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Naïve CD4+ T helper (Th) cells differentiate into distinct lineages to achieve successfuladaptive immune responses to diverse categories of pathogens. However, the molecularregulation of this development to the subset cells is still unresolved. Conventionalmethods to explore gene expression in Th cells are based on populations of cells, so theexpression profiles are averages over large numbers of cells, which obscures whetherthere are one or more distinct cell types within a population. In this research, wedetermine the molecular changes during Th cell differentiation upon the Nippostrongylusbrasiliensis immune response by monitoring gene expression at the single cell level.Single Th cells are monitored from different mouse tissues: mediastinal lymph nodes(LN), mesenteric LN, lungs, and gut, from infected and uninfected mice, and at differenttimes points: 0,3,5, and 7 days. We first calibrated the accuracy of the BioMark qPCRprotocol in a cell line, and found that the technical errors are low. We then establishedthat there is a high correlation between transcript levels and their protein products insingle cells for a panel of cell surface markers. When looking at the differences betweenCD4+ T cells during type-2 response, we recapitulated expected gene expression patternsin naïve, Th2 and Treg cells, and several new and unexpected patterns of geneexpression. We show that the activated populations contain three populations Th2-regand early activated cells (Th2-act), which are located at lymph nodes. In addition, we findanother unknown sub-population that expresses RORA together with GATA3 (Th2-efc),which is located mostly in lungs and gut. Finally, we suggest the role of this population incytokine secretion. |