Rcan1 negatively regulates FcεRI-mediated signaling and mast cell function

Aggregation of the high affinity IgE receptor (FcεRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off FcεRI-mediated mast cell activation. FcεRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor κB activation, increased cytokine production, and enhanced immunoglobulin E–mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced FcεRI-mediated cytokine production. Rcan1 deficiency also led to increased FcεRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in FcεRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in FcεRI signaling.     

IgE Enhances Mouse Mast Cell FcεRI Expression In Vitro and In Vivo: Evidence for a Novel Amplification Mechanism in IgE-dependent Reactions

The binding of immunoglobulin E (IgE) to high affinity IgE receptors (FcεRI) expressed on the surface of mast cells primes these cells to secrete, upon subsequent exposure to specific antigen, a panel of proinflammatory mediators, which includes cytokines that can also have immunoregulatory activities. This IgE- and antigen-specific mast cell activation and mediator production is thought to be critical to the pathogenesis of allergic disorders, such as anaphylaxis and asthma, and also contributes to host defense against parasites. We now report that exposure to IgE results in a striking (up to 32-fold) upregulation of surface expression of FcεRI on mouse mast cells in vitro or in vivo. Moreover, baseline levels of FcεRI expression on peritoneal mast cells from genetically IgE-deficient (IgE −/−) mice are dramatically reduced (by ~83%) compared with those on cells from the corresponding normal mice. In vitro studies indicate that the IgE-dependent upregulation of mouse mast cell FcεRI expression has two components: an early cycloheximide-insensitive phase, followed by a later and more sustained component that is highly sensitive to inhibition by cycloheximide. In turn, IgE-dependent upregulation of FcεRI expression significantly enhances the ability of mouse mast cells to release serotonin, interleukin-6 (IL-6), and IL-4 in response to challenge with IgE and specific antigen. The demonstration that IgE-dependent enhancement of mast cell FcεRI expression permits mast cells to respond to antigen challenge with increased production of proinflammatory and immunoregulatory mediators provides new insights into both the pathogenesis of allergic diseases and the regulation of protective host responses to parasites.     

Mast Cells Can Secrete Vascular Permeability Factor/ Vascular Endothelial Cell Growth Factor and Exhibit Enhanced Release after Immunoglobulin E–dependent Upregulation of Fcε Receptor I Expression

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcε receptor I (FcεRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the FcεRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of FcεRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.     

Serum IgE clearance is facilitated by human FcεRI internalization

The high-affinity IgE receptor FcεRI is constitutively expressed in mast cells and basophils and is required for transmitting stimulatory signals upon engagement of IgE-bound allergens. FcεRI is also constitutively expressed in dendritic cells (DCs) and monocytes in humans; however, the specific functions of the FcεRI expressed by these cells are not completely understood. Here, we found that FcεRI expressed by human blood DC antigen 1–positive (BDCA1⁺) DCs and monocytes, but not basophils, traffics to endolysosomal compartments under steady-state conditions. Furthermore, IgE bound to FcεRI on BDCA1⁺ DCs was rapidly endocytosed, transported to the lysosomes, and degraded in vitro. IgE injected into mice expressing human FcεRIα (FCER1A-Tg mice) was endocytosed by conventional DCs and monocytes, and endocytosis was associated with rapid clearance of circulating IgE from these mice. Importantly, this rapid IgE clearance was dependent on monocytes or DCs but not basophils. These findings strongly suggest that constitutive internalization of human FcεRI by DCs and monocytes distinctively contributes to serum IgE clearance.     

Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation

T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation.T cell, or transmembrane, immunoglobulin domain and mucin domain (Tim-3) is a type I membrane protein expressed on a variety of innate and adaptive immune cell types. Tim-3 is often referred to as a checkpoint receptor due to its apparent inhibitory function on T cells and its association with activation-induced T cell exhaustion in tumors and chronic viral infection (Sánchez-Fueyo et al., 2003; Jones et al., 2008; Fourcade et al., 2010; Jin et al., 2010; Sakuishi et al., 2010). Recent studies, however, suggest a more nuanced picture of Tim-3 function in T cells, depending on the setting, e.g., acute versus chronic stimulation (Ferris et al., 2014; Gorman and Colgan, 2014). In addition to CD4 and CD8 T cells, Tim-3 is also expressed on other immune cell types, such as NK cells, macrophages, DCs, and mast cells, but its function on these cell types is less clear. Tim-3 blockade was shown to enhance macrophage function in response to sepsis (Yang et al., 2013), and also to regulate antigen (Ag) presentation by DCs, partly through Btk and c-Src (Maurya et al., 2014). On the other hand, Tim-3 expression on monocytes infiltrating the CNS during EAE was shown to promote inflammation (Anderson et al., 2007).Mast cells are first-line defenders against allergens and invading pathogens as a result of their proximity to the external environment. Cross-linking of IgE bound to the high-affinity IgE receptor FcεRI by Ag leads to the release of preformed mediators and de novo synthesis of proinflammatory and antiinflammatory mediators and cytokines, which together serve to regulate hypersensitivity, autoimmunity, cardiovascular disease, and tumor progression (Kalesnikoff and Galli, 2008). In addition to their well-known pathological roles in allergic responses, mast cells also contribute to defense against bacteria, helminthes, and tumors (Abraham and St John, 2010). It was reported that mast cells constitutively express cell surface Tim-3, and that cross-linking of Tim-3 could enhance cytokine production of IgE-sensitized and Ag-stimulated BM-derived mast cells (BMMCs) and peritoneal mast cells (pMCs) without affecting degranulation (Nakae et al., 2007). TGF-β has been shown to up-regulate expression of Tim-3 in tumor-infiltrating mast cells and a human mast cell line, through a mitogen-activated protein kinase Erk-kinase (MEK)–dependent pathway (Wiener et al., 2006; Yoon et al., 2011). Although previous data suggest that Tim-3 is a positive regulator of mast cell activation, the molecular mechanisms behind the contribution of Tim-3 to mast cell function are still unknown. Importantly, there was until now no genetic evidence addressing the function of Tim-3 in these cells. Given the important role of mast cells as sentinels in both allergic and nonallergic diseases, it is of interest to explore Tim-3 activity on this cell type and how antibody (Ab) modulation can affect its function.Here, we demonstrate through multiple approaches that Tim-3 functions to enhance proximal FcεRI signaling in mast cells. Cross-linking of Tim-3 with multiple independent antibodies enhanced mast cell degranulation and cytokine release in a dose-dependent manner. Acute knock-down or genetic deficiency of Tim-3 rendered mast cells less responsive to Ag cross-linking of FcεRI, resulting in decreased degranulation and cytokine production. The cytoplasmic tail of Tim-3 was required for co-stimulatory signal transduction in mast cells, together with FcεRI signaling pathways. This was shown in part with the use of recently reported Nur77-GFP transgenic models, which have not previously been used for the study of FcεRI signaling. Collectively, our data demonstrate that Tim-3 acts at a receptor-proximal level to intensify activation of FcεRI-dependent signaling pathways upon Ag cross-linking, while maintaining the threshold for negative signaling of Lyn.     

IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking

Although it has long been hypothesized that allergen immunotherapy inhibits allergy, in part, by inducing production of IgG Abs that intercept allergens before they can cross-link mast cell FcεRI-associated IgE, this blocking Ab hypothesis has never been tested in vivo. In addition, evidence that IgG-allergen interactions can induce anaphylaxis by activating macrophages through FcγRIII suggested that IgG Ab might not be able to inhibit IgE-mediated anaphylaxis without inducing anaphylaxis through this alternative pathway. We have studied active and passive immunization models in mice to approach these issues and to determine whether any inhibition of anaphylaxis observed was a direct effect of allergen neutralization by IgG Ab or an indirect effect of cross-linking of FcεRI to the inhibitory IgG receptor FcγRIIb. We demonstrate that IgG Ab produced during the course of an immune response or administered passively can completely suppress IgE-mediated anaphylaxis; that these IgG blocking Abs inhibit IgE-mediated anaphylaxis without inducing FcγRIII-mediated anaphylaxis only when IgG Ab concentration is high and challenge allergen dose is low; that allergen epitope density correlates inversely with the allergen dose required to induce both IgE- and FcγRIII-mediated anaphylaxis; and that both allergen interception and FcγRIIb-dependent inhibition contribute to in vivo blocking Ab activity.     

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.     

Tespa1 negatively regulates FcεRI-mediated signaling and the mast cell–mediated allergic response

Antigen-mediated cross-linking of IgE on mast cells triggers a signaling cascade that results in their degranulation and proinflammatory cytokine production, which are key effectors in allergic reactions. We show that the activation of mast cells is negatively regulated by the newly identified adaptor protein Tespa1. Loss of Tespa1 in mouse mast cells led to hyper-responsiveness to stimulation via FcεRI. Mice lacking Tespa1 also displayed increased sensitivity to IgE-mediated allergic responses. The dysregulated signaling in KO mast cells was associated with increased activation of Grb2-PLC-γ1-SLP-76 signaling within the LAT1 (linker for activation of T cells family, member 1) signalosome versus the LAT2 signalosome. Collectively, these findings show that Tespa1 orchestrates mast cell activation by tuning the balance of LAT1 and LAT2 signalosome assembly.Allergic responses are Th2- and IgE-mediated immune responses that evolved to protect against environmental substances and parasites. However, these responses can also lead to allergy when inadvertently activated by noninfectious environmental antigens and cause harmful symptoms such as itching, inflammation, or tissue injury. As to our current understanding, mast cells are at the center of allergic responses (Galli et al., 2005; Bischoff, 2007). Mast cells are BM-derived hematopoietic cells localized under surfaces exposed to the external environment, such as the skin, airways, and intestine. They function as sentinel cells in host defense reactions including immediate hypersensitivity responses and allergic responses (Galli et al., 2005). Activated mast cells trigger allergic responses by releasing preformed granule-associated chemical mediators, producing multiple cytokines and chemokines, and secreting de novo synthesized arachidonic acid metabolites and various proteins (Metcalfe et al., 1997; Bischoff, 2007).Mast cells recognize antigens via IgE and specific Fc receptors, termed FcεRI. Binding of multivalent antigen to FcεRI-bound IgE induces receptor aggregation and triggers mast cell activation (Kinet, 1999; Siraganian, 2003). FcεRI is expressed on the surface of mast cell as a tetrameric complex consisting of an IgE-binding α subunit, a signal-modulating β subunit, and two signal-transduction γ subunits (Kraft and Kinet, 2007). The signaling cascades elicited by FcεRI aggregation in mast cells have been extensively studied (Kalesnikoff and Galli, 2008). Briefly, the conserved immunoreceptor tyrosine-based activation motifs (ITAMs) within the cytoplasmic tails of the β and γ subunits are rapidly phosphorylated upon FcεRI stimulation in a Lyn-dependent manner (Garman et al., 1999; Kinet, 1999). Then another tyrosine kinase, Syk, binds to the tyrosine-phosphorylated ITAMs and initiates the principal axis pathway that includes Grb2, PLC-γ1, and SLP-76 (Gilfillan and Tkaczyk, 2006; Rivera and Gilfillan, 2006), which ultimately lead to the activation of downstream signaling cascades including mitogen-activated protein kinases, protein kinase C pathways, and calcium flux.During this signaling process, two similar adaptor proteins, linker for activation of T cells family, member 1 (LAT1) and LAT2, are both phosphorylated, resulting in the formation of two complementary and competitive signalosome complexes known as the LAT1 signalosome and the LAT2 signalosome. Both of these membrane adaptors recruit principal axis–related molecules including Grb2, SOS, PLC-γ1, SLP-76, and Vav1. The essential role of LAT1 in mast cell activation is clear because LAT1 deficiency markedly attenuates mast cell responsiveness. However, the function of the LAT2 signalosome in RcεRI signaling is still an enigma to many immunologists. In general, LAT2 may down-regulate antigen-mediated signaling in mast cells by either competing with LAT1 for a limited pool of signaling molecules or recruiting of phosphatases and ubiquitin-ligases such as SHP-1 and c-Cbl (Gu et al., 2001; Brdicka et al., 2002; Volná et al., 2004; Rivera, 2005). On the contrary, LAT2 has also been found to compensate for the loss of LAT1 in the Lat1^−/− background (Volná et al., 2004; Zhu et al., 2004), either through activation of the principal axis pathway with lower efficiency, or by recruiting another adaptor protein, Gab2, to facilitate Fyn-Gab2-PI3K-Akt signaling termed the complementary axis (Gilfillan and Tkaczyk, 2006; Kalesnikoff and Galli, 2008; Alvarez-Errico et al., 2009).The current model of LAT1 and LAT2 activation raises another unavoidable question of what determines the recruitment of the same pool of signaling molecules to two different adaptor proteins, namely LAT1 and LAT2, with such high similarity in sequence and structure. It is still unclear whether there is a shunt to modulate the allocation of the signal molecules to these different signalosomes. If such a regulator were found, it would be an attractive target for pharmacological intervention for fine-tuning allergic responses and the treatment of mast cell–driven disorders such as allergic asthma.Recently, we identified a novel adaptor protein, Tespa1 (thymocyte-expressed, positive selection-associated 1), which plays a critical role in the positive selection of thymocytes (Wang et al., 2012). KO mice have fewer mature thymic CD4⁺ and CD8⁺ T cells due to impaired thymocyte development. We later demonstrated that Tespa1 associates with the TCR signaling components Grb2 and PLC-γ1, which in turn facilitates TCR signal transduction (Wang et al., 2012). Other than in T cells, Tespa1 is also highly expressed in mast cells, as demonstrated by both real-time PCR analysis and the BioGPS gene expression atlas database (Su et al., 2004), suggesting a potential role of Tespa1 in mast cells.To our great surprise, KO mast cells showed hyper-responsiveness to FcεRI stimulation, which is evidenced by enhanced cytokine production, degranulation, calcium mobilization, and elevated activation of downstream signaling pathways. Consistently, KO mice developed exacerbated anaphylactic response and allergic asthma. Our data revealed an unexpected function of Tespa1 as a negative regulator of FcεRI-mediated mast cell activation through fine-tuning of LAT1 and LAT2 signalosome assembling.     

