T regulatory cells from people with asthma show a Th2-like phenotypeKirstin Jansen1, Oliver F. Wirz1, Willem van de Veen1,2, Ge Tan1,3, Milena Sokolowska1, Simon D. Message4, Tatiana Kebadze4, Nicholas Glanville4, Patrick Mallia4, Cezmi A. Akdis1,2, Sebastian L. Johnston4, Kari Nadeau5 and Mübeccel Akdis1*1 Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland.2 Christine Kühne – Center for Allergy Research and Education (CK-CARE), Davos, Switzerland.3 Functional Genomics Center Zürich, ETH Zürich/University of Zürich, Zürich, Switzerland.4 National Heart and Lung Institute, Imperial College London, United Kingdom.5 Sean N. Parker Center for Allergy and Asthma Research, Department of Medicine, Stanford University, Palo Alto, CA, USA.* Corresponding author:Mübeccel Akdis, MD, PhD.Swiss Institute of Allergy and Asthma Research (SIAF)Herman-Burchard-Strasse 9CH-7265 Davos-Wolfgang, SwitzerlandE-mail: email@example.comTel.: +41 81 410 08 48Declaration of fundingM. Akdis has received research support from the Swiss National Science Foundation No. 320030-159870/310030-179428 and PREDICTA (No: 260895) and the Sean N Parker Center for Allergy and Asthma Research at Stanford University. C.A. Akdis is employed by the Swiss Institute of Allergy and Asthma Research, University of Zurich; the Swiss National Science Foundation No. 310030-156823, and the Christine Kühne – Center for Allergy Research and Education (CK-CARE). M. Sokolowska received research grant from the Swiss National Science Foundation No. 310030_189334/1 and from the GSK. The experimental infection study was supported by a Medical Research Council Clinical Research Fellowship (to S.D.M.), a British Medical Association H.C. Roscoe Fellowship (to S.D.M.), British Lung Foundation/Severin Wunderman Family Foundation Lung Research Program Grant P00/2, Asthma UK Grants 02/027 and 05/067, Welcome Trust Grants 063717 and 083567/Z/07/Z for the Centre for Respiratory Infection, Imperial College, and the National Institute for Health Research (NIHR) Biomedical Research Center funding scheme. S. L. Johnston is the Asthma UK Clinical Professor (grant CH11SJ), is an NIHR Emeritus Senior Investigator and was supported by MRC Centre Grant G1000758, Asthma UK Centre Grant AUK-BC-2015-01 and European Research Council Advanced Grant 788575. K.C. Nadeau is supported by NIH grant U19 AI104209 (Asthma and Allergic Diseases Cooperative Research Center), U01 AI140498 and R01 AI140134 and the Naddisy Foundation.To the editor,Asthma is the most common chronic inflammatory disease of the lung, characterised by wheezing, shortness of breath and variable airflow obstruction. It is a heterogeneous disease that can be classified into different endotypes of which T2-high - allergic asthma is one of the most common forms, especially in children. Allergic asthma is characterised by increased IgE and type-2 cytokines, including IL-5, IL-4 and IL-131.Thus far, it is not completely understood why these type-2 responses are poorly controlled in asthma. T regulatory cells (Treg cells) are key mediators in controlling type 2 responses. However, under certain conditions, Treg cells can display a pathogenic and proinflammatory phenotype and contribute to disease pathogenesis2. Treg cells of food allergic children showed a T helper 2 (Th2)-like phenotype. Whether this Th2-like phenotype of Treg cells is also present in asthmatic individuals is unknown.Therefore, in this exploratory study, we compared the gene-expression profile of Tregs from people with stable allergic-asthma to non-allergic controls without asthma. We isolated PBMCs from 5 people with asthma and 4 controls (Table S1) and sorted Treg cells with flow cytometry (CD3+CD4+D25hiCD127low). Then, we isolated RNA from the sorted Treg cells and performed RNA-seq (See Supplemental information for detailed methods). In total, 369 genes were differentially expressed between Treg cells from asthmatic individuals and controls (P<0.01) (Supplemental Figure 1). We clustered the genes into different groups: Treg cell markers, cytokine receptors, virus related, transcription factors, cytokines and others (Figure 1A). Interestingly, we found that the expression of FOXP3was reduced in Treg cells from asthmatic individuals (Figure 1B). This is in line with a previous study that observed a lower expression ofFOXP3 in Treg cells from individuals with asthma3. Interestingly FOXP3 expression inversely correlated with the IgE levels found in the serum (Figure 2A), supporting the finding that Treg cells can suppress IgE production4.In addition, we found a significant upregulation of IL13 mRNA expression and a trend to increased expression of IL4 andIL5 mRNAs in Tregs in asthma, indicating a Th2-like phenotype as was reported in Tregs from children with food allergies2. Furthermore, we found an upregulation of the prostaglandin D2 receptor (PTGDR2 ) or CRTH2, in line with a previous study that reported an increased amount of CRTH2+ Tregs in asthma5.Interestingly, several cytokine receptors were differentially expressed between Tregs from