Surface Fc Research Articles

Abstract 5135: Standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies

Abstract In the treatment of solid tumors, chimeric antigen receptor(CAR)-engineered NK (CAR-NK) cells have distinct advantages over CAR-T cells, such as a lack graft-versus-host disease in allogenic setting to make “off the shelf” medicine; much safer because they are less likely to cause cytokine storms; and NK cells have their own activated receptors that recognize tumor surface antigens and thus have a natural ability to kill a wide range of tumors. In addition, NK cells are the main performers of antibody-dependent cell-mediated cytotoxicity (ADCC), which binds to the Fc-terminus of antibodies through their surface Fc receptor CD16A, thereby killing antibody-targeted tumor cells. Enhancing the ADCC action of NK cells themselves enhances the therapeutic efficacy of monoclonal antibodies targeting tumor surface antigens and is therefore of great importance in the treatment of tumors, especially solid tumors. We screened two optimal structures among nine different Fc chimeric receptors, and NK cells overexpressing these two receptors killed K562 and Daudi six times more intensely than normal NK cells when combined with rituximab, while the killing ability was comparable to that of normal NK without rituximab, indicating that NK cells expressing chimeric Fc receptors have stronger ADCC effects. Moreover, these NK cells can kill target cells in multiple rounds. In vivo experiments in mice demonstrated that NK cells expressing chimeric Fc receptors in combination with EGFR monoclonal antibodies had a stronger inhibitory effect on tumor growth than monoclonal antibodies or NK cells alone. Here we provide a novel broad “off the shelf” NK cells that can significantly enhance the killing ability of hematological and solid tumors in combination with different monoclonal antibodies. Citation Format: Yanan Lin, Shengbang Zhang, Jie Ran, Can Song, Shengcang Zhu, Hui Shi, Ping Guo, Shengjiang Tan, Yuchun Gu, Lida Wu. Standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5135.

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

Research Highlights

Research Highlights

Antigen-Induced Allosteric Changes in Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of cross-linking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over forty years and remains unresolved. Here we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcR) for IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21.

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKβ and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.

Analysis of the uptake of chlorotrimethylsilane on glass from toluene solution-phase depositions

The uptake of chlorotrimethylsilane on glass from toluene is proposed to follow a defined reaction model. Static contact angles θm are measured on glass exposed to a range of solution concentrations for t=20 min. The uptake is potentially sensitive to residual moisture. Fractional surface coverage fc is calculated from three models, direct conversion fc∝θm, a patchy layer fc∝cos (θm), and a micro-heterogeneous surface fc∝(1+cos(θm))2. Phase contrast images in atomic force microscopy show uniform coverage and thereby favor the micro-heterogeneous model. Calibrations of fc against the intensity of an R-Si peak from x-ray photoelectron spectroscopy favor the direct or micro-heterogenous model. The uptake reaction is a two step process. The first step is equilibrium Langmuir adsorption/desorption to physisorbed silane. The second is an irreversible, first-order, rate-determining step. The equation fc=1−exp(−krdstKeqC(1+KeqC)) is applied using weighted regression analysis. The parameters weighted from all three coverage models are <Keq>=35±14 M−1 and <krds>=0.219±0.053 min−1.

Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

Antibody-dependent enhancement (ADE) of Ebola virus (EBOV) infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa)-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs) is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

The Chosen Few: Only a Subset of Memory B Cells Responds to Secondary Dengue Virus Infections

The Chosen Few: Only a Subset of Memory B Cells Responds to Secondary Dengue Virus Infections

Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis.

Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions.

Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR)

Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses.