Type II and III Receptors for Immunoglobulin G (IgG) Control the Presentation of Different T Cell Epitopes from Single IgG-complexed Antigens

T cell receptors on CD4⁺ lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcγRIIb2 and FcγRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcγRIIb2 or FcγRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcγRIIb2 and FcγRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcγRIII, but not through FcγRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcγRIIb2 by addition of the FcγRIII-associated γ chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the γ chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.Antigen receptors on CD4⁺ helper T lymphocytes recognize short peptides presented by class II molecules of MHC (1, 2). Antigenic peptides are generated by proteolytic degradation in the endocytic pathway, where they associate with MHC class II molecules. Only a very limited set of peptides among all the potential peptides is actually loaded onto MHC class II molecules in APCs (3). The mechanisms underlying the selection of peptides for MHC class II–restricted antigen presentation are yet unclear. However, we know that two independent complex processes of intracellular transport towards endosomes are crucial for MHC class II–restricted antigen presentation: the traffic of MHC class II molecules and the delivery of antigens (4, 5).MHC class II intracellular transport has been analyzed in detail. Newly synthesized MHC class II molecules reach endosomes, either directly from the trans-golgi network or after a short appearance at the plasma membrane, in association to the invariant (Ii)¹ chain (5). Ii is then degraded and the class II–associated Ii chain peptide (CLIP) is replaced by an antigenic peptide under the control of HLA-DM (6). It has recently become clear that an alternative, Ii chain–independent pathway for MHC class II transport to endosomes also exists. Indeed, MHC class II molecules may reach the endocytic pathway from the cell surface by endocytosis (7), due to internalization signals present in the cytosolic domain of the MHC class II β chain (8). Newly synthesized and recycling MHC class II molecules may present different peptides (9). Accordingly, we have previously shown that different antigen receptors may also selectively target antigens for presentation by either of these MHC class II presentation pathways (10).In contrast, very little is known about the endocytic transport of antigen receptors. Physiologically, antigens are delivered to the endocytic pathway by different families of receptors, which strongly increase the efficiency of MHC class II–restricted antigen presentation (11). In B lymphocytes, surface Ig mediates both cell activation and the uptake of specific antigens (12), while the expression of a particular endocytosis-deficient receptor for the Fc portion of IgG (FcγRIIb1) prevents efficient presentation of irrelevant IgG-complexed antigens (13). Interestingly, the epitope specificity of surface Ig positively and negatively influences the presentation of various T cell epitopes (14, 15).In monocytes and dendritic cells, receptors for IgG (FcγRs), in addition to mannose receptors, mediate antigen internalization and strongly increase the efficiency of presentation to specific T cells (16). Two different FcγRs, type IIb2 and type III (FcγRIIb2 and FcγRIII) are expressed in dendritic cells. FcγRIIb2 is a monomeric receptor that, like FcγRIIb1, mediates the inhibition of cell activation when cocrosslinked to surface Ig (13). FcγIII is an heterotrimer consisting of an α chain and a dimer of γ chains, which couple the receptor to cytoplasmic effectors of signal transduction (17).To individually analyze the function of these two receptors, we expressed them by cDNA transfection into an FcγR-negative B cell lymphoma cell line (13, 18, 19). We have previously shown that the amino acid sequence, called immunoreceptor tyrosine kinase activation motif (ITAM), in the FcγIII-associated γ chain responsible for cell activation is also involved in receptor internalization (18, 19). In addition, mutation of either of the two tyrosine residues in the ITAM of the γ chain inhibits both cell activation and ligand internalization (19). Thus, in contrast to FcγRIIb2, which contains no ITAM, is not tyrosine phosphorylated, and does not induce cell activation, FcγRIII associates with and activates cytosolic tyrosine kinases after engagement by its ligand (18). In addition to this functional diversity of the two receptors, their expression is also selectively regulated, since TNF-α and IFN-γ increase the expression of FcγRIII and inhibit that of FcγRIIb2 in monocytes (20).The diversity in the signals required of FcγRIIb2 and FcγRIII internalization, as well as the differential regulation of their expression in APCs, suggest that the two receptors may have different antigen-presenting functions. We here examine the ability of FcγRIIb2 and FcγRIII to induce the presentation of various T cell epitopes from two different antigens, CI λ repressor and hen egg lysozyme (HEL). Internalization of antigen–antibody complexes through FcγRIII induced the efficient presentation of all the T cell epitopes tested, whereas FcγRIIb2 only induced the presentation of a few. Point mutation of leucine 35 to alanine (L35A) in the cytoplasmic tail of FcγRIII γ chain blocked signal transduction without affecting the internalization of immune complexes. This mutation also blocked the presentation of the epitopes that were only presented after internalization by FcγRIII. In contrast, the presentation of all the epitopes that were efficiently generated after internalization by FcγRIIb2 was not affected. Thus, the nature of the receptor that mediates antigen internalization influences the selection of the epitopes presented to T lymphocytes.     

