Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation

Abstract

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab′)2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

IgG-aggregates rapidly up-regulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in the generation of reactive oxygen species

“…The FcgRIIIb-specific 11.5 blocking antibody, actually enhanced FcgRI up-regulation, as did PI-PLC, a FcgRIIIb-specific shedding enzyme. Moreover, the 3G8 blocking antibody (36), which does not discriminate between the two FcgRIII isoforms, did not affect FcgRI up-regulation, supporting the concept of a decoy role for that receptor (10,37,38). Results leave the possibility of a role for FcgRIIIa.…”

“…FcgRIIa, with its integrated signaling Immunoreceptor Tyrosine-based Activation Motif (ITAM), is an essential mediator of destructive antibody-based inflammation in autoimmunity (5) and is necessary for neutrophil recruitment within glomerular capillaries following IgG deposition (8,9). Also, a relatively modest expression of FcgRIIIa (CD16a) on neutrophils was recently demonstrated (10). FcgRIIIa is a classic activating receptor, which, upon stimulation, associates with an ITAM-signaling system.…”

Harnessing the immune system via FcγR function in immune therapy: a pathway to next‐gen mAbs

“…The FcγRIIIa is expressed on NK cells and professional phagocytes, particularly macrophages. It is only recently apparent that FcγRIIIa is expressed on neutrophils, albeit at low levels, but plays a role in their function 23. FcγRIIIb is unique to humans and unlike other FcγRs it is attached to cell membrane via a glycophosphatidyl anchor.…”

“…Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68,88,89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa 23. However, certain neutrophil functions via FcγRIIa may be compromised 90,91…”

Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors

“…FcγRIIIb is known to be very abundant on human neutrophils, while FcγRIIIa is not present 19,34 . However the absence of FcγRIIIa from neutrophils has recently been challenged 35 . The western blot showed a smear from 50 to 80 kDa when probed with an anti-CD16 antibody (Figure 1).…”