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The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

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Background: Symptoms in patients with systemic mastocytosis (SM) are associated with an increase in mast cell burden and release of mast cell-derived mediators. The most frequent presentation of SM is indolent SM (ISM), with moderate symptoms and prognosis. Basophil numbers in these patients are generally normal. However, when examining basophil activation in patients with ISM, we noted an abnormal response to N-formylmethione-leucyl-phenylalanine (fMLP). Objective: Our aim was to compare basophil responsiveness to fMLP and anti-IgE in healthy volunteers and patients with ISM and relate the findings to fMLP receptor (FPR) expression. Methods: Basophils isolated from peripheral blood of 15 patients with ISM and 14 healthy volunteers were stimulated with fMLP or anti-IgE. CD63 expression to assess basophil activation and expression of FPRs were assessed by flow cytometry. Results: Baseline expression of CD63 on basophils was similar between the healthy volunteers and patients with ISM. fMLP induced higher expression of CD63 on basophils from patients with ISM, whereas responses to anti-IgE were similar between groups. Basophils from patients with ISM also had higher fMLP1 receptor (FPR1) expression, wheresas FPR2 and FPR3 were not detected. fMLP blocked the binding of anti-FPR1 antibody to FPR1, consistent with the conclusion that fMLP signals through FPR1. Conclusions: Level of fMLP-induced basophil activation is higher in patients with ISM, which is associated with an increase in FPR1 expression. Further investigation is needed to determine why FPR1 expression is elevated, whether such expression might serve as an additional surrogate marker in the diagnosis of ISM, and whether enhanced responses of basophils to fMPL might have some relationship to unexplained episodes of mediator release.

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Mastocytosis is a clonal myeloid disorder defined by an increase and accumulation of mast cells (MC) in one or multiple organ systems. The complex pathology of mastocytosis results in variable clinical presentations, courses, and outcomes. The World Health Organization (WHO) divides the disease into cutaneous mastocytosis (CM), several forms of systemic mastocytosis (SM), and MC sarcoma. In most SM patients, a somatic KIT mutation, usually D816V, is identified. Patients diagnosed with CM or non-advanced SM, including indolent SM, have a near-normal life-expectancy, whereas those with advanced SM, including aggressive SM and MC leukemia, have limited life-expectancy. Since 2001, a multidisciplinary consensus group consisting of experts from the European Competence Network on Mastocytosis (ECNM) and American Initiative in Mast Cell Diseases (AIM), has supported the field by developing diagnostic criteria for mastocytosis. These criteria served as the basis for the WHO classification of mastocytosis over two decades. More recently, an international consensus group (ICC) proposed slightly modified diagnostic criteria and a slightly revised classification. In this article, these changes are discussed. Furthermore, we propose harmonization among the proposals of the AIM/ECNM consensus group, WHO, and ICC. Such harmonization will facilitate comparisons of retrospective study results and the conduct of prospective trials.

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Activating mutations in KIT, particularly D816V, have been associated with mastocytosis. Additionally, expression of heterozygous KIT M541L has been primarily reported in patients with pediatric mastocytosis. We thus examined the prevalence of this variant in pediatric and adult patients with mastocytosis (n = 100) compared to ancestry-matched 1000 genomes controls (n = 500) and patients with idiopathic anaphylaxis (n = 23). We then compared clinical symptoms and laboratory data on patients with systemic and cutaneous mastocytosis and bone marrow histopathology on a matched cohort with and without the KIT M541L variant. Overall, the KIT M541L variant was identified in 19 individuals; the majority were diagnosed with systemic mastocytosis (89.4%) with an associated KIT D816V mutation. There were no significant differences in peripheral blood parameters between groups. Patients with mastocytosis carrying the KIT M541L variant did not demonstrate significant differences in symptomatology compared to a matched reference cohort (n = 13/81) without KIT M541L. In patients with idiopathic anaphylaxis, no significant associations were observed. This study uniquely examines the prevalence and impact of the KIT M541L variant in both adult and pediatric patients with mastocytosis further stratified by disease variant. To our knowledge, this is the first case/control study to show a significant genetic association with mastocytosis at the KIT M541L locus.