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.Activating Fc γ receptors (FcγRs) stimulate immune cell effector mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP), which combine to facilitate antibody-mediated tumor cell killing (Nimmerjahn and Ravetch, 2008; Hogarth and Pietersz, 2012). The importance of FcγR-mediated immune effector cell function has been demonstrated in preclinical efficacy studies for antibodies targeting a range of tumor cell–expressed receptors, including trastuzumab (HER2) and rituximab (CD20; Clynes et al., 2000; Nimmerjahn and Ravetch, 2012). The inhibitory FcγR, FcγRIIB, functions to modulate activating FcγR-mediated effector mechanisms in immune cells that coexpress both FcγR classes, such as macrophages and dendritic cells. FcγRIIB has recently been implicated in augmenting antibody-mediated receptor forward signaling through a mechanism of cross-linking in target cells expressing the TNF receptor (TNFR) family members TNFRSF10, TNFRSF10B (DR4 and DR5, respectively), and TNFRSF5 (CD40; Wilson et al., 2011; Li and Ravetch, 2012). It remains unclear what contribution FcγR biology has in the modality of antibody therapeutics that target other cell surface receptors. In particular, the emerging clinical benefit of agonistic antibodies targeting the T cell–APC interface raises the possibility that FcγR coengagement may contribute to their in vivo mechanism of action (Mellman et al., 2011).Preclinical studies in mice using agonistic antibodies targeted to glucocorticoid-induced TNFR-related protein (GITR)—a costimulatory TNFR expressed by regulatory and activated T cells—have shown compelling antitumor activity in syngeneic mouse tumor models (Turk et al., 2004; Ko et al., 2005). In vitro, stimulation of GITR with agonist antibodies can induce forward signaling into T cells, which promotes proliferation and cytokine production (Kanamaru et al., 2004; Ronchetti et al., 2007). In vivo, several mechanisms have been proposed to contribute to the antitumor activity of antibodies targeting GITR; however, the current paradigm stipulates that agonist properties of these antibodies promotes cytotoxic effector T cell generation, while dampening the immunosuppressive effects by FoxP3⁺ CD4⁺ T reg cells (Ronchetti et al., 2012; Schaer et al., 2012). The recent findings that antibodies targeted to TNFR family members require FcγRIIB interaction for their in vivo activities led us to explore a common mechanism for antibodies targeting TNFRs expressed by T cells, using GITR to test this paradigm.     

A New Class of Human Mast Cell and Peripheral Blood Basophil Stabilizers that Differentially Control Allergic Mediator Release

Treatments for allergic disease block the effects of mediators released from activated mast cells and blood basophils. A panel of fullerene derivatives was synthesized and tested for their ability to preempt the release of allergic mediators in vitro and in vivo. The fullerene C₇₀‐tetraglycolic acid significantly inhibited degranulation and cytokine production from mast cells and basophils, while C₇₀‐tetrainositol blocked only cytokine production in mast cells and degranulation and cytokine production in basophils. The early phase of FcɛRI inhibition was dependent on the blunted release of intracellular calcium stores, elevations in reactive oxygen species, and several signaling molecules. Gene microarray studies further showed the two fullerene derivatives inhibited late phase responses in very different ways. C₇₀‐tetraglycolic acid was able to block mast cell‐driven anaphylaxis in vivo, while C₇₀‐tetrainositol did not. No toxicity was observed with either compound. These findings demonstrate the biological effects of fullerenes critically depends on the moieties added to the carbon cage and suggest they act on different FcɛRI‐specific molecules in mast cells and basophils. These next generation fullerene derivatives represent a new class of compounds that interfere with FcɛRI signaling pathways to stabilize mast cells and basophils. Thus, fullerene‐based therapies may be a new approach for treating allergic diseases. Clin Trans Sci 2010; Volume 3: 158–169     

Fcγ receptors enable anticancer action of proapoptotic and immune-modulatory antibodies

