CD4-CD8 differentiation in the thymus: connecting circuits and building memories

Abstract

The proper choice of the CD4-helper or CD8-cytotoxic lineages by developing T cells is crucial for the generation of an antigen-responsive and functionally fit T cell repertoire. Here we present a brief overview of the transcriptional control of this process, with emphasis on two issues. The study of Cd4 expression, that had previously generated important paradigms for transcriptional regulation in eukaryotic cells, now brings new twists to the concept of 'epigenetic memory'. And connections are emerging between transcriptional regulators critical for commitment to either lineage. The present review attempts to integrate these findings and discusses the still elusive mechanisms that match CD4-CD8 lineage differentiation to MHC specificity.

Figure 1. Specification and commitment to the CD4 and CD8 lineages

DP thymocytes have rearranged genes encoding TCRα and TCRβ chains and express surface TCR complexes. These cells are programmed to undergo apoptotic cell death in the thymic cortex unless their TCR is productively engaged by MHC molecules expressed by the thymic epithelium, an event referred to as positive selection. Rescued thymocytes differentiate into CD4 or CD8 T cells, depending on whether they are MHC II- or MHC I-restricted, respectively. Lineage differentiation includes two conceptually distinct steps, specification and commitment. For the CD4 lineage, specification involves Gata3, Tox and E-proteins E2A and HEB (not shown), whereas commitment requires Thpok, which represses CD8-lineage genes including Runx3. For CD8 cells, specification involves Runx3, which also contributes to commitment by repressing Thpok and Cd4. Stat5 and Ets1 contribute to Runx3 expression, the latter binding the Runx3 locus. Note that the CD4⁺CD8^int cells has precursor activity for both CD4 and CD8 lineages and is thought to include truly bi-potent cells [1]. In contrast the CD4^intCD8⁺ subset only has CD8 precursor activity.

Figure 2. Cd4 cis-regulatory elements

Schematic representation (not on scale) of the Cd4 locus, with exons 1 and 2 (black boxes), the silencer (red-filled oval) and positive regulatory elements (green-filled rectangles), including the proximal enhancer (E4P), promoter (Pr) and a downstream enhancer known as ‘thymic enhancer’ (E4T) even though it is now known to be active in LTi cells, not in thymocytes. Transcription factors important for the activity of each element are indicated, as are cell subsets in which each element is active, or determines ‘epigenetic memory’ despite having no intrinsic activity in the subset. Note that while AP4 does not bind the silencer, it interacts with Runx molecules and could therefore ‘bridge’ that element with E4P. Factors distinct from Runx proteins are thought to contribute to Cd4 silencing because the silencer contains functionally important motifs in addition to Runx binding sites [21].

Figure 3. Thpok cis-regulatory elements

The Thpok locus is schematically represented (not on scale). The two transcription start sites, defined by alternate exons 1a and 1b, are shown as arrows and exons as black boxes; thick areas depict coding regions in exons 2 and 3. Boxes (top) indicate the distal regulatory element (DRE, including the silencer), the general T lymphoid element (GTE), the proximal enhancer (PE), and a Gata3 binding site, using the same color code as in Fig. 2. Binding areas of repressors (Runx molecules, Mazr and Bcl11b) and activators (Gata3) are schematically depicted. While the DRE is depicted as including distinct activating (green) and silencing (red) elements, it is not clear whether these activities are physically separated [19, 22, 23]. Note that Runx molecules also bind the proximal enhancer [22], although it is not know whether that binding serves an activating or repressive function. In CD4-lineage cells, which express Thpok, Thpok molecules bind the silencer [15].