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The receptor tyrosine kinase (RTK) KIT and its ligand stem cell factor (SCF) are essential for human mast cell (huMC) survival and proliferation. HuMCs expressing oncogenic KIT variants secrete large numbers of extracellular vesicles (EVs). The role KIT plays in regulating EV secretion has not been examined. Here, we investigated the effects of stimulation or inhibition of KIT activity on the secretion of small EVs (sEVs). In huMCs expressing constitutively active KIT, the quantity and quality of secreted sEVs positively correlated with the activity status of KIT. SCF-mediated stimulation of KIT in huMCs or murine MCs, or of transiently expressed KIT in HeLa cells, enhanced the release of sEVs expressing exosome markers. In contrast, ligand-mediated stimulation of the RTK EGFR in HeLa cells did not affect sEV secretion. The release of sEVs induced by either constitutively active or ligand-activated KIT was remarkably decreased when cells were treated with KIT inhibitors, concomitant with reduced exosome markers in sEVs. Similarly, inhibition of oncogenic KIT signalling kinases like PI3K, and MAPK significantly reduced the secretion of sEVs. Thus, activation of KIT and its early signalling cascades stimulate the secretion of exosome-like sEVs in a regulated fashion, which may have implications for KIT-driven functions.

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Mast cell activation syndrome (MCAS) is a term applied to several clinical entities that have gained increased attention from patients and medical providers. Although several descriptive publications about MCAS exist, there are many gaps in knowledge, resulting in confusion about this clinical syndrome. Whether MCAS is a primary syndrome or exists as a constellation of symptoms in the context of known inflammatory, allergic, or clonal disorders associated with systemic mast cell activation is not well understood. More importantly, the underlying mechanisms and pathways that lead to mast cell activation in MCAS patients remain to be elucidated. Here we summarize the known literature, identify gaps in knowledge, and highlight research needs. Covered topics include contextualization of MCAS and MCAS-like endotypes and related diagnostic evaluations; mechanistic research; management of typical and refractory symptoms; and MCAS-specific education for patients and health care providers.

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Background: Autoantibodies to type I interferons have been identified in association with a variety of inflammatory and autoimmune diseases. Type I interferons have demonstrated inhibitory effects on mast cell proliferation and degranulation. Systemic mastocytosis (SM) is a disease characterized by increased mast cell burden and mediator release. Whether autoantibodies to type I interferon are present in the sera of patients with SM, and if so, whether they correlate with characteristics of disease, is unknown. Objective: The purpose of this study was to determine whether autoantibodies to type I interferons are observed in the sera of patients with SM, and if so, whether they correlate with biomarkers of disease severity. Methods: We analyzed sera from 89 patients with SM for concentrations of autoantibodies to type I interferon by using a multiplex particle-based assay and signal neutralization capacity by using a STAT1 activity assay and then compared these measurements with those in a database of information on 1284 healthy controls. Results: Our cohort was predominantly female (57.3%), with a median age of 56 years. Of the cohort members, 13 produced autoantibodies to IFN-ß, 3 to IFN-ω, and 0 to IFN-α. None of the 13 sera demonstrated signal neutralization. Neither autoantibody concentration nor signaling inhibition measurements correlated with tryptase concentrations or D816V allele burden. Conclusion: Although a small subpopulation of patients with SM have autoantibodies to type I interferons, there was no correlation between autoantibody production and signaling inhibition. These data are consistent with the conclusion that autoantibodies to type I interferon do not play a significant role in the pathogenesis or severity of SM.

