Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling.

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Sertoli cells are the source of stem cell factor for spermatogenesis.

Several cell types have been proposed to create the required microenvironment for spermatogenesis. However, expression patterns of the key growth factors produced by these somatic cells have not been systematically studied and no such factor has been conditionally deleted from its primary source(s), raising the question of which cell type(s) are the physiological sources of these growth factors. Here, using single-cell RNA sequencing and a series of fluorescent reporter mice, we found that stem cell factor (Scf), one of the essential growth factors for spermatogenesis, was broadly expressed in testicular stromal cells, including Sertoli, endothelial, Leydig, smooth muscle and Tcf21-CreER+ stromal cells. Both undifferentiated and differentiating spermatogonia were associated with Scf-expressing Sertoli cells in the seminiferous tubule. Conditional deletion of Scf from Sertoli cells, but not any other Scf-expressing cells, blocked the differentiation of spermatogonia, leading to complete male infertility. Conditional overexpression of Scf in Sertoli cells, but not endothelial cells, significantly increased spermatogenesis. Our data reveal the importance of anatomical localization for Sertoli cells in regulating spermatogenesis and that SCF produced specifically by Sertoli cells is essential for spermatogenesis.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging.

ABSTRACT: Intrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by adiponectin, kit ligand (KITL) and insulin-like growth factor 1 (IGF1) from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF-FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF-FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular ß-sheet around a highly divergent ß-strand of the BTB domain. Complex dissociation allows SCF-FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF-FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor.

c-Kit is a classic proto-oncogene either mutated or upregulated in cancer cells, and this leads to its constitutive kinase activation and, thus, to uncontrolled proliferation. Although the pro-oncogenic role of c-Kit is of no doubt, some observations do not fit well with c-Kit solely as a tumor-promoting moiety. We show here that c-Kit actively triggers cell death in various cancer cell lines unless engaged by its ligand stem cell factor (SCF). This pro-death activity is enhanced when the kinase activation of c-Kit is silenced and is due to c-Kit intracellular cleavage by caspase-like protease at D816. Moreover, in vivo, overexpression of a c-Kit kinase-dead mutant inhibits tumor growth, and this intrinsic c-Kit tumor-suppressive activity is dependent on the D816 cleavage. Thus, c-Kit acts both as a proto-oncogene via its kinase activity and as a tumor suppressor via its dependence receptor activity.

Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

Cancer cell plasticity, stem cell factors, and therapy resistance: how are they linked?

Cellular plasticity can occur naturally in an organism and is considered an adapting mechanism during the developmental stage. However, abnormal cellular plasticity is observed in different diseased conditions, including cancer. Cancer cell plasticity triggers the stimuli of epithelial-mesenchymal transition (EMT), abnormal epigenetic changes, expression of stem cell factors and implicated signaling pathways, etc., and helps in the maintenance of CSC phenotype. Conversely, CSC maintains the cancer cell plasticity, EMT, and epigenetic plasticity. EMT contributes to increased cell migration and greater diversity within tumors, while epigenetic changes, stem cell factors (OCT4, NANOG, and SOX2), and various signaling pathways allow cancer cells to maintain various phenotypes, giving rise to intra- and inter-tumoral heterogeneity. The intricate relationships between cancer cell plasticity and stem cell factors help the tumor cells adopt drug-tolerant states, evade senescence, and successfully acquire drug resistance with treatment dismissal. Inhibiting molecules/signaling pathways involved in promoting CSCs, cellular plasticity, EMT, and epigenetic plasticity might be helpful for successful cancer therapy management. This review discussed the role of cellular plasticity, EMT, and stem cell factors in tumor initiation, progression, reprogramming, and therapy resistance. Finally, we discussed how the intervention in this axis will help better manage cancers and improve patient survivability.

Loss of endothelial membrane KIT ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells.

A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

Identifying the niche controlling melanocyte differentiation.