Single Chain Fragment Variable Recombinant Antibody as a Template for Fc Sensors

A label free immunosensor for detection of Fc receptors expressed on cell surface was developed and characterized using a Quartz Crystal Microbalance (QCM) transducer. Taking advantage of the characteristics of single chain fragment variable (scFv) recombinant antibody and the multivalency of an antibody, the engineered recombinant scFv was immobilized onto preformed functionalized self-assembled monolayers (SAMs) template surface. The monomeric scFv can bind with the CH1 region of any rabbit IgG to form a highly oriented IgG layer with its Fc portion pointing toward a solution phase. This results in a highly oriented Fc sensor that can be used to study the thermodynamics and kinetics of binding between the Fc portion of immunoglobulin and the cell surface Fc receptor (FcR), an important area of the immune system. The Fc sensor was used to study the binding between Staphylococcus aureus and the Fc receptor on macrophage. Parallel characterization of cell surface Fc receptors in the same samples by ELISA was also performed.

FCER1A genetic variability and serum IgE levels

We read with keen interest a very recent study by Chen et al. (1), which further expands on the recent findings byWeidinger et al. (2) who reported on the results of large genome-wide scan that demonstrated IgE receptor (FceRI) a-chain (Fc RIa) gene (FCER1A) variability to be a major marker of serum IgE levels in population. Chen et al. (1) successfully showed an association between FCER1A variants and serum IgE levels present in cord blood/small children to be independent of environmental stimuli, which seems to be an important genetic input on our understanding of multi-factorial etiology of allergic disorders.We would like to congratulate the authors of both the above very analytically powerful studies (1, 2) for scientific and statistical power as well as for elegance and excellence of their work. The large and powerful trials by Weidinger et al. (2) and Chen et al. (1) demonstrated two common variants FCER1A rs2427837 and rs2251746 to be strongly associated with serum IgE levels in general and paediatric populations. While direct evidence for strong functional activity of rs2251746 FCER1A proximal promoter polymorphism was previously demonstrated (3, 4), showing it to be a potent regulator of FCER1A/FceRIa expression in mast cells and/or basophils, no such data were yet provided for rs2427837 variant. Therefore, an association between rs2427837 polymorphism and IgE levels (1, 2) might possibly result from its almost complete linkage disequilibrium (1, 2) with functional (3, 4) rs2251746 variant. One might also cautiously speculate that some more functional FCER1A variants could by their haplotypic relationships/interplay with rs2251746 polymorphism, at least in small part, functionally contribute to the association with serum IgE levels observed for rs2251746 variant in population (1, 2). For example, rs2427827 polymorphism, close neighbour of rs2251746 variant in FCER1A proximal promoter, was also demonstrated to affect FCER1A transcriptional activity/FceRIa expression in mast cells and/or basophils (4, 5) in an additive manner with rs2251746 polymorphism (4). While the regulatory mechanism by which (serum) IgE upregulates cell surface Fc RI expression is well known (6), no direct evidence for the causal role of FceRI(a), FCER1A or FCER1A genetic variants in regulation of IgE synthesis and/ or levels was yet provided. Therefore, it might be only speculated that any alterations in FceRI expression, including also those genotype-related, might affect IgE synthesis and/or levels. Thus, previous results showing how FCER1A proximal promoter polymorphisms regulate FceRI(a) expression in mast cells and/or basophils (3–5) might provide only a partial mechanistic background for previously observed FCER1A genetic variants-serum IgE levels genotype-phenotype associations (1, 2, 5). Further functional studies focused on the analysis evaluating the putative effects of (genotype-related) alterations in FceRI expression level on IgE production/ levels seem to be required to reveal the underlying mechanism(s).

