A consensus protocol for the Basophil Activation Test for multicenter collaboration and External Quality AssuranceAuthors: Pascal, M# 1, Edelman SM#2, Nopp, A#3, Möbs, C4, Geilenkeuser, WJ5, Knol, EF6, Ebo, DG7, Mertens C7, Shamji, MH8, Santos, AF9,10, Patil, S11, Eberlein, B*12, Mayorga, C*13, Hoffmann HJ14*Affiliations1 Immunology Department, Centre de Diagnòstic Biomèdic, Hospital Clínic de Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Spain.2 Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland, present address Aimmune Therapeutics, Finland3 Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, and Sachs´ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden4 Department of Dermatology and Allergology, Philipps-Universität Marburg, Marburg, Germany5 Reference Institute for Bioanalytics, Bonn, Germany6 Center of Translational Immunology and Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.7 Faculty of Medicine and Health Sciences, Department of Immunology-Allergology- Rheumatology, University of Antwerp, Antwerp, Belgium8 National Heart and Lung Institute, Imperial College London, UK and NIHR Imperial Biomedical Research Centre, UK9 Department of Women and Children’s Health (Pediatric Allergy) & Peter Gorer Department of Immunobiology, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom10 Children’s Allergy Service, Evelina London Children’s Hospital, Guy’s and St Thomas’ Hospital, London, United Kingdom11 Division of Allergy and Immunology, Departments of Medicine and Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States12 Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University Munich, Munich, Germany13 Allergy Clinical Unit, Hospital Regional Universitario de Málaga and Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA-BIONAND, Malaga, Spain;14 Department of Clinical Medicine, Aarhus University, Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark# shared first authors, * shared senior authorsCOIM Pascal, SM Edelman, A Nopp, C Möbs, EF Knol, SU Patil and C Mayorga have no conflict of interest regarding this work. B Eberlein received methodological and technical support from the company BUEHLMANN Laboratories AG (Schönenbuch, Switzerland) outside the submitted work. Dr Hoffmann reports grant from the Innovation Fund of Denmark, outside the submitted work. Dr Shamji reports grants awarded to institution from the Immune Tolerance Network, UK Medical Research Council, Allergy Therapeuitics, LETI Laboratories, Revolo biotherapeutics and Angany Inc. He has received consulting fees from Bristol Meyers Squibb and lecture fees from Allergy Therapeutics and LETI laboratories, all outside the submitted work. Dr. Santos reports grants from Medical Research Council (MR/M008517/1; MC/PC/18052; MR/T032081/1), Food Allergy Research and Education (FARE), the NIH, Asthma UK (AUK-BC-2015-01), the Immune Tolerance Network/National Institute of Allergy and Infectious Diseases (NIAID, NIH) and the NIHR through the Biomedical Research Centre (BRC) award to Guy’s and St Thomas’ NHS Foundation Trust, during the conduct of the study; speaker or consultancy fees from Thermo Scientific, Nutricia, Infomed, Novartis, Allergy Therapeutics, IgGenix, Stallergenes, Buhlmann, as well as research support from Buhlmann and Thermo Fisher Scientific through a collaboration agreement with King’s College London, outside the submitted work. Dr Geilenkeuser is an employee of Referenizinstitut für Bioanalytik, DE that provided logistic assistance and reagent support for the study.To the editorThe basophil activation test (BAT) has significant potential as a diagnostic tool to better phenotype and manage patients with IgE-mediated allergies, so that only a small proportion of patients need to be challenged. Sample, reagent, laboratory procedure, analysis protocols, and population characteristics can influence BAT performance (1,2). Regulatory approval and clinical implementation require extensive standardization of laboratory protocols, cytometer settings, and results interpretation (3). European national authorities require External Quality Assurance (EQA) of the performance of modern diagnostic laboratories by agencies independent of test suppliers to meet ISO 15189:2012, 15189:2013 and 9001:2015.Based on an online survey among 59 responding European laboratories performing BAT in 2017 (4,5) (Online Supplement; Results of the online survey), a Task Force was launched in 2018 to create the basis for a BAT-EQA. Round Robins (RR) were organized with seven shipments of 2 donors each to 7-10 European centers with overnight courier service from Bonn, DE. To minimize variation, prior to shipment, blood basophils were activated with 1 ul FcεRI antibody/ml of blood and stabilized with 0.2 mL Transfix (Cytomark, UK) per mL of blood to stabilize activated basophils up to 48 hours for staining (6). Fresh blood was included for stimulation and staining at the participating