SLP-76 interaction with PLC-γ1 fine-tunes TCR signal strength for appropriate thymocyte selection, T cell homeostasis, and Ag-specific T cell response

Abstract

TCR signal strength regulates T cell fate at various stages of thymocyte development and the differentiation of peripheral T cells into distinct T cell subsets. We previously showed that TCR engagement forms a heterotetrametric complex composed of LAT, Gads, SLP-76, PLC-γ1 and that this complex is essential to activate PLC-γ1 to further transduce the TCR signal. The interaction of the SLP-76 proline-rich region (PRR) with the PLC-γ1 SH3 domain is relatively weak, and these two domains are highly conserved amongst many mammalian species. Therefore, we hypothesized that the SLP-76 PRR sequence with low affinity for PLC-γ1 might be evolutionarily favored to prevent excess TCR signals. To test this hypothesis, we developed mice carrying a conditional T cell-specific knock-in mutation in the SLP-76 PRR with a 2.5-fold increased affinity for PLC-γ1 by CRISPR/Cas9-mediated gene editing and Cre/LoxP system (cKI). Notably, cKI increased the expression of active caspase-3 in signaled SP4 and SP8 thymocytes and decreased the numbers of mature medullary thymocytes, suggestive of enhanced negative selection. Under steady-state conditions, cKI enhanced the generation of CD8 +central memory T cells, implying an increased sensitivity to self-Ag/MHC I in the periphery. Moreover, cKI rendered CD8 +T cells more sensitive to OVA altered peptide ligands with a weaker affinity for OT-I TCR. Finally, cKI enhanced priming of OT-I CD8 +T cells that had not undergone massive cell divisions for IFNγ-producing effectors in vivo, implying an increased TCR signal strength in weakly activated CD8 +T cells by cognate Ag. Our data indicate that the SLP-76 PRR/PLC-γ1 SH3 interaction plays a critical role in fine-tuning TCR signal strength to avoid excess signals via TCR. NCI/NIH Intramural Support