Human Neutrophil Peptides and Phagocytic Deficiency in Bronchiectatic Lungs

A well-known clinical paradox is that severe bacterial infections persist in the lungs of patients with cystic fibrosis (CF) despite the abundance of polymorphonuclear neutrophils (PMN) and the presence of a high concentration of human neutrophil peptides (HNP), both of which are expected to kill the bacteria but fail to do so. The mechanisms remain unknown. This study examined several possible mechanisms to understand this paradox. PMN were isolated from sputum and blood of subjects with and without CF or non-CF bronchiectasis for phagocytic assays. HNP isolated from patients with CF were used to stimulate healthy PMN followed by phagocytic tests. PMN isolated from the sputum of the bronchiectatic patients display defective phagocytosis that correlated with high concentrations of HNP in the lung. When healthy PMN were incubated with HNP, decreased phagocytic capacity was observed in association with depressed surface Fc gamma RIII, actin-filament remodeling, enhanced intracellular Ca(2+), and degranulation. Treatment of PMN with an intracellular Ca(2+) blocker or alpha1-proteinase inhibitor to attenuate the activity of HNP largely prevented the HNP-induced phagocytic deficiency. Intratracheal instillation of HNP in Pallid mice (genetically deficient in alpha1-proteinase inhibitor) resulted in a greater PMN lung infiltration and phagocytic deficiency compared with wild-type mice. HNP or PMN alone exert antimicrobial ability, which was lost as a result of their interaction. These effects of HNP may help explain the clinical paradox seen in patients with inflammatory lung diseases, suggesting HNP as a novel target for clinical therapy.

Effect of LDL-apheresis on plasma lipids, chitotriosidase and anti-oxLDL antibodies in heterozygous familial hypercholes-terolemia

Forty four consecutive subjects aged 29-58 years (21 males and 23 females) with a clinical diagnosis of heterozygous familial hypercholesterolemia period- ically treated every 30 days with LDL-apheresis for statin resistance, were enrolled in this study. A lipid profile was obtained immediately before starting LDL-apheresis, a second profile was obtained within four hours after LDL-apheresis. Chit activity and anti-oxLDL levels were determined with appropriate methods in all patients before and after LDL- apheresis. Total cholesterol, LDL-cholesterol, HDL- cholesterol and triglycerides decreased significantly after LDL-apheresis, while the variations of Chit activity and anti-oxLDL were not significant after LDL-apheresis. The correlation between Chit and total cholesterol was negative (r= -0.44 and -0.50 res- pectively) before and after LDL-apheresis as between Chit and LDL-cholesterol (r= -0.45 and -0.55 respectively). Anti-oxLDL concentration before and after LDL-apheresis positively correlated with Chit activity (r= 0.52 and r = 0.63 respectively), negatively with total cholesterol (r= -0.33 and r = -0.35 res- pectively) and with LDL (r = -0.32 and r = -0.21 respectively). We think that removing LDL with LDL-apheresis the anti-oxLDL/oxLDL ratio could increase and the excess of anti-oxLDL could induce macrophage activation through the surface Fc receptors. Alternatively with high levels of LDL- cholesterol, the deposition of foam cells represent the characteristic evolution of atherosclerosis process. Macrophage activation in the heterozygous familial

Effect of LDL-apheresis on plasma lipids, chitotriosidase and anti-oxLDL antibodies in heterozygous familial hypercholes-terolemia

Forty four consecutive subjects aged 29-58 years (21 males and 23 females) with a clinical diagnosis of heterozygous familial hypercholesterolemia periodically treated every 30 days with LDL-apheresis for statin resistance, were enrolled in this study. A lipid profile was obtained immediately before starting LDL-apheresis, a second profile was obtained within four hours after LDL-apheresis. Chit activity and anti-oxLDL levels were determined with appropriate methods in all patients before and after LDL- apheresis. Total cholesterol, LDL-cholesterol, HDL- cholesterol and triglycerides decreased significantly after LDL-apheresis, while the variations of Chit activity and anti-oxLDL were not significant after LDL-apheresis. The correlation between Chit and total cholesterol was negative (r= –0.44 and –0.50 res- pectively) before and after LDL-apheresis as between Chit and LDL-cholesterol (r= –0.45 and –0.55 respectively). Anti-oxLDL concentration before and after LDL-apheresis positively correlated with Chit activity (r= 0.52 and r = 0.63 respectively), negatively with total cholesterol (r= –0.33 and r = –0.35 res- pectively) and with LDL (r = –0.32 and r = –0.21 respectively). We think that removing LDL with LDL-apheresis the anti-oxLDL/oxLDL ratio could increase and the excess of anti-oxLDL could induce macrophage activation through the surface Fc receptors. Alternatively with high levels of LDL- cholesterol, the deposition of foam cells represent the characteristic evolution of atherosclerosis process. Macrophage activation in the heterozygous familial hypercholesterolemia could represent an attempt for re-modeling the vessel wall, reducing the growth of lipid plaques.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon‐α via toll‐like receptor 9