Melanocytes present in hair follicles are responsible for their pigmentation. Melanocyte differentiation and hair pigmentation depend on the stem cell factor (SCF)/c-Kit signaling pathway, but the niche that regulates melanocyte differentiation is not well characterized. In this issue of Genes & Development, Liao and colleagues (pp. 744-756) identify Krox20+-derived cells of the hair shaft as the niche and the essential source of SCF required for melanocyte maturation. This study delineates the niche factors regulating melanocyte differentiation and hair pigmentation and opens up new avenues to further characterize the cross-talk between the hair follicle and melanocytes that controls melanocyte maintenance and differentiation.

Identification of hair shaft progenitors that create a niche for hair pigmentation.

Hair differentiates from follicle stem cells through progenitor cells in the matrix. In contrast to stem cells in the bulge, the identities of the progenitors and the mechanisms by which they regulate hair shaft components are poorly understood. Hair is also pigmented by melanocytes in the follicle. However, the niche that regulates follicular melanocytes is not well characterized. Here, we report the identification of hair shaft progenitors in the matrix that are differentiated from follicular epithelial cells expressing transcription factor KROX20. Depletion of Krox20 lineage cells results in arrest of hair growth, confirming the critical role of KROX20+ cells as antecedents of structural cells found in hair. Expression of stem cell factor (SCF) by these cells is necessary for the maintenance of differentiated melanocytes and for hair pigmentation. Our findings reveal the identities of hair matrix progenitors that regulate hair growth and pigmentation, partly by creating an SCF-dependent niche for follicular melanocytes.

Therapeutic modulation of KIT ligand in melanocytic disorders with implications for mast cell diseases.

KIT ligand and its associated receptor KIT serve as a master regulatory system for both melanocytes and mast cells controlling survival, migration, proliferation and activation. Blockade of this pathway results in cell depletion, while overactivation leads to mastocytosis or melanoma. Expression defects are associated with pigmentary and mast cell disorders. KIT ligand regulation is complex but efficient targeting of this system would be of significant benefit to those suffering from melanocytic or mast cell disorders. Herein, we review the known associations of this pathway with cutaneous diseases and the regulators of this system both in skin and in the more well-studied germ cell system. Exogenous agents modulating this pathway will also be presented. Ultimately, we will review potential therapeutic opportunities to help our patients with melanocytic and mast cell disease processes potentially including vitiligo, hair greying, melasma, urticaria, mastocytosis and melanoma.

The MicroRNA-132 and MicroRNA-212 Cluster Regulates Hematopoietic Stem Cell Maintenance and Survival with Age by Buffering FOXO3 Expression.

MicroRNAs are critical post-transcriptional regulators of hematopoietic cell-fate decisions, though little remains known about their role in aging hematopoietic stem cells (HSCs). We found that the microRNA-212/132 cluster (Mirc19) is enriched in HSCs and is upregulated during aging. Both overexpression and deletion of microRNAs in this cluster leads to inappropriate hematopoiesis with age. Enforced expression of miR-132 in the bone marrow of mice led to rapid HSC cycling and depletion. A genetic deletion of Mirc19 in mice resulted in HSCs that had altered cycling, function, and survival in response to growth factor starvation. We found that miR-132 exerted its effect on aging HSCs by targeting the transcription factor FOXO3, a known aging associated gene. Our data demonstrate that Mirc19 plays a role in maintaining balanced hematopoietic output by buffering FOXO3 expression. We have thus identified it as a potential target that might play a role in age-related hematopoietic defects.

SCF/C-kit drives spermatogenesis disorder induced by abscopal effects of cranial irradiation in mice.

