An ITK-substrate site SLP-76 p-Y173 regulates thymocyte differentiation and activation
2National Institutes of Allergy and Infectious Diseases, National Institutes of Health
3Immunology & Microbiology Department, Anschutz School of Medicine, University of Colorado
Introduction: The T cell antigen receptor (TCR) signaling pathway is partially-dependent on the Tec-family tyrosine kinase ITK. ITK is activated downstream of the TCR, and phosphorylates PLC-γ1, leading to calcium influx and the activation of crucial transcription factors. ITK is known for its role in the differentiation and function of effector and regulatory T cells. Within the thymus, ITK regulates the balance between conventional and innate-like developmental pathways.
In addition to phosphorylating PLC-γ1, ITK also phosphorylates the TCR pathway adaptor protein SLP-76 at tyrosine 173 (Y173). We previously showed that SLP-76 p-Y173 can mediate TCR-induced activation of the NF-κB pathway.
Research Question: Since ITK can phosphorylate multiple substrates, we did not know which of its signaling functions may be mediated by SLP-76 p-Y173. Moreover, the in vivo regulatory roles of this phosphorylation site were unknown.
Materials and Methods: To address these questions, we developed a novel mouse model, SLP76(Y173F), lacking the Y173 phosphorylation site, and compared the thymic phenotypes of this mouse model to known phenotypes of ITK-deficient mice.
Results and Discussion: SLP76(Y173F) mice exhibited multiple phenotypes closely resembling ITK-deficient (ITK-/-) mice. Among the ITK(-/-)-like phenotypes, SLP76(Y173F) mice exhibited: (i) decreased thymic cellularity, (ii) decreased activation markers on double-positive (CD4+CD8+) thymocytes, (iii) reduced TCR-induced Ca2+ flux, (iv) a skewing of thymic development towards innate-like lineages and (v) markedly increased representation of innate-like lineages in peripheral lymphoid tissues. Remarkably, virtually all SLP76(Y173F) CD8 SP thymocytes and many CD4 SP thymocytes were EOMES+ memory-like cells, a trait that closely resembles ITK(-/-) thymocytes.
Conclusions: Our results demonstrate that key biological functions of ITK depend on its downstream substrate, SLP-76 Y173. Further experiments will be required to determine whether the ITK- and SLP-76 Y173-dependent phenotypes are mediated by PLC-γ1 and/or by NF-κB.
Acknowledgements: This research was supported in part by the Division of Intramural Research of NIAID, NIH, and by a grant from the U.S-Israel Binational Science Foundation.