Fc gamma RIIa, not Fc gamma RIIb, is constitutively and functionally expressed on skin-derived human mast cells

The expression of FcgammaR by human skin-derived mast cells of the MC(TC) type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of beta-hexosaminidase, PGD(2), leukotriene C(4) (LTC(4)), IL-5, IL-6, IL-13, GM-CSF, and TNF-alpha. FcgammaRIIa was consistently detected along with FcepsilonRI at the mRNA and protein levels; FcgammaRIIc was sometimes detected only by RT-PCR; but FcgammaRIIb, FcgammaRI, and FcgammaRIII mRNA and protein were not detected. FcgammaRIIa-specific mAb caused skin MC(TC) cells to degranulate and secrete PGD(2), LTC(4), GM-CSF, IL-5, IL-6, IL-13, and TNF-alpha in a dose-dependent fashion. FcepsilonRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC(4), which was not released by this agonist. Simultaneous but independent cross-linking of FcepsilonRI and FcgammaRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MC(TC) cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding

Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.     

Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions.

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure.     

The structure and expression of the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2R).

A cDNA clone encoding the receptor for guinea pig immunoglobulin G was isolated from a guinea pig peritoneal macrophage cDNA library. The cloned cDNA encoded 271 amino acids containing an N-terminal signal sequence. The deduced amino acid sequence is most homologous to murine Fc gamma RII beta 2. The receptor protein could be expressed in COS-7 and L cells transfected with the cDNA, suggesting that the expression of this receptor does not require the co-expression of a second chain such as gamma chain of Fc epsilon RI or CD3 zeta chain. The transformant L cells showed the binding to both the guinea pig IgG1 and IgG2 antibodies complexed with antigen, indicating that the cDNA we cloned was the one for guinea pig Fc gamma 1/gamma 2R.     

Soluble antigens of IgG receptor Fc gamma RIII in human seminal plasma

Human seminal plasma (SP) has been shown to affect several immunologic reactions in vitro. This might be due in part to the presence of proteins that specifically bind the Fc domain of IgG. By using mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 we showed that the Fc binding of SP is associated with a molecule that antigenically resembles Fc gamma RIII. This molecule manifests specific affinity for solid phase-coupled IgG-Fc, and appears not be be cell membrane-associated. When compared with serum or blood plasma, its highest concentration was found in SP. Western blot analysis of SP performed with mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 showed distinct bands at approximately 70 and 35 kDa, which contrasts with the broad area of electrophoretic mobility reported for membrane-bound Fc gamma RIII. These molecules in SP could influence maternal immune responses to paternal Ag during fertilization and pregnancy.     

Cloned and expressed human Fc receptor for IgG mediates anti-CD3-dependent lymphoproliferation

We have utilized gene transfer experiments to investigate the role of a human monocyte receptor for IgG (Fc gamma RII) in mouse IgG1 anti-CD3 (Leu 4)-induced lymphoproliferation in vitro. Mouse Ltk- cells expressing human Fc gamma RII or a mutant of Fc gamma RII lacking the entire cytoplasmic domain of the receptor mediate anti-CD3-induced lymphoproliferation in cultures of adherent cell-depleted human PBMC. Expression of an Fc gamma RII mutant lacking transmembrane and cytoplasmic domains (soluble Fc gamma RII) in COS7 cells yielded a secreted receptor which retained affinity for IgG, even in the absence of the mutant receptor's N-linked oligosaccharides. Soluble Fc gamma RII inhibits rosette formation by human IgG-sensitized RBC and the Fc gamma RII-bearing cell line K562, but does not sitmulate anti-CD3-induced lymphoproliferation under the conditions tested.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.     

The two binding-site models of human IgG binding Fc gamma receptors

Fc receptors (FcR) are immunoglobulin-binding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and with their ligands. Recent data on the structure of IgG binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity Fc gamma RI possess one active binding site specific for contact residues that is located at the N-proximal end of the CH2 domain and is able to mediate both binding and signal transfer. The low-affinity Fc gamma RIII has two active binding sites: the CH3 domain-specific site, which mediates only binding; and the CH2 domain-specific site, which is responsible for binding and signaling. Similarly, the low-affinity Fc gamma RII on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc gamma RII on B cells correlates with the cell cycle.     