Cranial radiotherapy is a major treatment for leukemia and brain tumors. Our previous study found abscopal effects of cranial irradiation could cause spermatogenesis disorder in mice. However, the exact mechanisms are not yet fully understood. In the study, adult male C57BL/6 mice were administrated with 20 Gy X-ray cranial irradiation (5 Gy per day for 4 days consecutively) and sacrificed at 1, 2 and 4 weeks. Tandem Mass Tag (TMT) quantitative proteomics of testis was combined with bioinformatics analysis to identify key molecules and signal pathways related to spermatogenesis at 4 weeks after cranial irradiation. GO analysis showed that spermatogenesis was closely related to oxidative stress and inflammation. Severe oxidative stress occurred in testis, serum and brain, while serious inflammation also occurred in testis and serum. Additionally, the sex hormones related to hypothalamic-pituitary-gonadal (HPG) axis were disrupted. PI3K/Akt pathway was activated in testis, which upstream molecule SCF/C-Kit was significantly elevated. Furthermore, the proliferation and differentiation ability of spermatogonial stem cells (SSCs) were altered. These findings suggest that cranial irradiation can cause spermatogenesis disorder through brain-blood-testicular cascade oxidative stress, inflammation and the secretory dysfunction of HPG axis, and SCF/C-kit drive this process through activating PI3K/Akt pathway.

Expression and regulation of Siglec-6 (CD327) on human mast cells and basophils.

BACKGROUND: Mast cells (MC) and basophils are effector cells of allergic reactions and display a number of activation-linked cell surface antigens. Of these antigens, however, only a few are functionally relevant and specifically expressed in these cells. OBJECTIVE: We sought to identify MC- and basophil-specific surface molecules and to study their cellular distribution and regulation during cytokine-induced and IgE-dependent activation. METHODS: Multicolor flow cytometry was performed to recognize surface antigens and to determine changes in antigen expression upon activation. RESULTS: We identified Siglec-6 (CD327) as a differentially regulated surface antigen on human MC and basophils. In the bone marrow, Siglec-6 was expressed abundantly on MC in patients with mastocytosis and in reactive states, but it was not detected on other myeloid cells, with the exception of basophils and monocytes. In healthy individuals, allergic patients, and patients with chronic myeloid leukemia (CML), Siglec-6 was identified on CD203c+ blood basophils, a subset of CD19+ B lymphocytes, and few CD14+ monocytes, but not on other blood leukocytes. CML basophils expressed higher levels of Siglec-6 than normal basophils. IL-3 promoted Siglec-6 expression on normal and CML basophils, and stem cell factor increased the expression of Siglec-6 on tissue MC. Unexpectedly, IgE-dependent activation resulted in downregulation of Siglec-6 in IL-3-primed basophils, whereas in MC, IgE-dependent activation augmented stem cell factor-induced upregulation of Siglec-6. CONCLUSIONS: Siglec-6 is a dynamically regulated marker of MC and basophils. Activated MC and basophils exhibit unique Siglec-6 responses, including cytokine-dependent upregulation and unique, cell-specific, responses to IgE-receptor cross-linking.

Hematopoietic Growth Factors Regulate the Entry of Monocytes into the Adult Brain via Chemokine Receptor CCR5.

Monocytes are circulating macrophage precursors generated from bone marrow hematopoietic stem cells. In adults, monocytes continuously replenish cerebral border-associated macrophages under physiological conditions. Monocytes also rapidly infiltrate the brain in pathological settings. The mechanisms of recruiting monocyte-derived macrophages into the brain under pathological conditions have been extensively studied. However, it remains unclear how monocytes enter the brain to renew border-associated macrophages under physiological conditions. Using both in vitro and in vivo approaches, this study reveals that a combination of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), complementarily and synergistically enhances the adhesion of monocytes to cerebral endothelial cells in a dose-dependent manner. Cysteine-cysteine chemokine receptor 5 (CCR5) in brain endothelial cells, but not the cell adhesion molecules mediating neuroinflammation-related infiltration of monocyte-derived macrophages, modulates SCF+G-CSF-enhanced monocyte-endothelial cell adhesion. Blocking CCR5 or genetically deleting CCR5 reduces monocyte-endothelial cell adhesion induced by SCF+G-CSF. The SCF+G-CSF-enhanced recruitment of bone marrow-derived monocytes/macrophages into the cerebral perivascular space is also reduced in adult CCR5 knockout mice. This study demonstrates the role of SCF and G-CSF in regulating the entry of monocytes into the adult brain to replenish perivascular macrophages.

Epigenetic histone modification by butyrate downregulates KIT and attenuates mast cell function.

