T-bet high and low sorted CD4 T cells after acute infection

The purpose of this study was to understand in detail how T-bet+ CD4 T cells differ depending on their quantitative T-bet levels after acute LCMV infection. For this, the murine T-bet ZsGreen reporter system was used for sorting cells ex vivo without manipulation according to their T-bet brightness into high and low expressors. The bulk RNA Sequencing of these cells showed that T-bet high cells (n=3) have a rather cytotoxic profile, while T-bet low cells (n=3) upregulate multiple Tfh associated markers while still strongly depicting a Th1 phenotype. Overall, only up to 5% of the expressed genes differed between high and low expressors. Furthermore, the bulk ATAC-sequencing also confirmed strong overlap in chromatin accessability of high (n=2) and low (n=2) expressors. Only a couple of the highly differentially expressed genes from the RNA-Seq data also showed differences in chromatin accessability at the TSS. This study provides a further insight into the quantitative differences of T-bet expressing CD4 T cells. Overall design: Naive Smarta CD4 T cells from T-bet ZsGreen donors (Thy1.1+) were transferred into T-bet ZsGreen recipients (Thy1.2+), which were infected shortly after with LCMV Arm (200pfu). On day 10 p.i. the cells were harvested from the spleen and lymph nodes of the mice. The cells were FACS sorted according to their T-bet ZsGreen brightness into High and Low sorted fractions and bulk RNA- and ATAC-Sequencing was performed. Cells generated in 3 (RNA-seq) or 2 (ATAC-seq) independent experiments were used for the analysis.