Plasticity and lineage commitment of individual Th1 cells are determined by stable T-bet expression quantities

T helper 1 (Th1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated Th1 cells based on their quantitative expression of T bet and interferon ?. Heterogeneous T bet expression states were regulated by virus induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the Th2 lineage: T bet quantities were inversely correlated with the ability to express the Th2 lineage-specifying transcription factor GATA 3 and Th2 cytokines. Reprogramed Th1 cells acquired graded, but stable mixed Th1+2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated Th1 cells was essential to ensure Th1 cell stability. Thus, innate cytokine signals regulate Th1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges. Overall design: Adoptively transferred WT, Tbx21+/-, Tbx21-/- LCMV-TCRtg CD4+ Thy1.1+ cells from LCMV-challenged mice were isolated and MACS-sorted on day 10 of infection. Cells were cultured for two rounds under neutral (5 ng/ml IL-2 (Peprotech), 10 µg/ml anti–IL-4 (11B11), 10 µg/ml anti–IL-12 (C17.8), and 10 µg/ml anti–IFN-g (AN18.17.24)) or Th2 conditions (30 ng/ml IL-4 (Sigma-Aldrich), 5 ng/ml IL-2 (Peprotech), 10 µg/ml anti–IL-12 (C17.8), and 10 µg/ml anti–IFN-g (AN18.17.24)). On day 4 of the second round of stimulations, the cells were harvested and processed for RNA-seq. Two biological replicates per condition were sequenced using Illumina HiSeq 2000 paired-end 100 bp sequence type.