Single-Cell RNA Sequencing Approaches for Tracing T Cell Development

Abstract

T cell development occurs in the thymus, where uncommitted progenitors are directed into a range of sublineages with distinct functions. The goal is to generate a TCR repertoire diverse enough to recognize potential pathogens while remaining tolerant of self. Decades of intensive research have characterized the transcriptional programs controlling critical differentiation checkpoints at the population level. However, greater precision regarding how and when these programs orchestrate differentiation at the single-cell level is required. Single-cell RNA sequencing approaches are now being brought to bear on this question, to track the identity of cells and analyze their gene expression programs at a resolution not previously possible. In this review, we discuss recent advances in the application of these technologies that have the potential to yield unprecedented insight to T cell development.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential

“…The γδ lineage has been proposed to bifurcate from the main developmental pathway at the DN2>DN3 transition, when Tcrb/g/d gene rearrangement occurs ( 9 – 12 ), because clonal assays of ETPs and DN2 thymocytes show that a large proportion at these cells can give rise to both lineages, but this biopotency is lost by the DN3 stage ( 10 ). As thymocytes do not express cell surface TCR until late DN3, it suggests that the αβ vs. γδ commitment occurs independently of a TCR signal.…”

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential

“…These are further subdivided into DN1 (CD44 + CD25 - ), DN2 (CD44 + CD25 + ), DN3 (CD44 - CD25 + ) and DN4 (CD44CD25 - ) stages. αβ and γδ development is thought to bifurcate at the DN2b>DN3 transition, when TCRB/G/D gene rearrangement occurs to generate a functional pre-TCR or γδ TCR (Ciofani, Knowles, Wiest, von Boehmer, & Zuniga-Pflucker, 2006; Fehling, Krotkova, Saint-Ruf, & von Boehmer, 1995; Kreslavsky, Gleimer, Garbe, & von Boehmer, 2010; Oh, Gray, & Chong, 2021). Evidence also suggests that effector outcomes of γδ T cells may also be determined in the thymus rather than in the periphery because the expression of IL-17A and IFNγ by mature cells correlates with Vγ chain usage.…”

“…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).…”

“…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).…”

“…Thus, even within ETPs, there is significant heterogeneity. The γδ lineage has been proposed to bifurcate from the main developmental pathway at the DN2>DN3 transition, when Tcrb/g/d gene rearrangement occurs (9)(10)(11)(12), because clonal assays of ETPs and DN2 thymocytes show that a large proportion at these cells can give rise to both lineages, but this biopotential is lost by the DN3 stage (10). Because thymocytes don't express cell surface TCR until late DN3, it suggests that the αβ versus γδ commitment occurs independently of a TCR signal.…”