laboratory sites.We met after the third shipment to reach consensus on a protocol for BAT (Online Supplement; Proposed SOP for in house BAT). The threshold set on an unstimulated control sample was determined empirically on an independent data set as equal or greater than 2.5% with ROC curves based on data from patients with hypersensitivity to amoxicillin and patients with peanut allergy, (Online supplement, tables S1 and S2). This proposal did not find universal consensus among the authors.Data analysis started with identification of the relevant region in a scatter plot, followed by identification of basophils with the relevant markers, for instance, using low SSC and CD193 only or CD193 and CD123. Finally, the threshold was set at 2.5% of CD63 expression on resting basophils (Figure 1A). >5% CD63+basophils above that threshold in an activated sample was considered a positive response. This setting was used to obtain the percentage of CD63+ cells in centrally preactivated and locally activated blood samples; however, it was not adopted in all labs. Data from participating labs analyzed with their proprietary and the above standardized analysis compared well (online supplement, figure S4).The first two RR were used to establish coherence between participating laboratories. Data from RR3–RR7 were comparable. The standard deviation of activation measured at all participating centers was 16.8% in preactivated blood (Figure 1B) compared with 49.2% for samples activated and analyzed locally, illustrating the utility of using preactivated blood for EQA. Shipment to Málaga took 48h, and local activation of blood basophils was consistently suboptimal, consistent with a preliminary round robin from 2012, where the clinical outcome was robust up to 24 h. Centrally activated basophils performed as well in Málaga as in other centers.EQA for BAT is critical to facilitate routine implementation of this assay in the field of in vitro allergy diagnostics. The variability of the responses to our survey highlighted the importance and need for multicenter validation. Full validation and standardization of the BAT protocol and analysis is essential and possible for setting the grounds for controlled multicenter research studies as well as EQA. The BAT-EQA Task Force provides a standard operating protocol (Online supplement; Proposed SOP for in house BAT) and reference materials for the test to standardize and enhance the accuracy of BAT for both clinical and research collaborations and EQA.

Transcriptome changes during peanut oral immunotherapy and omalizumab treatmentTo the Editor,Peanut allergy is a common food allergy and the main cause of anaphylaxis among children1. In recent years, oral immunotherapy has emerged as a promising treatment for children with different IgE-mediated food allergies, although safety issues must be considered2. The main aim of immunotherapy is to induce tolerance or desensitization to an allergen which otherwise causes an allergic reaction. For oral immunotherapy this means ingesting the allergen in a controlled manner with gradually increasing dosages. Specifically, peanut oral immunotherapy (pOIT) is able to induce tolerance/desensitization3. While the pathogenesis of food allergy in general is relatively well-studied4, mechanisms of OIT-induced tolerance are not well understood. Omalizumab (anti-IgE) used as treatment for severe allergic asthma and other IgE-driven allergies, can facilitate OIT initiation5, however, little is known about the involved mechanisms, including possible changes at the transcriptional level. We therefore investigated transcriptional changes in whole blood using RNA-sequencing profiles during omalizumab treatment and pOIT in participants from the FASTX (Food Allergen Suppression Therapy with Xolair ®) study previously described in detail elsewhere5.In brief, peanut-allergic adolescents (n=23 of whom 17 completed the study, age 12-18 years) were started on omalizumab (baseline) and treated for at least 8 weeks before starting pOIT (pOIT start) while on omalizumab. The peanut-dose was gradually increased during the 8 weeks until reaching a maintenance dose. Guided by a basophil activation test (BAT/CD-sens)6 after 8 weeks on the maintenance dose, participants decreased the omalizumab dose by 50% (maintenance) and continued to decrease the omalizumab dose if pOIT was tolerated. Eleven patients were able to tolerate pOIT without omalizumab protection for >8 weeks and then passed an open peanut food challenge (final); 6 patients could not discontinue omalizumab, but blood samples were obtained for analysis after 2-3 years of omalizumab treatment (final); 6 patients dropped out of the study. RNA-sequencing was performed on whole blood at baseline, pOIT start, maintenance and final time-points using the NovaSeq 6000 platform. DESeq2 was used for differential expression analysis of the omalizumab effect and a linear mixed-effect model