Title Page:Article Type: Letter to the EditorTitle: Anaphylaxis to a blood feeding leechAuthors: Carmen H. Li MSc1,2*, Maggie Jiang MD3*, Gabriele Gadermaier PhD4, Sebastian Kvist PhD5,6,7 Julia E. M. Upton MD8,9, Xiaojun Yin PhD1, Jennifer A. Hoang MSc MDS1,10, Mikhail Monteiro BSc1, Lisa Hung PhD1,10, Akash Kothari MSc1,2, Theo J. Moraes MD PhD1,9, Peter Vadas MD PhD3,10**, Thomas Eiwegger MD1,10,11,12***Co-first author**Co-senior author(1) Translational Medicine Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada(2) Institute of Medical Science, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada(3) Division of Clinical Immunology and Allergy, St. Michael’s Hospital(4) Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria(5) Department of Ecology and Evolutionary Biology, University of Toronto, ON, Canada(6) Department of Invertebrate Zoology, Royal Ontario Museum, Toronto, ON, Canada(7) Swedish Museum of Natural History, Stockholm, Sweden(8) Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Hospital for Sick Children(9) Department of Pediatrics, University of Toronto, Toronto, ON, Canada(10) Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Ontario, Toronto, Canada(11) Karl Landsteiner University of Health Sciences, Krems, Austria(12) Department of Pediatric and Adolescent Medicine, University Hospital St. Pölten, St. Pölten, AustriaCorrespondence:  Thomas Eiwegger, MDChair, Department of Pediatric and Adolescent MedicineUniversity Hospital St. PöltenDunant-Platz 1, 3100 St. Pölten, AustriaEmail: [email protected].: +43 2742-9004-11740

Background: Peanut allergy is among the most severe and common food allergies. The diagnosis has a significant impact on the quality of life for patients and their families. An effective management approach depends on accurate, safe, and easily implementable diagnostic methods. We previously developed a cell-based assay using Hoxb8 mast cells (Hoxb8 MCs) aimed at improving clinical allergy diagnosis. In this study we assessed its diagnostic performance by measuring blinded sera from a prospectively enrolled and pre-validated peanut allergy cohort. Methods: Hoxb8 MCs were passively sensitized with sera from peanut-allergic and peanut tolerant children and adolescents (n=96). Degranulation of Hoxb8 MCs was quantified upon stimulation with dose-titrated peanut extract by means of flow cytometry, using CD107a as activation marker. The results from the Hoxb8 mast cell activation test (Hoxb8 MAT) were compared to established diagnostic assays such as the skin prick test (SPT), specific IgE (sIgE) levels, and the basophil activation test (BAT). Additionally, serum samples from BAT non-responders were assessed with the Hoxb8 MAT. Results: Hoxb8 MAT displayed a robust dose-dependent activation to peanut extract, with a cut-off value of ≤5.2% CD107a positive cells. The diagnostic accuracy was highest at allergen concentrations ≥100 ng/ml, with an area under the receiver operating characteristic curve (AUROC) of 0.97, 93% sensitivity, and 96% specificity, outperforming traditional SPT and sIgE tests. When compared to BAT, Hoxb8 MAT exhibited comparable diagnostic efficacy. However, sera from BAT non-responders were accurately classified into allergics and non-allergics by the Hoxb8 MAT. Conclusions: The Hoxb8 MAT demonstrated a very good diagnostic precision in patients prospectively assessed for peanut allergy comparable to the fresh blood based BAT. It also demonstrated its value for accurate classification of BAT non-responders into allergic and non-allergic individuals. Further investigations into its utility in the routine clinical setting are warranted.

Background: Food allergy affects up to 8% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). Methods: PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0-24h. Distribution of relevant cell subsets was confirmed using single cell nuclear sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. Results: PCIS viability was maintained for 24h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. Conclusion: PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.

Article Type : Letter to the EditorTitle : Whole blood high dimensional single cell functional profiling of basophils in peanut allergy