asthmatic individuals compared to controls. The IL-4 receptor alpha transcript IL4RA was significantly reduced in asthma. The expression of IL4RA also strongly correlated with the levels of IgE in the serum (Figure 2A). Previously, it was shown in mice that IL-4 receptor signalling is essential in controlling Th2 responses and airway inflammation6. Our data suggest a similar role of IL4RA in humans. Likewise, we observed a downregulation of TNF receptor superfamily member 25 (TNFRSF25 ), which was shown to contribute to preventing allergic lung inflammation7 and downregulation of OX40 (TNFRSF4 ).Additionally, we observed a difference in virus/type-I interferon(IFN)-related genes in asthma, which was also observed in single-cell transcriptomic data of allergen-specific Tregs from individuals with asthma8. Curiously, the expression of the type 1 IFN receptors IFNAR1/2 were lower expressed in asthma, which could indicate a deficiency against respiratory viruses and chronicity.Lastly, we performed an enrichment analysis to see up or downregulation of pathway maps, process networks and go processes with MetaCore (Table 1). The pathway maps and process networks included upregulation of pathways related to immune functions already described. However, the affected GO processes were mostly related to epigenetic mechanisms including nucleosome organisation, nucleosome assembly and chromatin organisation. With the tool STRING, we performed a pathway analysis that showed a cluster of histone genes (Figure 2B). So far, there is no data reporting the function of histone genes in Tregs or related to asthma, but perhaps this finding could be related to changes in epigenetics. It was reported that in asthma Tregs have increased CpG methylation of theFOPX3 locus compared to individuals without asathma3.In conclusion, Tregs from individuals with asthma show reduced expression of several molecules related to Treg suppressive functionality, while having increased expression of Th2-like characteristics that could lead to their reduced control of allergic airway inflammation. Further studies are needed to confirm these findings in a larger population and investigate their contribution to disease pathology.References1. Kuruvilla, M. E., Lee, F. E. H. & Lee, G. B. Understanding Asthma Phenotypes, Endotypes, and Mechanisms of Disease. Clinical Reviews in Allergy and Immunology (2019). doi:10.1007/s12016-018-8712-12. Noval Rivas, M. & Chatila, T. A. Regulatory T cells in allergic diseases. Journal of Allergy and Clinical Immunology138 , 639–652 (2016).3. Runyon, R. S. et al. Asthma Discordance in Twins Is Linked to Epigenetic Modifications of T Cells. PLoS One (2012). doi:10.1371/journal.pone.00487964. Meiler, F., Klunker, S., Zimmermann, M., Akdis, C. A. & Akdis, M. Distinct regulation of IgE, IgG4 and IgA by T regulatory cells and toll-like receptors. Allergy Eur. J. Allergy Clin. Immunol.(2008). doi:10.1111/j.1398-9995.2008.01774.x5. Boonpiyathad, T. et al. Impact of high-altitude therapy on type-2 immune responses in asthma patients. Allergy Eur. J. Allergy Clin. Immunol. 75 , 84–94 (2020).6. Khumalo, J., Kirstein, F., Hadebe, S. & Brombacher, F. IL-4Rα signaling in CD4+CD25+FoxP3+ T regulatory cells restrains airway inflammation via limiting local tissue IL-33. JCI Insight (2020). doi:10.1172/jci.insight.1362067. Schreiber, T. H. et al. Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation. J. Clin. Invest.(2010). doi:10.1172/JCI429338. Seumois, G. et al. Single-cell transcriptomic analysis of allergen-specific T cells in allergy and asthma. Sci. Immunol.(2020). doi:10.1126/SCIIMMUNOL.ABA60879. Message, S. D. et al. Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production. Proc Natl Acad Sci U S A105 , 13562–13567 (2008).10. Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner.Bioinformatics 29 , 15–21 (2013).11. Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: Fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41 , e108 (2013).12. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26 , 139–140 (2009).13. Szklarczyk, D. et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 45 , D362-d368 (2017).Figure 1: Tregs from asthmatic individuals show a distinct phenotype compared to controls. (A) Genes that are significantly changed in Tregs cells from asthmatic individuals compared to controls (log 2 ratio)– clustered in the groups: Treg markers, cytokine receptors, virus related, transcription factors, cytokines and others. (B) Fragments per kilo base per million mapped reads (FPKM) values of genes of interest (FOXP3, IL13, IL5, IL4, IL4R, PTGDR2, TNFRSF25, TNFRSF4, IFNAR1, IFNAR2) of all donors. N = 4 (healthy), 5 (asthma). *** p<0.001 , ** p<0.01, * p<0.05Figure 2: Phenotype of Tregs might be associated to Treg function . (A) Correlation between expression of FOXP3 (left) and IL4RA (right) with IgE serum levels. (B) Satellite plot showing a cluster of known interactions related to nucleosome assembly. Genes higher expressed in asthmatic individuals are shown in red, and lower expression in blue.