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

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. 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 Chip-seq. Cells were incubated for 10min at room temperature with 1% formaldehyde. Cross-linking was terminated by addition of glycine to a final concentration of 125mM. ChIP was performed using iDeal ChIP-seq Kit (Diagenode) according to the manufacturer’s instructions. Antibodies against H3K4me3 (ab8580, Abcam), H3K4me1 (ab8895, Abcam), H3K27ac (ab4729, Abcam), H3K27me3 (ab6002, Abcam) and H3K9me3 (ab8898, Abcam) were used. Library preparation for ChIP-seq was performed using NEBNext ChIP-Seq library Prep Master Mix set kit. Two biological replicates per condition were sequenced using Illumina HiSeq 2000 (50bp single-end) sequencing technology. 12 indexes were used per sequencing lane.