Convergence of Fc gamma receptor IIA and Fc gamma receptor IIIB signaling pathways in human neutrophils

Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcgammaRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcgammaRIIIB. Cross-linking of FcgammaRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcgammaRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcgammaRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcgammaRIIA colocalizes with cross-linked FcgammaRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcgammaRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcgammaRIIIB signaling is conducted by molecules of FcgammaRIIA that are recruited to protein/lipid domains induced by clustered FcgammaRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs.     

The valence for ligand of the human mononuclear phagocyte 72 kD high-affinity IgG Fc receptor is one

The valence for ligand of the 72 kD high-affinity IgG FcR present on human mononuclear phagocytes was evaluated. Lysates of U937 cells whose high-affinity FcR had been saturated with equivalent quantities of 125I-IgG1 kappa and unlabeled IgG1 lambda or with 125I-IgG1 lambda and unlabeled IgG1 kappa were incubated with Sepharose-anti-kappa. Eighty-nine percent of the applied 125I-IgG1 kappa was bound, whereas 0.35% of the applied 125I-IgG1 lambda bound (mean of two experiments), indicating that if the receptors are occupied with ligand, the receptors bind only one ligand molecule at a time. Two experiments were performed to show that the receptors were ligand-occupied. First, a monoclonal antibody directed against the 72 kD FcR (FcRmab32) was added to lysates of U937 cells saturated with equal quantities of 125I-IgG1 lambda and IgG1 kappa. This anti-FcR antibody caused a dose-dependent sevenfold increase in the amount of 125I-IgG1 lambda bound to the anti-kappa immunoadsorbent (presumably by cross-linking receptors bearing 125I-IgG1 lambda with receptors bearing IgG1 kappa), whereas monoclonal antibodies (MMA and IV3) directed against two other determinants on U937 caused no such increase. In the second experiment, Sepharose-FcRmab32 adsorbed 60% of the 125I-IgG1 kappa and 46% of the 125I-IgG1 lambda applied in a U937 lysate (bearing high-affinity FcR), whereas only 3% of 125I-IgG1 kappa and 6% of 125I-IgG1 lambda applied in a K562 lysate (bearing no high-affinity FcR) were adsorbed. We interpret these data to indicate that in detergent solution the valency of the high-affinity FcR on U937 cells is one.     

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.     

Affinity of human IgG subclasses to mouse Fc gamma receptors

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to FcγR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcγRs is available. The orthologous mouse and human FcγRs share roughly 60–70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcγR. Human IgGs bound to mouse FcγR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcγRI>>FcγRIV>FcγRIII>FcγRIIb. This suggests human IgG subclasses to have similar relative FcγR-mediated biological activities in mice.     

Neonatal Fc receptor expression in macrophages is indispensable for IgG homeostasis

ABSTRACT

The maintenance of the homeostasis of immunoglobulin G (IgG) represents a fundamental aspect of humoral immunity that has direct relevance to the successful delivery of antibody-based therapeutics. The ubiquitously expressed neonatal Fc receptor (FcRn) salvages IgG from cellular degradation following pinocytic uptake into cells, conferring prolonged in vivo persistence on IgG. However, the cellular sites of FcRn function are poorly defined. Pinocytic uptake is a prerequisite for FcRn-mediated IgG salvage, prompting us to investigate the consequences of IgG uptake and catabolism by macrophages, which represent both abundant and highly pinocytic cells in the body. Site-specific deletion of FcRn to generate mice harboring FcRn-deficient macrophages results in IgG hypercatabolism and ~threefold reductions in serum IgG levels, whereas these effects were not observed in mice that lack functional FcRn in B cells and dendritic cells. Consistent with the degradative activity of FcRn-deficient macrophages, depletion of these cells in FcRn-deficient mice leads to increased persistence and serum levels of IgG. These studies demonstrate a pivotal role for FcRn-mediated salvage in compensating for the high pinocytic and degradative activities of macrophages to maintain IgG homeostasis.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Production of TNF-alpha by human V gamma 9V delta 2 T cells via engagement of Fc gamma RIIIA, the low affinity type 3 receptor for the Fc portion of IgG, expressed upon TCR activation by nonpeptidic antigen

Human lymphocytes expressing the gammadelta TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vgamma9Vdelta2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vgamma9Vdelta2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vgamma9Vdelta2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vgamma9Vdelta2 T cells express FcgammaRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vgamma9Vdelta2 T cells. Indeed, ligation of CD16 on Vgamma9Vdelta2 T cells leads to TNF-alpha production. This TNF-alpha production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vgamma9Vdelta2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcgammaRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vgamma9Vdelta2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.