Multilayered specification of the T-cell lineage fate

Abstract

T-cell development from stem cells has provided a highly accessible and detailed view of the regulatory processes that can go into the choice of a cell fate in a postembryonic, stem cell-based system. But it has been a view from the outside. The problems in understanding the regulatory basis for this lineage choice begin with the fact that too many transcription factors are needed to provide crucial input: without any one of them, T-cell development fails. Furthermore, almost all the factors known to provide crucial functions during the climax of T-lineage commitment itself are also vital for earlier functions that establish the pool of multilineage precursors that would normally feed into the T-cell specification process. When the regulatory genes that encode them are mutated, the confounding effects on earlier stages make it difficult to dissect T-cell specification genetically. Yet both the positive and the negative regulatory events involved in the choice of a T-cell fate are actually a mosaic of distinct functions. New evidence has emerged recently that finally provides a way to separate the major components that fit together to drive this process. Here, we review insights into T-cell specification and commitment that emerge from a combination of molecular, cellular, and systems biology approaches. The results reveal the regulatory structure underlying this lineage decision.

Fig. 1. Stages in T-cell specification and commitment

Double negative (CD4⁻CD8⁻ surface TCR⁻) stages between thymic entry and the first TCR-dependent selection events are highlighted. Signals from Notch-Delta interaction are required throughout both ‘phase 1’ and ‘phase 2’. Proliferation is strongest in phase 1 and in cells that successfully rearrange TCRβ genes, after β-selection. γδ cells have a somewhat different program that is not discussed in this review. Mac, macrophage; gran, granulocyte; Meg, megakaryocyte; eryth, erythrocyte. The DN1 cells that have actual T-cell precursor activity are Kit^High, i.e. ETPs.

Fig. 2. Regulatory gene expression changes during T-cell specification

Curves show approximate timing of increases and decreases in expression of the indicated regulatory genes and key T-cell identity genes, selected from the groups discussed in the text. Data are from ref. (58) and J. Zhang et al., manuscript in preparation. RNA transcripts encoding Myb, Ikaros, and E2A (bottom) are expressed at high but relatively stable levels throughout the indicated stages.

Fig. 3. Effects of Bcl11b deletion on pro-T cells: increased self renewal, prolonged access to myeloid and dendritic fates, and enhanced, long-term access to NK cell fates

Bcl11b is not required for Notch signals to inhibit myeloid differentiation, but Bcl11b deletion weakens the ability of Notch signals to inhibit NK differentiation. Gene regulatory alterations proposed to underlie these shifts are shown in Fig. 6.

Fig. 4. Enhanced upregulation of NK-cell specific transcription factor Zfp105 in pro-T cell populations with reduced Bcl11b gene dosage

This experiment shows efficient upregulation of this normally silent gene even when experimental deletion of Bcl11b is incomplete. (A) Primary cell populations derived from fetal liver, differentiating in T-cell conditions with OP9-DL1 stroma) for a week after treatment with retroviral Cre. Left and right panels show control and Bcl11b-deleted precursors, respectively (from ref. 84). Positions of populations sorted for gene expression and returned to secondary culture are labeled in red. (B) Secondary cultures derived from the populations shown in (A), after 12 days of further OP9-DL1 culture. Populations sorted for further RNA analysis from each culture came from gated regions labeled in blue. (C) Real-time PCR analysis of gene expression in the populations derived from primary and secondary cultures in A and B, from control (WT) and Bcl11b-deleted (KO) samples. Measurement of spliced transcripts of Bcl11b, as a measure of exon 4 deletion efficiency, and of the NK-specific transcription factor Zfp105. Note the selection for cells with reduced levels of intact Bcl11b in the secondary cultures of Bcl11b-deleted cells (KO, compare red-labeled and blue-labeled populations) and the marked upregulation of Zfp105.

Fig. 5. Fate of Bcl11b-deleted bone marrow cells in the thymus of radiation chimeric host after adoptive transfer

Donor cells were Lin⁻ bone marrow cells from Bcl11b-floxed and Bcl11b wildtype mice, each with a Cre-dependent ROSA26-YFP marker to monitor successful Cre exposure. Cells were treated with retroviral Cre in vitro and then used to reconstitute irradiated hosts. (A) Diagram of donor alleles. (B) Experimental scheme: in this version of the experiment, Rag-deficient hosts were used. (C) Phenotype of donor-type YFP⁺ cells in the thymus of recipient mice. Note that the Cre treatment marks a cohort of cells that tend not to persist in the thymus normally after 12 weeks (R26R-YFP only, right panels). However, the loss of Bcl11b (Bcl11b^L2/L2 R26R-YFP) consistently increases the accumulation of donor cells in the thymus and the retention of Kit on the surfaces of NK-like and pro-T cell-like (arrow) CD4⁻CD8⁻ populations.

Fig. 6. Separate regulatory subroutines in pro-T-cell commitment: selective effects of loss of Bcl11b in the phase 1 to phase 2 transition

The figure relates the functional effects shown in Fig. 3 to the gene expression patterns shown in Fig. 2 and their alteration in Bcl11b-deficient pro-T cells. Groups of genes that interact in coordinated segments of the T-cell specification process are depicted in ‘layers’, as well as groups of genes that are activated specifically when the cells escape the T-cell program to enter NK or myeloid/dendritic cell programs. Genes shared between two regulatory states are in parentheses. Cd3g, Cde3, and Ptcra are upregulated in two steps, where the first (violet layer) but not the second (pink layer) can occur without Bcl11b. Note that Bcl11b locus activation does not depend on Bcl11b function (84). Not shown: whereas CD3 and Bcl11b gene activation is sustained in later T-cell development, some genes induced in the DN2 stage (Ptcra, Rag1) are downregulated at β-selection.