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ABSTRACT: Timely diagnosis of systemic mastocytosis (SM) remains challenging because of care heterogeneity. We implemented a standardized approach for SM screening and diagnosis using a novel health care system-wide international screening registry. A retrospective analysis assessed rates of SM, cutaneous mastocytosis (CM), and molecular diagnoses before and 2 years after care standardization. The accuracy of individual and combined SM screening tests, basal serum tryptase (BST) ≥11.5 and ≥20.0 ng/mL, REMA ≥2, monomorphic maculopapular CM (MPCM), and elevated BST based upon tryptase genotype, was analyzed. Tryptase genotyping and high-sensitivity KIT p.D816V testing increased substantially 2 years after care standardization. SM diagnoses doubled from 47 to 94, and KIT p.D816V molecular diagnoses increased from 24 to 79. Mean BST and KIT p.D816V variant allele frequency values were significantly lower in patients diagnosed after standardization. Hereditary-alpha tryptasemia prevalence was increased in SM before care standardization (4/30 [13.3%]) but reflected the general population prevalence 2 years later at (5/76 [6.6%]). Elevated BST based upon genotype and BST ≥11.5 ng/mL had the highest sensitivities at 84.2% and 88.3%, respectively. The presence of monomorphic MPCM, elevated BST based upon tryptase genotype, and the combination of REMA ≥2 with elevated BST based upon tryptase genotype had specificities >90%. BST >20.0 ng/mL had low sensitivity and specificity and was not required to establish any indolent SM (ISM) diagnosis. Care standardization increased SM diagnosis rates, particularly in patients with low BSTs. Stratifying BST based upon genotype had the best overall sensitivity and specificity of any ISM screening test and improved the REMA score specificity.

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Physiological levels of basal serum tryptase vary among healthy individuals, depending on the numbers of mast cells, basal secretion rate, copy numbers of the TPSAB1 gene encoding alpha tryptase, and renal function. Recently, there has been a growing debate about the normal range of tryptase because individuals with the hereditary alpha tryptasemia (HαT) trait may or may not be symptomatic, and if symptomatic, uncertainty exists as to whether this trait directly causes clinical phenotypes or aggravates certain conditions. In fact, most HαT-positive cases are regarded as asymptomatic concerning mast cell activation. To address this point, experts of the European Competence Network on Mastocytosis (ECNM) and the American Initiative in Mast Cell Diseases met at the 2022 Annual ECNM meeting and discussed the physiological tryptase range. Based on this discussion, our faculty concluded that the normal serum tryptase range should be defined in asymptomatic controls, inclusive of individuals with HαT, and based on 2 SDs covering the 95% confidence interval. By applying this definition in a literature screen, the normal basal tryptase in asymptomatic controls (HαT-positive persons included) ranges between 1 and 15 ng/mL. This definition should avoid overinterpretation, unnecessary referrals, and unnecessary anxiety or anticipatory fear of illness in healthy individuals.

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The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.

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BACKGROUND: Intestinal epithelial integrity compromise has been identified in gastrointestinal (GI), atopic, and autoimmune diseases. OBJECTIVE: Episodes of idiopathic anaphylaxis (IA) are often accompanied by GI manifestations. We, therefore, sought to determine whether surrogate markers of GI permeability were aberrant in this patient population. METHODS: Serum concentrations of zonulin, intestinal fatty acid binding protein (I-FABP), and soluble CD14 (sCD14) measured in 54 patients with IA were compared with concentrations in healthy controls (HCs); and correlated with clinical and laboratory parameters. RESULTS: The I-FABP was elevated in sera of patients with IA compared with HCs (median 1,378.0 pg/mL vs 479.0 pg/mL, respectively; P < .001). The sCD14 was also elevated compared with HCs (median 2,017.0 ng/mL and 1,189.0 ng/mL, respectively; P < .001), whereas zonulin was comparable between patients with IA and HCs (median 49.6 ng/mL vs 52.4 ng/mL, respectively; P = .40). The I-FABP was elevated in patients with IA who experienced vomiting and/or diarrhea compared with patients with IA who did not (P = .0091). CONCLUSIONS: The I-FABP and sCD14 are elevated in the serum of patients with IA. Elevations in these biomarkers of IA provides evidence that increased GI permeability, as is observed in other allergic conditions such as food allergy, is a common finding in those with IA and offers possible insight into the pathogenesis of this disease.

