IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3.

BACKGROUND: IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES: We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS: HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS: IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION: IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Activated mast cells synthesize and release soluble ST2-a decoy receptor for IL-33.

IL-33 released from damaged cells plays a central role in allergic inflammation by acting through its membrane-bound receptor, ST2 receptor (ST2L). IL-33 activity can be neutralized by the soluble spliced variant of ST2 (sST2) that has been associated with allergic inflammation but its source is not well defined. We investigated whether mast cells (MCs) are a significant source of sST2 following activation through FcεRI or ST2. We find that antigen and IL-33 induce substantial production and release of sST2 from human and mouse MCs in culture and do so synergistically when added together or in combination with stem cell factor. Moreover, increases in circulating sST2 during anaphylaxis in mice were dependent on the presence of MCs. Human MCs activated via FcεRI failed to generate IL-33 and IL-33 produced by mouse bone marrow-derived MCs was retained within the cells. Therefore, FcεRI-mediated sST2 production is independent of MC-derived IL-33 acting in an autocrine manner. These results are consistent with the conclusion that both mouse and human MCs when activated are a significant inducible source of sST2 but not IL-33 and thus have the ability to modulate the biologic impact of IL-33 produced locally by other cell types during allergic inflammation.

The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling.

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases.

Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production.

BACKGROUND: Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. OBJECTIVES: We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. METHODS: Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. RESULTS: Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. CONCLUSION: Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.

Assay of mast cell mediators.

Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well as cytokine release.

Flow cytometry-based monitoring of mast cell activation.

Mast cell activation is a central process in the initiation of allergic disorders. As described elsewhere in this volume, this process can be readily monitored by biochemical, antibody-based, and enzyme-based formats when the cell population examined is homogenous. When dealing with mixed and transfected cell populations however, such approaches may not be appropriate. Hence alternative methods are required. Here we describe flow-cytometry-based assays that can be utilized to examine signaling processes and degranulation in both pure mast cell populations and, following appropriate selection, in populations where the mast cells of interest may only represent a fraction of the total cell population.

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype.

BACKGROUND: Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. OBJECTIVES: We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. METHODS: Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. RESULTS: KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. CONCLUSION: Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing.

Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation.

BACKGROUND: Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. OBJECTIVE: Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. METHODS: We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. RESULTS: Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. CONCLUSION: This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.

KIT GNNK splice variants: expression in systemic mastocytosis and influence on the activating potential of the D816V mutation in mast cells.

Stem cell factor-dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK(-) and GNNK(+) transcripts from bone marrow mononuclear cells was developed. The GNNK(-)/GNNK(+) copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK(-)/GNNK(+) copy number ratio. Relative expression of only the GNNK(-) variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK(-) isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK(-) isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK(-) variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis.

A truncated splice-variant of the FcεRIß receptor subunit is critical for microtubule formation and degranulation in mast cells.

Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIß) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIß (t-FcεRIß) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIß is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIß in the presence of Ca2+ could be critical for t-FcεRIß function. In addition, gene targeting of t-FcεRIß attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIß mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIß has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation.

Prevention of F-actin assembly switches the response to SCF from chemotaxis to degranulation in human mast cells.

Following antigen/IgE-mediated aggregation of high affinity IgE-receptors (FcεRI), mast cells (MCs) degranulate and release inflammatory mediators leading to the induction of allergic reactions including anaphylaxis. Migration of MCs to resident tissues and sites of inflammation is regulated by tissue chemotactic factors such as stem cell factor (SCF (KIT ligand)). Despite inducing similar early signaling events to antigen, chemotactic factors, including SCF, produce minimal degranulation in the absence of other stimuli. We therefore investigated whether processes regulating MC chemotaxis are rate limiting for MC mediator release. To investigate this issue, we disrupted actin polymerization, a requirement for MC chemotaxis, with latrunculin B and cytochalasin B, then examined chemotaxis and mediator release in human (hu)MCs induced by antigen or SCF. As expected, such disruption minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. In contrast, SCF, in the absence of other stimuli, induced substantial degranulation in a concentration-dependent manner following actin disassembly. It also moderately enhanced antigen-mediated human mast cell degranulation which was further enhanced in the presence of SCF. These observations suggest that processes regulating cell migration limit MC degranulation as a consequence of cytoskeletal reorganization.

Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production.

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1⁻/⁻ and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1⁻/⁻ mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1⁻/⁻ mice. Furthermore, contrary to expectations, Trpc1⁻/⁻ BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1⁻/⁻ BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1⁻/⁻ mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.

Usage of sphingosine kinase isoforms in mast cells is species and/or cell type determined.

FcεRI engagement in mast cells (MCs) induces the activation of two distinct sphingosine kinase isoforms (SphK1 and SphK2) to produce sphingosine-1-phosphate, a mediator essential for MC responses. Whereas embryonic-derived SphK2-null MCs showed impaired responses to Ag, RNA silencing studies on other MC types indicated a dominant role for SphK1. Given the known functional heterogeneity of MCs, we explored whether the reported differences in SphK1 or SphK2 usage could be reflective of phenotypic differences between MC populations. Using lentiviral-based short hairpin RNA to silence SphK1 or SphK2, we found that SphK2 is required for murine MC degranulation, calcium mobilization, and cytokine and leukotriene production, irrespective of the tissue from which the MC progenitors were derived, the stage of MC granule maturity, or the conditions used for differentiation. This finding was consistent with the lack of a full allergic response in SphK2-null mice challenged to undergo passive cutaneous anaphylaxis. A redundant role for both SphKs was uncovered, however, in chemotaxis toward Ag in all MC types tested and in TNF-α production in certain MC types. In contrast, human MC responses were dependent only on SphK1, associating with a more robust expression of this isoform and a more varied representation of SphK variants relative to murine MCs. The findings show that the function of SphK1 and SphK2 can be interchangeable in MCs; however, an important determinant of SphK isoform usage is the species of origin and an influencing factor, the tissue from which MCs may be derived and/or their differentiation state.

A novel KIT-deficient mouse mast cell model for the examination of human KIT-mediated activation responses.

Activation of KIT, by its ligand, stem cell factor (SCF), results in the initiation of signal transduction pathways that influence mast cell survival and proliferation. Activating mutations in KIT have thus been linked to clonal MC proliferation associated with systemic mastocytosis. SCF also modulates MC function by inducing MC chemotaxis and by potentiating antigen (Ag)/IgE-mediated MC degranulation. Thus, mutations in KIT also have the potential to affect these processes in allergic and other mast cell-related diseases. Studies to determine how native and mutated KIT may modulate MC chemotaxis and activation have, however, been limited due to the lack of availability of a suitable functional MC line lacking native KIT which would allow transduction of KIT constructs. Here we describe a novel mouse MC line which allows the study of normal and mutated KIT constructs. These cells originated from a bone marrow-derived mouse MC culture out of which a rapidly dividing mast cell sub-population spontaneously arose. Over time, these cells lost KIT expression while continuing to express functional high affinity receptors for IgE (FcεRI). As a consequence, these cells degranulated in response to Ag/IgE but did not migrate nor show any evidence of potentiation of Ag/IgE degranulation in response to SCF. Retroviral transduction of the cells with a human (hu)KIT construct resulted in surface expression of huKIT which responded to huSCF by potentiation of Ag/IgE-induced degranulation and chemotaxis. This cell line thus presents a novel system to delineate how MC function is modulated by native and mutated KIT and for the identification of novel inhibitors of these processes.

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

IL-33 is elevated in afflicted tissues of patients with mast cell (MC)-dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ(1) and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

The adaptor 3BP2 is required for early and late events in FcεRI signaling in human mast cells.

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.

Stem cell factor programs the mast cell activation phenotype.

Mast cells, activated by Ag via FcεRI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of FcεRI-mediated degranulation and cytokine production. The hyporesponsive phenotype was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders.

Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions.

BACKGROUND: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).