD16A macrophages were observed by spinning-disc confocal microscopy under 63x objective oil immersion with differential interference contrast and DAPI stain on a Quorum multi-modal imaging system (Quorum Technologies, Ontario, Canada). Four images were taken at the center of each well with Z-stacking. Phagocytosis was quantified using Imaris v9.6.0. Scale bar = 3 μm. White arrows indicate examples of phagocytosed platelets.</p

SDS-PAGE analysis of blocking antibodies deglycosylation.

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Expression of human FcγRs on non-differentiated and PMA-differentiated THP-1-CD16A cells.

THP-1-CD16A cells were differentiated to macrophages by treatment with PMA (100 ng/mL) as described in the Materials and methods. Five hundred thousand cells were stained with direct-labelled monoclonal antibodies against (A) FcγRI (clone 10.1 conjugated to PE/Cy7), (B) FcγRIIA (clone IV.3 conjugated to FITC), (C) FcγRIIA/B/C (clone AT10 conjugated to AF647), or (D) FcγRIIIA (clone 3G8 conjugated to BV421). Stained cells were washed and analyzed by flow cytometry using a BD LSRFortessa X-20. Data analysis was performed using FlowJo v10. Data are presented as the mean ± the standard deviation from four independent experiments. MFI: Mean fluorescent intensity (arbitrary units). Ab concentration: Concentration of the fluorescent antibody for FcγR expression detection. The dashed line represents the MFI value of the corresponding isotype control at 10 μg/mL. The statistical analysis was performed by the non-parametric Mann-Whitney T-test, comparing the MFI of both conditions for each antibody concentration (*: p = 0.05).</p

Brightfield micrographs of THP-1 or THP-1-CD16A macrophages demonstrating phagocytosis of IgG-opsonized human erythrocytes.

Macrophages were incubated with human erythrocytes previously opsonized with a polyclonal anti-human RhD antibody (WinRho SDFTM). External, non-phagocytosed erythrocytes were removed by hypotonic (water) lysis. Black arrows indicate examples of phagocytosed erythrocytes. Micrographs are representative of five independent experiments.</p

Expression of human FcγRs on THP-1 and THP-1-CD16A cells.

Five hundred thousand cells were stained with direct-labelled monoclonal antibodies against (A) FcγRI (clone 10.1 conjugated to PE/Cy7), (B) FcγRIIA (clone IV.3 conjugated to FITC), (C) FcγRIIA/B/C (clone AT10 conjugated to AF647), and (D) FcγRIIIA (clone 3G8 conjugated to BV421). Stained cells were washed and analyzed by flow cytometry using a BD LSRFortessa X-20. Data analysis was performed using FlowJo v10. Data are presented as the mean ± the standard deviation from four independent experiments. MFI: Mean fluorescent intensity (arbitrary units). Antibody concentration: Concentration of the fluorescent antibody for FcγR expression detection. The dashed line represents the MFI value for the corresponding isotype control at 10 μg/mL. The statistical analysis was performed using the non-parametric Mann-Whitney T-test, comparing the MFI of both cell lines for each antibody concentration (*: p = 0.05).</p