High-affinity IgE receptor (Fc epsilon RI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro-inflammatory cytokines (IL-6, TNF-alpha) following Fc epsilon RI stimulation - a mode of activation that simultaneously reduces expression of Toll-like receptor 9 (TLR9). Whether or not TLR9 and/or Fc epsilon RI levels and their function on dendritic cells relate to allergic status is unknown. The aim of this study is to compare the innate (TLR9-mediated) immune response of human pDCs to TLR9 and Fc epsilon RI alpha receptor expression in allergic and non-allergic subjects. Basophil-depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non-allergic subjects. Intracellular TLR9 and surface Fc epsilon RI alpha expression in blood dendritic cell antigen-2-positive cells were determined by flow cytometry. Activating anti-IgE antibody, anti-Fc epsilon RI alpha antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA. No difference in the frequency of pDCs was detected among allergic (n=9) vs. non-allergic (n=11) subjects (P=0.261). While there was also no difference in the baseline expression of TLR9, pDCs from allergic subjects produced sixfold less IFN-alpha when stimulated with CpG (P=0.002). Conversely, there was higher Fc epsilon RI alpha expression (P=0.01) on the pDCs of allergic subjects. Impaired TLR9-dependent immune responses in human pDCs are associated with allergic status and inversely correlated with Fc epsilon RI alpha expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG-based immunotherapy.

Downregulation of FceRI Beta Chain During IgE Signaling

Mast cells are critical players in allergic disease mediated by IgE and FcεRI interactions. In mice, FcεRI functions as a tetramer, composed of an IgE binding alpha subunit, two ITAM bearing gamma subunits, and a signal enhancing/amplifying beta subunit. Little is known about beta chain regulation. We activated mast cells with IgE + antigen and determined the resulting effects on beta chain mRNA, protein levels and surface expression. IgE crosslinkage inhibited beta chain mRNA expression, with a nadir at 5 hours post‐stimulation. This was matched by reduced protein and surface expression over the following 24 hours. Similar effects were observed in either mouse bone marrow‐derived or human skin‐derived mast cells. We were able to classify the beta chain mRNA regulation pathway as primarily involving Syk, PI3K, and NFκB. Monomeric IgE is known to greatly increase surface Fc□RI expression. Interestingly, we found that IgE crosslinkage returned membrane Fc□RI to basal levels in cells previously incubated with monomeric IgE. These data suggest that FceRI signaling not only activates mast cells but may also convey a homeostatic signal returning FceRI expression to a reduced basal level, perhaps to suppress continued signaling. The role of beta chain regulation in mast cell homeostasis offers this subunit as a possible therapeutic target in allergic disease. Supported by NIH grant 1R01 AI59638.

Chapter 3 New Insights on Mast Cell Activation via the High Affinity Receptor for IgE

Mast cells are innate immune cells that function as regulatory or effector cells and serve to amplify adaptive immunity. In adaptive immunity these cells function primarily through cell surface Fc receptors that bind immunoglobulin antibodies. The dysregulation of their adaptive role makes them central players in allergy and asthma. Upon encountering an allergen (antigen), which is recognized by immunoglobulin E (IgE) antibodies bound to the high affinity IgE receptor (FcepsilonRI) expressed on their cell surface, mast cells secrete both preformed and newly synthesized mediators of the allergic response. Blocking of these responses is an objective in therapeutic intervention of allergic diseases. Thus, understanding the mechanisms by which antigens elicit mast cell activation (via FcepsilonRI) holds promise toward identifying therapeutic targets. Here we review the most recent advances in understanding antigen-dependent mast cell activation. Specifically, we focus on the requirements for FcepsilonRI activation, the regulation of calcium responses, co-stimulatory signals in FcepsilonRI-mediated mast cell activation and function, and how genetics influences mast cell signaling and responses. These recent discoveries open new avenues of investigation with therapeutic potential.

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.