Gene_expression_during_Th2_differentiation_in_RORA_knockout. Gene_expression_during_Th2_differentiation_in_RORA_knockout

Naïve CD4+ T helper (Th) cells differentiate into distinct lineages to achieve successful adaptive immune responses to diverse categories of pathogens. However, the molecular regulation of this development to the subset cells is still unresolved. Conventional methods to explore gene expression in Th cells are based on populations of cells, so the expression profiles are averages over large numbers of cells, which obscures whether there are one or more distinct cell types within a population. In this research, we determine the molecular changes during Th cell differentiation upon the Nippostrongylus brasiliensis 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 different times points: 0,3,5, and 7 days. We first calibrated the accuracy of the BioMark qPCR protocol in a cell line, and found that the technical errors are low. We then established that there is a high correlation between transcript levels and their protein products in single cells for a panel of cell surface markers. When looking at the differences between CD4+ T cells during type-2 response, we recapitulated expected gene expression patterns in naïve, Th2 and Treg cells, and several new and unexpected patterns of gene expression. We show that the activated populations contain three populations Th2-reg and early activated cells (Th2-act), which are located at lymph nodes. In addition, we find another 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 in cytokine secretion.