Understanding the role T helper cells during asthmatic inflammation at the single cell level
Chris Tibbitt1*, Lisebeth Martens2, Yvan Saeys2, Jonathan Coquet1
1Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm 171 77, Sweden; 2VIB-UGent Centre for Inflammation Research, Ghent B-9000, Belgium
T helper cells are critical to the development of asthma. These cells are known to acquire specific functions that can cause pathological inflammation in the lung, leading to asthma. How many functions those T helper cells can acquire during the development of asthmatic disease remains unclear. Furthermore, the critical genes and pathways that regulate T helper cell functions are yet to be fully elucidated. This project aims to understand the heterogeneity of T helper cell response, in a well-defined mouse model and subsequently identify those genes vital to pathogenic subsets. Single cell RNA sequencing (scRNA-Seq) was performed using the SMART-seq2 protocol on individual T helper cells isolated from the airways (Bronchial Alveolar Lavage) of mice sensitized and challenged with House dust mite (HDM) allergens. Index sorting yielded a total of 339 T helper cells with RNA of sufficient quality to be clustered by t-SNE machine learning following Principle Component Analysis (PCA). Using such an approach we can successfully identify both known and potentially novel asthmatic T helper cell subsets based on single cell transcriptomes. Previously implicated subpopulations such as T helper-2 (Th2) and regulatory T helper cells (Tregs) form discreet clusters based on differential gene expression while the Th1 and Th17 subsets may be present but are somewhat less defined by t-SNE. Further work has confirmed novel target genes previously not implicated in asthmatic T cells with flow cytometric anaylsis. Thus, our project has assessed the T helper response in HDM model at an unparalleled level of detail with implications for future therapeutic design.
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