Effect of Interleukin-2 Inducible Kinase (ITK) on the Temporal Dynamics of TCR Signaling in CD8 T cells

Abstract

Abstract Antigen affinity has been linked to the rate of downstream TCR activation and the activation of transcription factors such as NFAT and NF-kb. Interleukin-2 inducible T cell kinase (ITK) plays a key role in the differential rate and magnitude of TCR-dependent transcription factor activation, especially when T cells are stimulated with lower affinity ligands. The Nr4a3-Tocky Timer mouse model carries a construct for a fluorescent protein controlled by the Nr4a3 promoter. Nr4a3 is highly expressed following TCR activation and thus is a good metric for TCR signaling. When first produced, the Tocky protein fluoresces “blue,” at peak wavelength 466nm and then, after ~7hrs, degrades to a “red” fluorescence at peak wavelength 583nm. Splenocytes were harvested from OT-I RAG1KO mice crossed to Nr4a3-Tocky mice and stimulated using varying affinities and doses of ova peptide over 7 days in vitro. Cells were exposed to the ITK inhibitor, PRN694, to observe the effect of ITK on the rate of expression of Tocky fluorescence along with activation surface markers CD69, CD25, CD44, and CD122. With ITK inhibition, CD8 T cells expressed the “Blue” protein slower and in fewer cells compared to OT-I cells stimulated without PRN694 at 4hrs. At 48hrs after stimulation, sustained signaling was observed in control cells, whereas a subset of ITK-inhibited cells showed evidence of halted TCR-induced Tocky transcription. This decrease in the longevity of TCR signaling was more evident when lower peptide doses or lower affinity peptides were used for stimulation. These results suggest that ITK plays a role in amplifying the initial TCR signal, and is also required for sustained signaling after TCR activation in CD8 T cells, resulting in the tuning of early gene expression. Supported by grants from the NIH (Dissecting the pathways controlling tunable responses to TCR signaling: 5R01AI132419-06)