Single‐cell transcriptomics uncovers an instructive T‐cell receptor role in adult γδ T‐cell lineage commitment

Abstract

After entering the adult thymus, bipotent T-cell progenitors give rise to ab or cd T cells. To determine whether the cd T-cell receptor (TCR) has an instructive role in cd T-cell lineage commitment or only "confirms" a pre-established cd Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for cd TCR signaling. Although these mice showed a T-cell development block at the CD4 À CD8 À double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of cd TCR (further referred to as cd int ) at their surface. Single-cell transcriptomics of LAT-deficient DN3 cd int cells demonstrated no sign of commitment to the cd T-cell lineage, apart from cd TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 cd int cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the cd T-cell lineage. Therefore, the cd TCR-LAT signaling axis builds upon a cd T-cell uncommitted lineage state to fully instruct adult cd T-cell lineage specification.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential

“…High Trdc, Id3, Sox13 and Rorc identified mature γδ thymocytes (Fig 2D). This analysis revealed that multiple distinct populations correspond to each of the canonical DN stages and consistent with recent T cell development studies (Spidale et al, 2018;Sagar et al, 2020;Scaramuzzino et al, 2022), this suggests that DN thymocytes are much more heterogenous than previously thought.…”

“…The instructive model proposes that TCRγδ signals competes with pre-TCR(β) signals and the lineage decision is determined by cell specific interactions that activate key transcription factors, which in turn instructs gene expression program (Carpenter & Bosselut, 2010). In contrast, the stochastic selective model postulates that lineage commitment is determined prior to the onset of TCR gene rearrangement, and those thymocytes that receives appropriate TCR signaling that matches their hardwired identity further progress along the αβ versus γδ lineage and differentiate into a specific subset of γδ T cells (Scaramuzzino et al, 2022). Indeed, Scaramuzzino and colleagues showed that TCR signaling clearly has a role in γδ lineage maturation because LAT-deficient DN3 cells that express a γδ TCR are unable to complete the activation of the γδ T cell lineage, including expression of Sox13, Maf , and Cpa3 .…”

“…The predetermination model proposes that αβ versus γδ T cell lineage commitment is determined by lineage specific factors prior to the onset of TCR gene rearrangement. Consequently, only those thymocytes that receive a corresponding signal via the stochastically expressed pre-TCR or γδTCR can further develop (Oh, Gray, & Chong, 2021; Scaramuzzino et al, 2022). Evidence suggest that this Notch-independence is at least partially due to the transcription factor ID3 because a weak TCR signal in cooperation with Notch signaling is sufficient to inhibit E-protein activity (Lauritsen et al, 2009).…”

“…Evidence for an instructive role of the T-cell receptor in γδ T cell lineage commitment comes from a single-cell RNA sequencing (scRNA-seq) study that identified DN3 cells that expressed intermediate levels of γδ TCR in LAT deficient mice. LAT is an adaptor required for γδ TCR signaling and deficient thymocytes failed to turn on the transcriptional program associated with the γδ T cell lineage (Scaramuzzino et al, 2022). However, whether the lineage commitment is determined prior or at DN3 stage when TCR signaling remains unclear.…”

“…The predetermination model proposes that αβ versus γδ T cell lineage commitment is determined by lineage specific factors prior to the onset of TCR gene rearrangement. Consequently, only those thymocytes that receive a corresponding signal via the stochastically expressed pre-TCR or γδTCR can further develop (Oh, Gray, & Chong, 2021;Scaramuzzino et al, 2022).…”