Null donors favor prepared immunologists

In this issue of Blood, Golay et al carefully examine activation and effector functions of normal neutrophils and of FcγRIIIB (CD16B)–negative neutrophils isolated from the blood of a rare null donor.¹  Their work reveals, for the first time, that normal neutrophils also express FcγRIIIA (CD16A) and that the 2 receptors on neutrophils mediate distinctly different effector functions for processing antibody-opsonized substrates.

The role of the Fcγ receptor (FcγR) expressed on immune effector cells (eg, monocytes, macrophages, neutrophils, and natural killer cells) in disposing of soluble and particulate immunoglobulin G (IgG)–containing immune complexes (ICs) is well-established.^2-5  For example, effector cells use FcγR to mediate clearance and destruction of IgG-opsonized tumor cells. However, several important questions concerning the actions of these receptors have not been addressed. FcγRIIIB, expressed at very high levels on neutrophils (∼200 000 per cell), has no outright signaling capacity because it is anchored to the cell membrane via a phosphatidylinositol (PI) linkage.⁴  Therefore, its potential to mediate neutrophil activation is unclear. However, some evidence suggests that complement receptor CR3 (β-integrin CD11b/CD18) and FcγRIIIB on neutrophils may synergize in binding and processing of complement-opsonized IgG-containing IC, but no clear patterns have been elucidated.⁵  Moreover, considerable evidence indicates that trogocytosis decreases the efficacy of tumor-targeting monoclonal antibodies (mAbs).⁶  In this reaction, effector cells (monocytes, macrophages, and neutrophils) make use of FcγR to remove and internalize tumor-bound IgG mAb, chelated target antigen, and associated membrane. Tumor cells thereby lacking target antigen cannot be eliminated, thus severely compromising the efficacy of mAb treatment. The relative quantitative contribution of neutrophils and their individual FcγRs to this reaction are not presently known.

There have been numerous reports of rare null individuals whose neutrophils do not express FcγRIIIB.^4,7-9  In some instances, these deficiencies have been found to be associated with autoimmune diseases such as systemic lupus erythematosus.⁷  However, in most cases, there has been no disease-associated phenotype, that is, the lack of FcγRIIIB often does not presage infection or any other problem, which suggests normal neutrophil function. It should now be possible to address these seemingly disparate questions and observations on the basis of the seminal article by Golay et al who were able to demonstrate modest expression but substantial biological activity of FcγRIIIA on normal neutrophils. The investigators seem to have followed Pasteur’s maxim (“Chance favors only the prepared mind”) and were prepared to clarify the meaning of their chance observation of the FcγRIIIB-null normal individual.

Golay et al first found (on the basis of polymerase chain reaction) that 1 normal (null) individual in their study cohort lacked the FCGR3B gene. Further analyses based on western blots and flow cytometry confirmed the absence of FcγRIIIB and surprisingly revealed the expression of small amounts of FcγRIIIA. This receptor has an intracytoplasmic domain and can mediate signaling and promote cell activation.⁴  The investigators found equivalent (small) amounts of FcγRIIIA on neutrophils of normal individuals. The implications of these findings were made evident by tests for neutrophil activation initiated by reaction with B cells opsonized with glycoenginered rituximab or obinutuzumab. They noted that these 2 mAbs have previously been demonstrated (because they are defucosylated) to react most strongly with FcγRIIIA and FcγRIIIB. They found that normal neutrophils and neutrophils from the FcγRIIIB null donor were equally well activated by these substrates on the basis of increased expression of CD11b and decreased expression of CD62L.These findings suggest that it was FcγRIIIA on both cell types that was reacting with mAb-opsonized B cells.

Golay et al report similar patterns of activation of normal and null neutrophils based on stimulation with F(ab′)₂ CD16 antibodies, but they found that stimulation of neutrophil-associated FcγRIIA (CD32) with F(ab′)₂ CD32 antibodies did not induce activation, thus eliminating this receptor as a mediator of the phenomenon they were studying. Moreover, they found that phagocytosis of IgG-opsonized beads was demonstrable for normal and null neutrophils, and that this reaction could be blocked in part by F(ab′)₂ CD16 antibodies, but not by F(ab′)₂ CD32 antibodies. In future studies, controls such as these will be quite important to clearly define the relative contribution of FcγRIIA vs FcγRIIIA to neutrophil activity. Finally, they examined trogocytosis of RTX-opsonized B cells and demonstrated that both normal and null neutrophils mediated trogocytosis. They reported that in normal neutrophils, this reaction was inhibited by CD16-specific antibodies but not by CD32 antibodies, thus implicating FcγRIIIB as the primary mediator of trogocytosis by normal neutrophils. Curiously, in null neutrophils only CD32-specific antibodies could block trogocytosis, suggesting that in the absence of FcγRIIIB, FcγRIIA can promote trogocytosis.

What is next? The immunologic activity of FcγRIIIA on neutrophils reported by Golay et al is quite remarkable in view of the relatively low expression of this receptor on the cells. There are ∼200 000 FcγRIIIB and 20 000 to 40 000 FcγRIIA on neutrophils^2,4,10 ; extrapolation based on the data reported by Golay et al suggests that the number of FcγRIIIA expressed on neutrophils may be a few thousand, yet substantial activity is demonstrable. To clearly examine the action of FcγRIIIA in the absence of high levels of FcγRIIIB, it will be necessary to examine neutrophils from other (rare) FcγRIIIB null donors. Alternatively, neutrophils of patients with paroxysmal nocturnal hemoglobinuria have about 10% of normal levels of FcγRIIIB, and enzymatic treatment of neutrophils with PI-phospholipase C leads to almost quantitative removal of FcγRIIIB with no effect on FcγRIIA.^2,3 

Experiments that evaluate the activity of FcγRIIIA on neutrophils should focus on mechanisms of activation on the basis of assays for γ-chain phosphorylation as well as for downstream signaling.⁴  On the basis of the activity exhibited by FcγRIIIA on neutrophils, strategies that increase its expression might provide a reasonable approach to increasing defenses against infection. It might also be informative to revisit the synergy of CR3 with FcγR and determine the relative contribution of FcγRIIIA and FcγRIIIB to this phenomenon. Golay et al recognized that their findings may also pertain to the action of neutrophils in autoimmune disease. Both basic scientists and clinicians can look forward to additional progress in several areas of investigation on the basis of the article by Golay et al.