Antibodies have important roles in controlling cellular immunity through interaction with activating or inhibitory Fcγ receptors (FcγRs). FcγR engagement can facilitate receptor cross-linking on target cells, or induce retrograde FcγR signals to stimulate or suppress antibody-dependent, cell-mediated depletion of antigen-bearing target cells. Recent studies uncover unexpectedly important roles for FcγRs in the anticancer action of antibodies designed to trigger tumor cell apoptosis or enhance antitumor immunity. Here, we outline a conceptual framework for understanding these findings and discuss their mechanistic and translational implications.The adaptive immune system is capable of recognizing an essentially limitless number of antigens via the combinatorial assembly of gene segments that encode antibody variable domains. This diversity has been exploited successfully in a growing number of therapeutic antibodies that bind to a wide range of clinically validated targets. Antibodies that recognize soluble antigens, such as the cytokines tumor necrosis factor (TNF), vascular endothelial growth factor, or interleukin-6, act as antagonists by blocking the interaction of a target ligand with its cognate receptor. In some cases, such antibodies may also augment clearance of the target antigen.In contrast, antibodies that bind to cell surface antigens, often transmembrane receptors such as HER2, EGFR, or DR5, may act as antagonists or agonists, respectively, to block or stimulate the action of the cognate target. Alternatively, antibodies may bind a cell surface target that lacks signaling function, such as the CD20 antigen, and act as an anchor for FcγR-based recruitment of immune-effector cells to kill the antigen-expressing target by antibody-dependent, cell-mediated cytotoxicity (ADCC). Therefore, antibodies that recognize cell surface receptors can be categorized by their function of either mediating target cell killing or modulating target receptor signal transduction. However, two new studies in this issue demonstrate that these activities are not mutually exclusive and that antibodies harboring both properties may be advantageous for cancer immunotherapy. Due to shared expression of cell surface antigens, such as CTLA-4 or glucocorticoid-induced TNFR-related protein (GITR) on protumorigenic regulatory T (T reg) cells and antitumorigenic effector T (T eff) cells, antibodies that target such receptors are capable of inducing antitumor immunity both by depleting T reg cells and by stimulating T eff cells. However, antibodies that conform to this dual mechanism of action have the risk of depleting T eff cells, which are the final mediators of tumor cell killing. Therefore, understanding the principles that govern antibody–FcγR interactions is crucial for designing effective antibody-based immunotherapies.

Antibody–FcγR interactions

FcγRs fall into two functional classes: activating and inhibitory (Nimmerjahn and Ravetch, 2006). The FcγR family comprises three activating (mouse FcγRI, FcγRIII, and FcγRIV; human FcγRI, FcγRIIA, and FcγRIIIA) and one inhibitory (FcγRIIB) receptor. Activating FcγRs associate with a common signaling chain (FcRγ), containing an immunoreceptor tyrosine-based activation motif (ITAM) that recruits Syk family kinases to stimulate effector function. In contrast, FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that recruits specific phosphatases to oppose signaling by activating FcγRs. Innate-immune cells, such as macrophages, monocytes, dendritic cells, mast cells, and granulocytes, express both activating and inhibitory FcγRIIB (Amigorena et al., 1992; Nimmerjahn and Ravetch, 2008). IgG subtypes differ in FcγR affinity: human IgG1 and IgG3 have higher affinity for activating than inhibitory FcγR, as do mouse IgG2a and IgG2b (Dijstelbloem et al., 2001; Nimmerjahn and Ravetch, 2005, 2006).Antagonist antibodies may bind to a soluble ligand or a cell surface receptor to prevent signaling. Target inhibition per se typically does not require accessory FcγR-bearing cells; therefore, antagonist antibodies often act independently of FcγRs, and accordingly, IgG subtype. However, if the target is engaged at the cell surface and is sufficiently abundant, effector cells may be recruited via Fc–FcγR interactions to deplete the antigen-displaying cell, an outcome that can be desirable or undesirable depending on the context. Target cell depletion can be manipulated by selecting IgG subtypes that favor binding to activating or inhibitory FcγRs. Unwanted target cell depletion can be minimized by incorporating Fc mutations that decrease FcγR affinity (Presta et al., 2002; Carter, 2006; Lazar et al., 2006; Satoh et al., 2006; Jefferis, 2009). For example, asparagine 297, the site for N-linked glycosylation required for FcγR binding in the constant region, can be replaced by alanine. Further mutations to enhance or decrease specific FcγR interactions have also been reported. Alternatively, some antibody variants can be produced in Escherichia coli rather than mammalian cells to prevent Fc glycosylation. Fc effectorless antibodies have been demonstrated to be equally as potent at blocking ligand–receptor interactions as their wild-type counterparts.Recent work has revealed unexpectedly that agonist antibodies designed to stimulate the tumor necrosis factor receptor superfamily (TNFRSF) members DR4, DR5, or CD40 depend on FcγR interaction for robust agonist activity (Li and Ravetch, 2011; Wilson et al., 2011; Smith et al., 2012). As TNFRSF members usually require ligand-induced super-clustering for signal transmission, bivalent IgG molecules are unable to induce their efficient stimulation. In vitro activity can be enhanced by artificial cross-linking of the primary antibody, with secondary anti-Fc antibodies, or—perhaps more importantly—by providing contact with FcγR-bearing cells. Pretreating FcγR-expressing cells with actin polymerization inhibitors blocks this enhancement, suggesting that FcγR clustering is important for antibody-mediated stimulation of the target receptor (Wilson et al., 2011). Studies with mice deficient in specific FcγR subsets demonstrate that expression of the inhibitory FcγRIIB is sufficient—if not superior—for enabling in vivo efficacy of agonist antibodies targeting CD40, or the death receptors DR4 and DR5 (Li and Ravetch, 2011; Wilson et al., 2011). Reliance on FcγRIIB circumvents the potential for FcγR-mediated target cell depletion and is therefore advantageous for inducing signal transduction on target cells, such as CD40 signaling in dendritic cells. CD40 engagement on dendritic cells enhances their antigen presentation capabilities, thereby increasing T cell responses. Therefore, cross-linking CD40 via FcγRIIB on DCs enhances their T cell priming function, while minimizing depletion. Importantly, anti-CD40–induced adjuvant activity was unabated in mice deficient in activating FcγRs, yet was abrogated in FcγRIIB-deficient mice (Li and Ravetch, 2011). However, FcγR-dependent depletion of DR4- or DR5-expressing tumor cells could be useful to induce antitumor activity. These findings suggest that agonist antibodies targeting either proapoptotic or co-stimulatory TNFRSF members can rely on FcγRs as a dynamic cross-linking scaffold—a function that, at least in mice, may be supported more effectively by FcγRIIB.

