Regulation of TCR-induced Transcriptional Kinetics by Interleukin 2-inducible T Cell Kinase (ITK) in CD8⁺ T cells

The initial activation of naïve CD8⁺ T cells induces three major transcription factor pathways, NFAT, NFκB, and AP-1, predominantly regulated by T-cell receptor (TCR) signaling. Downstream of the TCR, the Tec family tyrosine kinase ITK modulates the transcriptional response in T cells by differentially affecting the kinetics and magnitude of activation of these three transcription factors. How signaling through ITK regulates the duration and/or termination of TCR signaling has not been investigated. To address this, we utilized the "Nr4a3-Tocky" reporter mouse which provides information on the kinetics of TCR signaling over time courses from hours to days. OT-I CD8⁺ Nr4a3-Tocky T cells were stimulated with peptide ligands of varying affinities, and cells were assayed for a panel of surface and intracellular markers of activation and differentiation at multiple time points post-stimulation. As previously reported, at early time points ITK signaling enhanced the kinetics and magnitude of expression of these markers. At later time points the absence of ITK led to persistent expression of several proteins, indicating an important role for ITK in the termination of TCR-dependent transcriptional responses. Using a dual ITK/ RLK inhibitor (PRN694) at 24h post OVA peptide stimulation it was found that this function of ITK was required in the first 24 hours. These findings reveal a possible key negative regulatory mechanism that is programmed in the first day of activation but impacts CD8⁺ T cell gene expression patterns days later.