Allergy discordant twins do not exhibit differences in gene expression in non-switched and switched B cellsStephan Schneider1, Pattraporn Satitsuksanoa1, Huseyn Babayev2, Willem van de Veen1, Iris Chang3, Minglin Yang1, Cezmi A. Akdis1, Kari Nadeau4, Mübeccel Akdis11 Swiss Institute of Allergy and Asthma Research, University of Zurich, Davos, Switzerland2 Abant Izzet Baysal University Hospital, Department of Medical Microbiology, Bolu, Türkiye3 Department of Pediatrics/ Division of Hematology, Oncology, Stem Cell Transplantation, and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA4 Harvard TH Chan School of Public Health, Department of Environmental Health, Boston, Mass. USA 02115To the Editor,Allergy is a globally spread affliction that is based on dysregulation of immune responses towards ‘harmless’ antigens.1 B cells and their regulation are at the center of allergic disease mechanisms.2,3 They produce IgE, which is essential for allergen-induced mast cell and basophil degranulation.4 The common consensus is, that the regulation of B cells is closely tied to tolerance and allergy.2,5 In allergy this regulation is likely dysfunctional, resulting in different B cell behaviour. Therefore we performed whole transcriptome analysis on peripheral B cells from twins who were discordant or concordant for allergic diseases to identify allergy-associated differential gene expression patterns (i.e. receptors to a type 2 response). We sorted switched and non-switched B cells from 16 twin pairs that were either healthy, allergy discordant or allergy concordant and performed bulk RNA sequencing to identify differences in gene expression patterns. We hypothesized that there would be significantly different expressions in pathways related to B cell activation, B cell regulation or B cell isotype switching correlating to allergic symptoms. The donors were monozygotic and mainly allergic to timothy grass, birch tree pollen and/or house dust mites; none had taken or were on allergen-immunotherapy (Supplementary Table 1-3). Samples were obtained via ethics approval and consent. We sorted switched and unswitched B cells from bio-banked PBMCs, extracted RNA, depleting the ribosomal RNA, and performed 100bp single-end RNA sequencing on the Illumina Novaseq 6000 platform.6 Unbiased clustering, excluding long non-coding RNA, (Figure 1A) showed that the main influences for clustering in descending order are switched vs non-switched B cells, twin pairs and then their concordance status. An individuals allergy status had little to no significant impact on the overall clustering. The same holds true for the PCA analysis of the top 300 significant genes across all samples (Figure 1B). Comparing healthy concordant to allergy concordant twins could be greatly influenced by confounding factors like twin pair similarities and grouping by concordance instead of their actual healthy status. For these reasons, we compared the healthy vs allergic within the discordant twin pairs to avoid these influences for gene expression analysis. In this comparison, neither switched nor non-switched B cell show pathways that would traditionally be associated with allergies or B cell regulation in particular (Supplementary Table 4). A log fold change of greater than 0.5 and a p-value of 0.05 gave a FDR of 0.9999. Adjusting for a FDR below 0.05 (p-value <0.00001) resulted no differentially expressed genes for switched and 5 genes without a common pathway in non-switched B cells. Using this methodology of analysis, we did not find significant differences in pathway regulations on the wider scale of B cells between allergy-discordant twins.PCA analysis for the top 300 genes by the p-value of the allergy-discordant twins confirmed that allergic vs healthy twins do not group by allergy status (Figure 2A). Pathway analysis of the top genes in allergy-discordant twins does not reveal any cohesive pathways (allergy vs healthy) in the switched B cells (Figure 2B). There is one pathway in the upregulated genes of the non-switched B cells, whose genes are associated with the cell cycle and not directly with immune functions.Our results show no indication that there is a general dysregulation of B cells as an underlying cause for allergies. Any differences that might exist are too subtle to be observed across B cells. We propose that distinctions between allergic and non-allergic individuals may only be noticeable in allergen-specific B cells. These effects are probably overshadowed by the variability among individuals due to the rarity of allergen-specific B cells in the overall B cell population.