Agonist GITR antibodies require activating FcγRs for antitumor efficacy

In this issue, Bulliard et al. report an apparent exception to the latter paradigm: agonist antibodies targeting the TNFRSF family member GITR require activating FcγR effector function to promote tumor regression in mice. GITR expression is induced on T eff cells upon T cell receptor (TCR) stimulation, and GITR cross-linking by GITR ligand or agonist antibodies co-stimulates TCR signaling (McHugh et al., 2002; Shimizu et al., 2002; Tone et al., 2003; Ronchetti et al., 2004; Stephens et al., 2004). In transplantable tumor models, anti-GITR treatment is hypothesized to induce tumor regression through T eff cell co-stimulation (Turk et al., 2004; Ko et al., 2005). Distinct from antibodies that target other TNFR superfamily members, anti-GITR activity was unaltered in Fcgr2b^−/− mice. However, anti-GITR treatment was ineffective in knockout mice lacking the ITAM-containing FcγR chain required for signaling by all activating FcγRs. Hence, an alternative (or additional) mechanism of action—distinct from FcγRIIB-mediated GITR cross-linking to promote co-stimulation—may be critical for antitumor efficacy. Given that GITR-expressing CD8⁺ T cells and CD4⁺ T eff cells are needed for tumor cell killing, this mechanism may involve FcγR-dependent depletion of GITR-expressing T reg cells.T reg cells frequently express T cell activation markers induced by TCR signaling (Gavin et al., 2002; McHugh et al., 2002). In addition to their well-characterized role in maintaining peripheral tolerance to self-antigens, T reg cells have been demonstrated to suppress tumor immunity (Nishikawa and Sakaguchi, 2010; Josefowicz et al., 2012). T reg cells are highly enriched in tumors, both in mouse models and in various human cancers. Furthermore, in cancer patients, abundance of T reg cells within tumors is associated with poor prognosis, suggesting that these cells play an important role in suppressing antitumor immunity. Therefore, strategies to deplete intratumoral T reg cells may enhance the generation of tumor-directed T eff cell responses.GITR is weakly expressed on naive T eff cells, but is present on resting T reg cells and up-regulated on activated T eff cells. Bulliard et al. (2013) observed a significant reduction in tumor-associated, but not peripheral, T reg cells upon anti-GITR treatment of tumor-bearing mice. Hence, anti-GITR antibodies may enhance antitumor immunity by depleting GITR-positive T reg cells, in addition to co-stimulating antigen-experienced T eff cells (Coe et al., 2010). However, the shared expression of GITR on T reg and T eff cells makes both populations potentially susceptible to antibody-dependent, FcγR-mediated depletion. Indeed, diminished effector CD4⁺ and CD8⁺ T cell numbers were reported to accompany T reg cell depletion in tumor tissues. Therefore, elimination of intratumoral T reg cells, coupled with T eff cell co-stimulation and minimal T eff cell depletion, might provide an integrated mechanism of action for anti-GITR antibodies. The relative contribution of each of these mechanistic components remains to be clarified.

CTLA-4 blocking antibodies mediate T reg cell depletion

CTLA-4 is an inhibitory receptor that is induced on antigen-experienced T eff cells as a negative feedback regulator (Walunas et al., 1994; Krummel and Allison, 1995). Whereas CTLA-4 expression is induced on activated T eff cells, CTLA-4 is constitutively expressed by T reg cells, which require CTLA-4 function to aggregate preferentially around dendritic cells and inhibit their antigen-presenting activity (Onishi et al., 2008). As such, the multiorgan autoimmunity detected in Ctla4 germline knockout mice is phenocopied in T reg cell–specific Ctla4 conditional knockout mice (Tivol et al., 1995; Waterhouse et al., 1995; Chambers et al., 1997; Wing et al., 2008). Although anti–CTLA-4 therapy is one of the first clinically validated examples of effective cancer immunotherapy, its mechanism of action is poorly understood (Hodi et al., 2010). It is hypothesized that antagonist anti–CTLA-4 antibodies enhance tumor immunity by relieving inhibitory CTLA-4 signals on antigen-experienced T eff cells, as well as curtailing the suppressive function of T reg cells. Following the paradigm of antagonist antibodies, CTLA-4–blocking antibodies would be expected to exert their efficacy in the absence of Fc effector function.However, studies from Bulliard et al. (2013) and Simpson et al. in this issue provide compelling evidence that the activity of anti–CTLA-4 antibodies requires activating FcγR engagement to deplete T reg cells. Similar to anti-GITR therapy, anti–CTLA-4–mediated tumor regression was abrogated in FcγR-deficient mice, suggesting that retrograde signaling by activating FcγRs and consequent T reg cell depletion is important for antitumor activity. Accordingly, intratumoral T reg cell numbers were dramatically reduced in response to anti–CTLA-4 treatment. Importantly, this reduction was not associated with dedifferentiation or impaired generation of T reg cells, thereby supporting cellular depletion as the most plausible mechanism of efficacy.Because activated T eff cells express CTLA-4, they are also susceptible to antibody-mediated depletion. In this regard, Bulliard et al. (2013) demonstrated that both T reg and T eff cell numbers were reduced in response to anti–CTLA-4 treatment. In contrast, Simpson et al. (2013) observed that in an adoptive T cell transfer model, intratumoral T eff cells were not depleted by anti–CTLA-4 treatment, but rather increased in number. Differences in tumor model, antibody isotype or clone, or vaccine co-administration could potentially account for these discrepant results. Nevertheless, T reg cells may be more sensitive to antibody-mediated depletion because of their increased expression of CTLA-4 within tumor tissues. Consistent with this notion, tumor-infiltrating T reg cells expressed nearly fourfold higher CTLA-4 levels than did tumor-associated T eff cells in the Colon26 carcinoma model. Additionally, in the B16-BL6 melanoma model, CTLA-4 surface expression was slightly elevated in tumor-associated versus peripheral T reg cells or intratumoral T eff cells. It remains to be investigated whether this variation in receptor expression is detected in human intratumoral T cells and leads to differential depletion in cancer patients.

