An ITK - SLP-76 Y173 Axis Regulates TCR Signal Persistence and Thymic Development

The TCR-induced tyrosine kinase cascade results in ITK-mediated phosphorylation of at least two substrates: PLC-γ1 Y783, an essential mediator of TCR signaling, and SLP-76 Y173. We previously provided evidence that SLP-76 Y173 supports proximal TCR and FcεRI signaling; but its role in thymocyte development was never explored. Here we demonstrate that SLP-76 Y173 was required for TCR/CD28-induced degradation of IκB; moreover, both ITK and SLP-76 Y173 were required for persistent TCR-induced phosphorylation of PLC-γ1, ERK and p38. To explore whether SLP-76 Y173 may mediate ITK-dependent events in vivo, we replaced SLP 76 Y173 with phenylalanine in a mouse model. Like ITK-deficient thymocytes, SLP-76 Y173F thymocytes exhibited reduced thymic cellularity, reduced CD4/CD8 ratios, and reduced surface expression of CD5. In ex vivo-stimulated thymocytes, mutation of SLP-76 Y173 or inhibition of ITK reduced the persistence of TCR-induced calcium flux; moreover, TCR-induced phosphorylation of ERK and p38 depended on SLP-76 Y173. Like ITK-deficient thymi, SLP-76 Y173F thymocytes exhibited decreased production of CD4 - iNKT 1 thymocytes, coupled with increased production of innate-like CD4 + and CD8 + SP thymocytes. Our results provide strong evidence that the ITK substrate site, SLP-76 Y173, promotes TCR signal persistence that is required to mediate the thymic developmental effects of ITK.