for analyses during pOIT in combination with omalizumab (pOIT+O) after adjustment for treatment outcome and cell type. A complete description of the treatment protocol and method is given in Appendix S1.General characteristics of the study participants at baseline can be found in Table S1 . To elucidate if omalizumab treatment alone induced alterations in peripheral blood gene expression, we investigated the two first timepoints, baseline and pOIT start, however no significant differences were observed (Figure S1 ). In the longitudinal analysis (pOIT start to final), 680 genes associated with pOIT+O at nominal p <0.005 (Table S2 ). The Gene Ontology (GO) biological process of the up- and down-regulation of these 680 genes are presented in Figure 1A,B . Upregulation of 337 genes were linked to GO terms “protein regulation and modification”, while “neutrophil degranulation, immune response, phagocytosis, and metabolic process” were among the top terms for the downregulated 343 genes. Out of the 680 genes, 16 were differentially expressed at false discovery rate (FDR) adjusted p<0.05 (Table 1, Figure S2 ). The three genes with the largest negative and positive coefficients, respectively, are displayed in Figure 1C,D ; downregulation of ASGR2 ,GPBAR1 and HM13, and upregulation of USP44 ,ICOS and CDKN2AIP . Finally, we evaluated the enrichment of 680 pOIT+O-associated genes, relative to peripheral blood gene expression associated with acute peanut allergic reactions in a recently published clinical study by Watson et al using the same p-value cut-off (p<0.005)7. Out of our 680 significant genes, 108 genes overlapped with the differentially expressed genes in Watson et al7, mostly with opposite direction, Penrichment = 0.0095 (Figure 2 ).Our results demonstrate that omalizumab treatment alone does not induce alterations in whole blood gene expression in patients with severe food allergy. This is not surprising given that these patients were unexposed to peanut allergen at the time of blood sampling, and any concomitant asthma, rhinitis or eczema were well controlled. However, the longitudinal analysis during pOIT+O identified up- and downregulation of several immune-related genes. CD278/ICOS (Inducible T-cell costimulatory) is expressed on activated T-cells and appears to play a role in directing effector T-cell differentiation and responses during inflammatory conditions8. ICOS-expression on T regulatory cells and T follicular helper cells may be involved in the allergic disease mechanism9. In the pathway analyses, we observed significant enrichment for several GO biological process terms related to T-cell function and immune responses. Notably, we have previously described alterations in T-cell polyclonal in vitroactivation during pOIT +O in the FASTX study10. Comparing our findings with data described by Watson et al7 , suggests that pOIT+O may alter the expression level of many genes that were found activated during an acute peanut allergy reaction.The main limitations of this study are lack of any control subjects and small sample size. Moreover, further studies are needed to evaluate the long-term biological effect of pOIT+O.In conclusion, omalizumab treatment alone does not alter the transcriptional signature in peripheral blood of peanut allergic patients, but during pOIT+O, several immune-related signatures were observed. These results may provide insights into mechanisms of allergen tolerance.

Background: IgA nephropathy (IgAN) advances from multiple pathogenic “hits” resulting in poorly O-galactosylated IgA1 glycoforms (Gd-IgA1), production of antibodies and glomerular deposition of immune complexes. A sequence of immune responses arising from plasma cells, T cells and antigen presenting cells (APCs), causes glomerular injury. Methods: This study was designed to phenotype subsets of B cells, monocytes and T cells in the peripheral circulation and their association with inflammatory cytokines and renal function in patients with IgAN, healthy controls (HC) and disease controls with autosomal dominant polycystic kidney disease (ADPKD). Results: We observed a significant decrease in the proportion of pre-switched B cells and plasmablasts, but an increase in long-lived plasma cells in the peripheral circulation of IgAN patients compared to HC. The proportion of non-classical monocytes was significantly higher in IgAN patients compared to both HC and ADPKD. We also report an association between sCD40L levels and the proportion of pre-switched B cells, as well as sCD40L and MCP-1 levels and albuminuria in IgAN patients. Conclusions: We applied an easy-access method to analyze subsets of immune cells as well as relevant inflammatory mediators in IgAN patients. Our data demonstrate an altered B cell profile that indicates a pathophysiological role of the B cell lineage and an increased proportion of non-classical monocytes that suggests their role in the disease process.