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Background: Patients with a low IgG level alone or with low IgA or IgM levels have been reported to be susceptible to respiratory tract infections and recurrent sinusitis. Patients diagnosed with CVID have a higher prevalence of autoimmune diseases and lymphoid malignancies. Mastocytosis is a myeloproliferative disease, not typically associated with autoimmune disease or frequent infections. Objective: We sought to determine the distribution of immunoglobulins in children and adults with mastocytosis. Evaluate the impact of low immunoglobulins on the clinical management of patients with mastocytosis. Methods: We performed a 10-year retrospective analysis on 320 adult and pediatric patients with mastocytosis for immunoglobulins using an electronic medical query. We identified 25 adults and 9 children with one or more low immunoglobulins. Patient records were examined for a history of infections and autoimmune disorders. Results: Serum immunoglobulins in children and adults with mastocytosis fell within a normal range. Among patients with low IgG levels alone or with low IgM and /or IgA, 20% had a history of infections and 20% of adults had autoimmune disorders. The most common infection was recurrent otitis media (OM). Conclusion: Patients with mastocytosis typically have normal immunoglobulins. With few exceptions, those with low immunoglobulins did not have frequent infections or autoimmune diseases. This data supports the conclusion that routine determination of immunoglobulins in patients with mastocytosis is not required and reserved for patients with clinical conditions, which might be related to an immunoglobulin deficiency.

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Anaphylaxis is an acute, potentially life-threatening systemic allergic reaction for which there are no known reliable preventative therapies. Its primary cell mediator, the mast cell, has several pathophysiologic roles and functions in IgE-mediated reactions that continue to be poorly understood. Recent advances in the understanding of allergic mechanisms have identified novel targets for inhibiting mast cell function and activation. The prevention of anaphylaxis is within reach with new drugs that could modulate immune tolerance, mast cell proliferation and differentiation, and IgE regulation and production. Several US Food and Drug Administration-approved drugs for chronic urticaria, mastocytosis, and cancer are also being repurposed to prevent anaphylaxis. New therapeutics have not only shown promise in potential efficacy for preventing IgE-mediated reactions, but in some cases, they are able to inform us about mast cell mechanisms in vivo. This review summarizes the most recent advances in the treatment of anaphylaxis that have arisen from new pharmacologic tools and our current understanding of mast cell biology.

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Mastocytosis is a heterogeneous group of neoplasms defined by a numerical increase and accumulation of clonal mast cells (MCs) in various organ systems. The disease may present as cutaneous mastocytosis or systemic mastocytosis (SM). On the basis of histopathological and molecular features, clinical variables, and organ involvement, SM is divided into indolent SM, smoldering SM, SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia. Each variant is defined by unique diagnostic criteria and a unique spectrum of clinical presentations. A key driver of MC expansion and disease evolution is the oncogenic machinery triggered by mutant forms of KIT. The genetic background, additional somatic mutations, and comorbidities also contribute to the course and prognosis. Patients with SM may also suffer from mediator-related symptoms or even an MC activation syndrome. This article provides an update of concepts on the genetics, etiology, and pathology of mastocytosis, with emphasis on diagnostic criteria and new treatment concepts.

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Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.

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Mast cells are present in all vascularized tissues, often associated with blood vessels, glandular structures, and nerves, and they tend to be more numerous in tissues that interface with the external environment, including the skin and gastrointestinal tract. These mast cells are involved in both innate and acquired immunity, as well as in other biologic processes, including wound healing. Mast cell numbers within tissues are remarkably consistent, although mast cell numbers somewhat increase in association with inflammation. Human mast cells have two principal receptors that when stimulated, lead to cellular activation.

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Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody-coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT-EVs. Immunoblot analysis demonstrated KIT-EVs have a distinct protein profile from KIT-depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT-EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT-EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle- and exosome-like EVs. Further, selective KIT-immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.

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