Resolving the instructions for αβ T cell development

Summary

In this issue of Immunity, Chopp et. al. use single cell transcriptomic and epigenomics in mice and human samples to delineate developmental trajectories of αβ T cell subsets and refine the kinetic selection model of CD4⁺ and CD8⁺ T cell lineage commitment.

αβT cells use their T cell antigen receptor (TCR) to recognize peptides and lipids derived from invading organisms presented by host MHC class I, MHC class II and MHC-like molecules expressed on antigen presenting cells. The central challenge of T cell selection and lineage commitment is to promote the development of thymocytes expressing TCRs that have specificity for host MHC ligands into mature T cells, and to properly match the expression of the CD4 or CD8 co-receptor with the class of MHC allele being recognized. T cell positive selection depends upon the quality of TCR/self-pMHC interactions, while the chain of events that culminate in CD4⁺ or CD8⁺ lineage choice can in part be described by the kinetic signaling model of T cell differentiation (Taniuchi, 2018). Following DP thymocyte recognition of self-pMHC, CD8 expression is diminished creating a TCR^hiCD4⁺CD8^lo intermediary, which results in MHC-I and MHC-II restricted thymocytes receiving abrogated or continuous TCR-derived positive selection signals, respectively. Asynchronous TCR signaling and the induced expression of key transcription factors, including Thpok and Runx3, contribute to CD4⁺ and CD8⁺ lineage commitment. However, whether the TCR^hi CD4⁺CD8^lo intermediary is indeed a lineage neutral state and how TCR signals regulate the expression of CD4⁺ and CD8⁺ lineage commitment factors and their target genes remain unresolved. To address these outstanding questions, Chopp et. al., (Chopp et al., 2020) performed comprehensive bulk and single cell RNAseq and ATACseq of developing mouse and human thymocytes, which with an impressive array of validation studies, allowed them to refine key aspects of T cell developmental branch points and gene regulatory networks that support CD4⁺ and CD8⁺ lineage differentiation.

Prior to the T cell lineage defining ‘big bang’ of αβTCR recombination and the discriminating pressures of positive and negative selection, thymic T cell precursor cells undergo multiple rounds of proliferation and differentiation to rewire stem cell transcription factor networks into the T cell lineage committed genetic program. This process induces TCRβ chain rearrangement and the expression of TCR complex signaling components, and in turn passage through β-selection and the expression of CD4 and CD8, as well as transcription factors that promote cell survival (Yui and Rothenberg, 2014). At the CD4⁺CD8⁺ double positive (DP) stage, TCRα rearrangement facilitates the creation of millions of distinct αβTCR clonotypes. Despite the complexities of this differentiation pathway, Chopp et. al., demonstrate that prior to selection, DP thymocytes are transcriptionally and epigenetically homogenous, DP-1 (Figure 1). This developmental-stage specific uniformity likely explains the high efficiency at which DP thymocytes can undergo positive selection into CD4⁺ or CD8⁺ T cells (Itano and Robey, 2000).

Figure 1. Major post-selection thymocyte subsets and precursor-product relationships identified by single cell RNAseq.

Classical phenotypic markers expressed by cells within each cluster; DP-1: TCR^lo Rag^pos CD5^lo Gata3^lo; DP-2: TCR^lo Rag^int CD5^pos Gata3^int; Sig-1: TCR^hi CD5^pos Gata3^pos Helios^neg; Sig-2: TCR^hi CD5^pos Helios^pos; Sig-3: TCR^hi CD4^lo CD8^lo CD122^neg Helios^pos PD-1^pos; Sig-4: TCR^hi CD4^neg CD8^neg CD122^pos Helios^pos; Sig-5a: TCR^int CD4^int CD8^int Bim^pos Helios^pos; Sig-5b: TCR^hi CD4^pos CD8^lo Ccr7^pos CD25^pos; ImmCD4: TCR^hi CD4^pos CD8^lo CD69^pos; MatCD4: TCR^hi CD4^pos CD8^neg CD69^lo; ImmCD8: TCR^hi CD4^lo CD8^pos CD69^pos; MatCD8: TCR^hi CD4^neg CD8^pos CD69^lo. MHC-I restricted subsets are colored purple, MHC-II restricted subsets are colored blue. Figure created with BioRender.

To gain insight into mechanisms that instruct αβ T cell development, Chopp et. al. used thymocytes from B2m^−/− and H2-Ab1^−/− mice to identify gene expression signatures and transcriptional activities of MHC-I- and MHC-II-restricted thymocyte subsets progressing through positive selection and lineage commitment. Unsupervised clustering of mouse thymic single cell RNAseq and ATACseq datasets revealed 16 relatively high frequency thymocyte clusters that arise at and following the auditioning step of positive selection, while 21 clusters were observed in human samples with no strict gene expression concordance between mouse and human clusters. In mouse, eight clusters are shared between MHC-I and MHC-II signaled thymocytes, with the rest exclusive or highly biased to selection on a particular MHC class. The identity of each clusters was determined in part using known gene expression patterns of previously defined thymic subsets (Mingueneau et al., 2013), and the expression of CD4⁺ and CD8⁺ lineage specific genes was used to identify the developmental staging of lineage commitment. To define the relationships between clusters, pseudotime mapping was used to infer developmental trajectories, revealing several branch points. Consistent with previous studies (Mingueneau et al., 2013), the gene expression signature of mouse and human DP thymocytes undergoing positive selection was largely overlapping, including between MHC-I and MHC-II signaled thymocytes (DP-2, Sig-1), albeit with MHC-II signaling inducing more robust expression.