Conclusions

Important roles have emerged for FcγRs in mediating the antitumor efficacy of a new generation of therapeutic antibodies aiming to attack cancer directly, by triggering tumor cell apoptosis, or indirectly, by enhancing antitumor immunity (Fig. 1). First, FcγRs on tumor-infiltrating innate immune cells provide a cross-linking scaffold to enhance antibody-mediated activation of proapoptotic TNFRSF members such as DR4 and DR5 on cancer cells. Similarly, FcγR-mediated cross-linking supports activation of T eff cells via co-stimulatory TNFRSF members such as GITR. In some instances, this cross-linking function can be performed redundantly by activating or inhibitory FcγRs; however, mouse studies suggest that the inhibitory FcγRIIB may provide a more effective molecular scaffold, for reasons that have yet to be defined. Fc mutations that enhance affinity for FcγRs may serve to improve the efficacy of agonist antibodies targeting various TNFRSF members. In addition to cross-linking cell surface receptors to support forward signaling in target cells, FcγRs can mediate antibody-driven reverse signaling to activate FcγR-bearing cells and promote target cell depletion. For cancer immunotherapy, antibodies that fulfill both functions may be beneficial to deplete immunosuppressive T reg cells and stimulate T eff cells. The high prevalence of T reg cells in tumor tissues poses a significant barrier to generating a productive antitumor T cell response. Expression of antigens such as GITR or CTLA-4 on T reg cells affords the opportunity for enhanced antibody-based depletion of such immune-suppressive cells from the tumor microenvironment, thereby augmenting antitumor immunity. A potential caveat is that desirable T eff cells also might express the same antigens and hence be subject to similar depletion. Thus, maximal therapeutic efficacy likely necessitates a delicate balance between depleting T reg cells while sparing T eff cells. Defining the FcγR interactions responsible for these different outcomes should help optimize the effectiveness of antibodies to biologically relevant T cell antigens, including GITR, CTLA-4, and beyond.Open in a separate windowFigure 1.How Fcγ receptors enable anticancer efficacy of proapoptotic and immune-modulatory antibodies. Inhibitory (Inh.) or activating (Act.) FcγRs can provide a dynamic scaffold for cross-linking agonist antibodies targeting proapoptotic receptors such as DR4 or DR5 on the surface of cancer cells, thereby promoting direct tumor cell apoptosis (A). Similarly, FcγRs can support agonist antibody-mediated cross-linking of immune cell co-stimulatory receptors, e.g., CD40 on antigen-presenting cells (B), or GITR on T eff cells (C), to augment antitumor immunity. T eff cell responses can be enhanced further, through direct antibody-based antagonism of the inhibitory molecule CTLA-4 (D). Finally, activating FcγRs can promote ADCC-based depletion of T reg cells upon antibody binding to GITR or CTLA-4, thus alleviating T reg suppression and strengthening antitumor immunity (E).     

DOCK5 functions as a key signaling adaptor that links FcεRI signals to microtubule dynamics during mast cell degranulation

Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcεRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcεRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3β. When DOCK5–Nck2–Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.Mast cells play a key role in induction of anaphylaxis, a life-threatening allergic reaction which occurs rapidly after exposure of certain antigens, such as foods, drugs, and insect venoms (Sampson et al., 2005). Mast cells express the high-affinity receptor for IgE, FcεRI, on their surface, and binding of multivalent antigens to FcεRI-bound IgE induces receptor aggregation and triggers mast cell activation (Kawakami and Galli, 2002; Kraft and Kinet, 2007). Activated mast cells secrete preformed chemical mediators, including proteases and vasoactive amines such as histamine, which are stored in cytoplasmic secretory granules (Kawakami and Galli, 2002; Lundequist and Pejler, 2011). This process involves the movement of secretory granules and their fusion with the plasma membrane followed by exocytosis to release the chemical mediators (Blott and Griffiths, 2002; Lundequist and Pejler, 2011). Degranulation of mast cells is therefore a complex and multistep process that is tightly regulated by FcεRI-mediated signals.Upon aggregation of FcεRI with IgE and antigens, two parallel signaling cascades operate. One cascade is initiated by activation of the Src family protein tyrosine kinase Lyn, which is bound to the FcεRI β subunit, and involves subsequent activation of the nonreceptor protein tyrosine kinase Syk (Kawakami and Galli, 2002; Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). The activated Syk then phosphorylates multiple substrates, including PLC-γ (Kawakami and Galli, 2002; Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). The other cascade uses Fyn, another FcεRI-associated Src family protein tyrosine kinase (Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). Fyn phosphorylates the adaptor protein Gab2, which leads to activation of phosphatidylinositol 3-kinase (PI3K) through association with the p85α regulatory subunit (Gu et al., 2001; Parravicini et al., 2002; Nishida et al., 2005, 2011). Several lines of evidence indicate that although the Lyn–Syk–PLC-γ axis regulates granule-plasma membrane fusion and exocytosis by controlling calcium response (Nishida et al., 2005; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009), the Fyn–Gab2 pathway plays a key role in translocation of secretory granules to the plasma membrane (Parravicini et al., 2002; Nishida et al., 2005, 2011). However, little is known about the distal events controlling mast cell degranulation. In particular, movement of secretory granules requires dynamic rearrangement of microtubules (Martin-Verdeaux et al., 2003; Smith et al., 2003; Nishida et al., 2005; Dráber et al., 2012), yet the signaling events regulating this step of mast cell activation are poorly understood.GSK3β is a serine/threonine kinase that negatively regulates microtubule dynamics (Cohen and Frame, 2001; Zhou and Snider, 2005). In resting cells, GSK3β phosphorylates many microtubule-binding proteins and inhibits their ability to interact with microtubules and to promote microtubule assembly (Zhou et al., 2004; Yoshimura et al., 2005; Kim et al., 2011). This inhibitory effect is relieved when GSK3β is phosphorylated at serine residue of position 9 (Ser9; Cohen and Frame, 2001). Although knockdown experiments revealed a role for GSK3β in cytokine production, chemotaxis, and survival of human mast cells (Rådinger et al., 2010; Rådinger et al., 2011), aggregation of FcεRI also induces GSK3β phosphorylation at Ser9 (Rådinger et al., 2010). Therefore, phosphorylation-dependent inactivation of GSK3β may be involved in FcεRI-mediated regulation of microtubule dynamics in mast cells.DOCK5 is a member of the atypical guanine nucleotide exchange factors (GEFs) for the Rho family of GTPases (Côté and Vuori, 2002). Although DOCK5 does not contain the Dbl homology domain typically found in GEFs (Schmidt and Hall, 2002), DOCK5 mediates the GTP–GDP exchange reaction for Rac through DOCK homology region 2 (DHR-2; also known as CZH2 or Docker) domain (Brugnera et al., 2002; Côté and Vuori, 2002; Meller et al., 2002). DOCK5 is widely expressed in various tissues and regulates multiple cellular functions, including myoblast fusion and bone resorption (Laurin et al., 2008; Vives et al., 2011), yet its roles in the immune system and immune responses remain unexplored. In this study, we demonstrate that DOCK5 regulates FcεRI-mediated anaphylactic responses in vivo and mast cell degranulation in vitro. Unexpectedly, this regulation by DOCK5 does not require its Rac GEF activity, but instead involves association with Nck2 and Akt. When this interaction was blocked in mast cells, FcεRI-mediated phosphorylation and inactivation of GSK3β were impaired, resulting in a marked reduction in microtubule dynamics and a severe defect in degranulation. Our results thus define a novel regulatory mechanism controlling degranulation response in mast cells.     

