Interlinked roles for HEB and Id3 in fetal gamma-delta T cell commitment and functional programming.

γδ T cells that produce IL-17 (γδT17) play essential roles in barrier immunity, but the gene networks that install their functions are not well understood. We previously linked T cell receptor (TCR) signal strength to the proportional upregulation of Id3 during T cell development, which antagonizes the activity of HEB (encoded by Tcf12), and showed that HEB is required for γδT17 development. To understand how HEB and Id3 regulate γδT17 cell development, we conducted single cell RNA-sequencing on fetal thymic γδ T cells from Tcf12-deficient mice. γδ T cells lacking HEB exhibited profound alterations in the expression of the genes encoding the TCRγ and TCRδ chains, accompanied by a decrease in expression of genes in the early γδ T cell specification network. Surprisingly, Id3 was among the most severely decreased genes in HEB-deficient γδ T cell precursors, suggesting a requirement for HEB in Id3 expression. Analysis of fetal thymic γδ T cells in Id3-deficient mice revealed that the TCRγ and TCRδ repertoires were unaffected, and expression of early γδ T cell genes was unperturbed. However, later stage regulators of γδT17 cell differentiation were decreased, and Id3-deficient γδ T cells were defective in IL-17 production. Therefore, our findings reveal an interlinked network in which HEB is initially required to upregulate Id3 expression, which in turn enables the later stages of γδT17 cell development and functional programming.