During mouse and human T cell development, the initial ‘common stem trajectory’ is bifurcated at the ‘selection step’, where DP agonist signaled cells separate from thymocyte subsets that lead t0 conventional T cells (Sig-1). Within the agonist signaled arm, the four mouse subsets (Sig-2–4, 5a) have limited expression of Ccr7, suggesting primarily a thymic cortex location. The agonist selected subsets include two branches from the Sig-2 stage; one leading to negative selection (Sig-5a) while the other leads to primarily MHC-I restricted non-conventional cells that include CD8αα IEL precursors (Sig-4). Interestingly, the Sig-5a subset, thymocytes undergoing cortical negative selection, is transcriptionally similar to an unrelated Ccr7⁺ subset (Sig-5b), with both expressing Nr4a1 (Nur77), Ikzf2 (Helios) and NF-Κb family members. However, the Sig-5b subset also expressing Tnfrsf4 (Ox40) and Tnfrsf18 (Gitr), suggesting they are regulatory T cell precursors and thymocytes undergoing late stage negative selection within the thymic medulla (Owen et al., 2019). Clusters of mouse iNKT and MAIT cells were not detected presumably due to their relatively low frequency in the thymus, and precursor-product relationships of several mouse (NC-1, NC-2) and human (Park et al., 2020) non-classical subsets could not be directly connected to the main developmental tree using the psuedotime analyses.

The developmental path leading to conventional and regulatory CD4⁺ and CD8⁺ T cells includes a post DP stage (Sig-1), at which the CD4⁺ and CD8⁺ lineages separate, resulting in the second major bifurcation. The kinetic selection model of lineage commitment posits that DP thymocytes undergoing positive selection reduce expression of the CD8 co-receptor causing a caseation of TCR signaling in MHC-I restricted, but not MHC-II restricted thymocytes (Taniuchi, 2018). At this stage, MHC-I restricted thymocytes use intra-thymic cytokines and additional factors to promote Runx3d expression, which in conjunction with Tcf1, silence CD4 gene expression and induce CD8 re-expression (Park et al., 2010). Runx3d further functions as a transcriptional silencer of Zbtb7b (Thpok), a key transcription factor required for CD4⁺ T cell differentiation (He et al., 2005; Sun et al., 2005; Taniuchi, 2018). In contrast, MHC-II restricted thymocytes maintain TCR signaling thereby allows Tcf1 and Lef1 to positively regulate Zbtb7b, which suppresses Runx3 expression, and induces c-Myb, Gata3 and other transcriptional regulators that promote the development of CD4⁺ T cells (Taniuchi, 2018). Curiously, during human T cell development, RUNX3 and THPOK are expressed in CD4⁺ lineage thymocytes, suggesting that despite the overall strong human-mouse conservation of transcriptional and epigenomic programs of T cell development, evolution has altered some of the nuclear machinery regulating lineage commitment.

Although it has been presumed that development of the post DP stage (Sig-1; CD4⁺CD8^lo) is lineage neutral, Chopp et. al. demonstrates that aspects of the CD4⁺ lineage gene signature arise at the DP-2 stage in mouse and human, and prior to the reduced expression of the CD8 gene. The CD8⁺ lineage gene signature, however, was only observed beginning at the mouse immature CD8SP stage corresponding to the stage of CD8 gene re-expression; this same signature did not distinguish human CD8 thymocytes. Thus, CD4⁺ lineage commitment may initiate at the time of positive selection, perhaps due to the increased TCR signal strength of MHC-II restricted thymocytes, whereas CD8⁺ lineage commitment is ‘learned’ later in development.

Our incomplete understanding of CD4⁺ and CD8⁺ lineage commitment led Chopp et. al. to use computational approaches of the mouse RNAseq and ATACseq datasets to identify transcriptional activities associated with, and putatively involved in, CD4⁺ or CD8⁺ lineage differentiation. This allowed for the generation of a low-resolution gene regulatory network map that controls Thpok and Runx3 expression. 27 transcription factors were identified whose activities are biased towards CD4⁺ lineage differentiation, most of which increase as cells differentiate following TCR signaling. This program involves expression or activation of signaling-induced transcription factors including Elk, Egr1 or JunB. In addition, 12 transcriptional activities biased towards CD8⁺ lineage differentiation were identified, most of which pre-exist in DP thymocytes, including E-proteins, Hbp1 and Tcf1, and do not increase (and in some instances decrease) as cells undergo differentiation. These data support the hypothesis that “less” TCR signaling is required for DP thymocytes to differentiate into CD8⁺ T cells as compared to CD4⁺ T cells.

How thymocytes use TCR signal quality and stage-specific asymmetry to establish the transcriptional circuits regulating CD4⁺ and CD8⁺ lineage choice is not yet fully understood. Through integrating transcriptomics and epigenomics, Chopp et. al., define the trajectories of mouse and human thymocytes undergoing selection and differentiation into conventional and agonist-selected T cell subsets. Their findings define gene regulatory networks that influence T cell development at various stages and show that these networks are largely conserved between mouse and human. Compelling data support the hypothesis that positive selection creates a ‘common stem trajectory’ for DP thymocytes differentiating into conventional CD4⁺ and CD8⁺ T cells, for which embedded are the initial genetic underpinnings of CD4⁺ or CD8⁺ lineage commitment. Moving forward, the advent of single cell -omics technologies will greatly facilitate our ability to deduce high resolution maps of human T cell development and the nuclear machinery that regulate the ‘helper’ and ‘killer’ T cell lineages, including the ability of CD4⁺ T cell to acquire cytotoxic functions during certain pathogen challenges.