Background: Oral and sublingual immunotherapies for peanut allergy have demonstrated efficacy in small clinical trials; however, mechanisms and biomarkers correlating with clinical outcomes remain elusive. Previous studies have demonstrated a role for IgG in post-OIT plasma in the suppression of IgE-mediated mast cell reactions. Objective: The aim of this study was to characterize the role that peanut oral and sublingual immunotherapy-induced plasma factors play in the inhibition of ex vivo basophil activation and whether inhibitory activity is associated with clinical outcomes. Methods: Plasma samples from subjects on placebo, peanut oral immunotherapy (OIT) or peanut sublingual immunotherapy (SLIT), and IgG-depleted plasma or the IgG fraction were incubated with sensitized basophils, and the inhibition of basophil activation following stimulation with peanut extract was measured. Basophil inhibition results were compared between the two routes of immunotherapy, time on treatment and clinical outcomes. Results: Plasma from subjects after 12 months of active peanut OIT, but not placebo, inhibits basophil activation ex vivo. Depletion of IgG abrogated the blocking effect of OIT plasma, while the IgG fraction substantially blocked basophil activation. Basophils are inhibited to a similar extent by undiluted OIT and SLIT plasma; however, diluted OIT plasma from the time of desensitization challenge inhibited basophils more than diluted SLIT plasma from time of desensitization challenge.Plasma from subjects who experienced sustained unresponsiveness following OIT inhibited basophils to a greater extent than plasma from subjects who were desensitized, but this was not true for SLIT. Conclusions and ClinicalRelevance: Peanut immunotherapy induces IgG-dependent functional changes in plasma that are associated with OIT but not SLIT clinical outcomes. Understanding the mechanisms of peanut OIT and SLIT may help derive informative biomarkers. K E Y W O R D S basophil activation, food allergy, IgG, immunoglobulin, immunotherapy, peanut allergy
Background10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms.ObjectiveWe used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells.MethodsTwo human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8.ResultsPeanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation.ConclusionAllergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.
RATIONALE: Currently, 1% of children in the US have a peanut allergy. With no existing cure for peanut allergy, treatments that inhibit allergic effector cell activation and degranulation may prove useful in preventing reactions. CD300c is an activating receptor known to cross-react with inhibitory receptor and family member CD300a. Engagement of CD300a has been shown to halt degranulation of mast cells. We are interested in utilizing CD300c in peanut allergy to determine its ability in inhibiting allergic effector cell degranulation. METHODS: Peanut allergy was confirmed in 10 subjects by specific-IgE > 10 kU/L and a positive double-blind, placebo-controlled, food challenge. Peanut allergic plasma from these subjects was used to sensitize Rat Basophilic Leukemia SX-38 cells in the presence or absence of anti-CD300c antibody at varying concentrations. Degranulation was then measured by beta-hexosaminidase release following peanut antigen stimulation of the cells. RESULTS: We showed clear and consistent trends in inhibition of mast cell degranulation by use of anti-CD300c. We reached an average percent inhibition of 22.17% after treatment with anti-CD300c at low concentrations. Low concentrations of anti-CD300c showed greater inhibition of beta-hexosaminidase release when compared with higher concentrations of anti-CD300c. CONCLUSIONS: Anti-CD300c antibodies at low concentrations have the ability to decrease degranulation of mast cells sensitized to peanut. This indicates that cross-reaction with CD300a may be occurring at lower concentrations, leading to decreased cellular degranulation. Further studies will elucidate the roles of other CD300 family members in mast cell inhibition and activation.
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