Short-chain fatty acid butyrate is produced from the bacterial fermentation of indigestible fiber in the intestinal lumen, and it has been shown to attenuate lung inflammation in murine asthma models. Mast cells (MCs) are initiators of inflammatory response to allergens, and they play an important role in asthma. MC survival and proliferation is regulated by its growth factor stem cell factor (SCF), which acts through the receptor, KIT. It has previously been shown that butyrate attenuates the activation of MCs by allergen stimulation. However, how butyrate mechanistically influences SCF signalling to impact MC function remains unknown. Here, we report that butyrate treatment triggered the modification of MC histones via butyrylation and acetylation, and inhibition of histone deacetylase (HDAC) activity. Further, butyrate treatment caused downregulation of SCF receptor KIT and associated phosphorylation, leading to significant attenuation of SCF-mediated MC proliferation, and pro-inflammatory cytokine secretion. Mechanistically, butyrate inhibited MC function by suppressing KIT and downstream p38 and Erk phosphorylation, and it mediated these effects via modification of histones, acting as an HDAC inhibitor and not via its traditional GPR41 (FFAR3) or GPR43 (FFAR2) butyrate receptors. In agreement, the pharmacological inhibition of Class I HDAC (HDAC1/3) mirrored butyrate's effects, suggesting that butyrate impacts MC function by HDAC1/3 inhibition. Taken together, butyrate epigenetically modifies histones and downregulates the SCF/KIT/p38/Erk signalling axis, leading to the attenuation of MC function, validating its ability to suppress MC-mediated inflammation. Therefore, butyrate supplementations could offer a potential treatment strategy for allergy and asthma via epigenetic alterations in MCs.

Protein Kinase C α and ß compensate for each other to promote stem cell factor-mediated KIT phosphorylation, mast cell viability and proliferation.

Mast cells (MCs) develop from hematopoietic progenitors and differentiate into mature MCs that reside within connective or mucosal tissues. Though the number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCß isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCß activate p38 mitogen activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cell proliferation. To ascertain the individual roles of PKCα and PKCß, we knocked down either PKCα or PKCß or both via short hairpin RNA (shRNA) and analyzed KIT phosphorylation, p38 MAPK phosphorylation, and MC viability and proliferation. To our surprise, downregulation of neither PKCα nor PKCß affected MC viability and proliferation. In contrast, blocking both PKCα and PKCß significantly attenuated SCF-induced cell viability and proliferation, suggesting that PKCα and PKCß compensate for each other downstream of SCF signaling to enhance MC viability and proliferation. Our results not only suggest that PKC classical isoforms are novel therapeutic targets for SCF/MC-mediated inflammatory and allergic diseases, but they also emphasize the importance of inhibiting both PKCα and ß isoforms simultaneously to prevent MC proliferation.

A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells.

Given that mast cells are pivotal contributors to allergic diseases, various allergy treatments have been developed to inhibit them. Omalizumab, an anti-immunoglobulin E antibody, is a representative therapy that can alleviate allergy symptoms by inhibiting mast cell degranulation. However, omalizumab cannot reduce the proliferation and accumulation of mast cells, which is a fundamental cause of allergic diseases. c-Kit is essential for the proliferation, survival, and differentiation of mast cells. Excessive c-Kit activation triggers various mast cell diseases, such as asthma, chronic spontaneous urticaria, and mastocytosis. Herein, we generated 2G4, an anti-c-Kit antibody, to develop a therapeutic agent for mast cell diseases. The therapeutic efficacy of 2G4 antibody was evaluated in LAD2, a human mast cell line. 2G4 antibody completely inhibited c-Kit signaling by blocking the binding of stem cell factor, known as the c-Kit ligand. Inhibition of c-Kit signaling led to the suppression of proliferation, migration, and degranulation in LAD2 cells. Moreover, 2G4 antibody suppressed the secretion of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, C-C motif chemokine ligand 2, brain-derived neurotrophic factor, and complement component C5/C5a, which can exacerbate allergy symptoms. Taken together, these results suggest that 2G4 antibody has potential as a novel therapeutic agent for mast cell diseases.