Marked Induction of the Helix-Loop-Helix Protein Id3 Promotes the γδ T Cell Fate and Renders Their Functional Maturation Notch Independent

Abstract

SUMMARY αβ and γδ T-cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and γδ T-cell receptor (γδTCR) play instructional roles in specifying the αβ and γδ T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify the fate and promote the development of these lineages remain poorly understood. Here we show that differential activation of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent IFNγ-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus.

Integration of T‐cell receptor, Notch and cytokine signals programs mouse γδ T‐cell effector differentiation

“…1C). This observation is consistent with Id3 levels being directly affected by γδTCR ligand exposure to weak or strong ligands 14 . A differential impact of T22 and T10 was also seen in KN6 cell maturation, in that KN6 cells co-cultured on T22 + MEFs showed a more efficient down-regulation of CD24, with a concomitant up-regulation of CD73, indicating a role for TCR signal strength in γδ T cell maturation as well as fate determination (Fig.…”

“…The mechanism by which Notch enables γδ17 development appears complex and may involve several distinct functions of the Notch signaling pathway. One key role of Notch signaling in γδ17 development appears to be enhancing expansion and survival of γδ T-cell precursors, consistent with previous reports 14, 53 . Specifically, Notch signaling appears to provide protection from the deleterious effects of cytokine signaling by inhibiting Il6r expression and inducing IL-6R shedding.…”

“…Retroviral constructs were generated, as previously described 14, 53, 58 , by subcloning the cDNAs of interest into the pMigR1 or pMIY plasmids, upstream of the internal ribosomal entry site 59, 60 . Stable retroviral-producing GP+E.86 packaging cell lines were generated for each construct.…”

“…Stable retroviral-producing GP+E.86 packaging cell lines were generated for each construct. The KN6 TCRγ and TCRδ subunits were cloned into pMiY as a fusion protein linked by the 2A Tescovirus linker peptide, as described previously 14 . OP9-DL1, OP9-DL4 and OP9-Ctrl cells were produced and maintained as previously described 59, 60 , and co-cultures were supplemented with 1 ng mL −1 mouse recombinant IL-7 and 5 ng mL −1 human recombinant Flt-3L (Peprotech).…”

“…The signal strength hypothesis asserts that the quantity of the signal downstream of the TCR can direct lineage choice 8 . TCR signaling activates the ERK-Egr3 pathway, which results in the up-regulation of Id3 in direct proportion to the strength of signal 14 . Moreover, high ERK signaling has been shown to repress the development of γδ17 cells 15 , and Id3 activity can repress the activity of HEB, which is required for γδ17 development 16 .…”