Reversal of Proinflammatory Responses by Ligating the Macrophage Fcγ Receptor Type I

Macrophages can respond to a variety of infectious and/or inflammatory stimuli by secreting an array of proinflammatory cytokines, the overproduction of which can result in shock or even death. In this report, we demonstrate that ligation of macrophage Fcγ receptors (FcγR) can lead to a reversal of macrophage proinflammatory responses by inducing an upregulation of interleukin (IL)-10, with a reciprocal inhibition of IL-12 production. IL-10 upregulation was specific to FcγR ligation, since the ligation of the Mac-1 receptor did not alter IL-10 production. The identification of the specific FcγR subtype responsible for IL-10 upregulation was determined in gene knockout mice. Macrophages from mice lacking the FcR γ chain, which is required for assembly and signaling by FcγRI and FcγRIII, failed to upregulate IL-10 in response to immune complexes. However, mice lacking either the FcγRII or the FcγRIII were fully capable of upregulating IL-10 production, implicating FcγRI in this process. The biological consequences of FcγRI ligation were determined in both in vitro and in vivo models of inflammation and sepsis. In all of the models tested, the ligation of FcγR promoted the production of IL-10 and inhibited the secretion of IL-12. This reciprocal alteration in the pattern of macrophage cytokine production illustrates a potentially important role for FcγR-mediated clearance in suppressing macrophage proinflammatory responses.     

The Inositol Polyphosphate 5-Phosphatase Ship Is a Crucial Negative Regulator of B Cell Antigen Receptor Signaling

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcγ receptor IIB (FcγRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)–FcγRIIB coligation. The function of Ship in lymphocytes was investigated in Ship^−/− recombination-activating gene (Rag)^−/− chimeric mice generated from gene-targeted Ship^−/− embryonic stem cells. Ship^−/−Rag^−/− chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship^−/− splenic B cells displayed prolonged Ca²⁺ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR–FcγRIIB coligation. These results demonstrate that Ship plays an essential role in FcγRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca²⁺ flux and MAPK activation.     

The glycosyl phosphatidylinositol-linked FcγRIII(PMN) mediates transmembrane signaling events distinct from FcγRII

To investigate the ability of FcγRIII(PMN), the GPI-anchored isoform of FcγRIII (CD16) in polymorphonuclear leukocytes (PMN), to mediate transmembrane signaling events, we measured changes in membrane potential with DiOC(5) and in intracellular calcium with indo-1. FcγR were ligated by anti-FcγRIII mAb 3G8 (IgG and Fab), anti-FcγRII mAb IV.3 (IgG and Fab), and human IgG aggregates. Cell bound mAbs were also crosslinked by goat F(ab’)(2) anti-mouse IgG. 3G8 IgG elicited a rapid change in [Ca(2+)](i), which was unaffected by EGTA, Vibrio cholerae toxin (CT), or Bordetella pertussis toxin (PT), and was abolished by BAPTA . Univalent receptor binding with 3G8 Fab gave no response but crosslinking with F(aV)2 GAM gave a rapid [Ca2,](i) response. Neither IV.3 Fab, IV.3 IgG, nor crosslinking of IV.3 Fab elicited a calcium signal. PI-PLC-treated PMN with the density of FcγRIII(PMN) reduced to that of FcγRII showed an unattenuated change in [Ca(2+)](i), with a 3G8 stimulus. The effects of IgG aggregates paralleled those of 3G8 mAb. These data indicate that multivalent ligation of FcγRIII(PMN) initiates an increase in [Ca(2+)];, derived from intracellular stores, that is distinct from both the FMLP- and FcγRII-induced responses. Ligand-dependent interaction with FcγRII is not required. Since FcγRIII(PMN) can internalize the FcγRIII-specific probe Con A-opsonized E and lyse anti-FcγRIII heteroantibody-opsonized chick E, this GPI-anchored molecule mediates both signal transduction and integrated cell responses.     

Differential Effects of B Cell Receptor and B Cell Receptor–FcγRIIB1 Engagement on Docking of Csk to GTPase-activating Protein (GAP)-associated p62

The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcγ receptors of the IIB1 type (FcγRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcγRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein–associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab′)₂ resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcγRIIB1 engagement on this association was abolished by blockade of FcγRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcγRIIB1-deficient cell line IIA1.6 and recovered when FcγRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcγRIIB1–mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor–mediated signals in B cells.     

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4⁺ and CD8⁺ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η^−/− and ζ/η^−/−–Fcγ^−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.