What happens in the thymus does not stay in the thymus: how T cells recycle the CD4⁺-CD8⁺ lineage commitment transcriptional circuitry to control their function

Abstract

MHC-restricted CD4⁺ and CD8⁺ T cell are at the core of most adaptive immune responses. Although these cells carry distinct functions, they arise from a common precursor during thymic differentiation, in a developmental sequence that matches CD4 and CD8 expression and functional potential with MHC restriction. While the transcriptional control of CD4⁺-CD8⁺ lineage choice in the thymus is now better understood, less was known about what maintains the CD4⁺- and CD8⁺-lineage integrity of mature T cells. In this review, we discuss the mechanisms that establish in the thymus, and maintain in post-thymic cells, the separation of these lineages. We focus on recent studies that address the mechanisms of epigenetic control of Cd4 expression and emphasize how maintaining a transcriptional circuitry nucleated around Thpok and Runx proteins, the key architects of CD4⁺-CD8⁺ lineage commitment in the thymus, is critical for CD4⁺ T cell helper functions.

Emergence of CD4⁺ and CD8⁺ lineages in the thymus

T lymphocytes constitute a critical arm of the immune system and serve multiple functions in responses against both external and internal offenses. Conventional T cells recognize MHC-peptide complexes (pMHC) through a heterodimeric T cell antigen receptor (TCR) comprising an α and a β chain (1, 2). Such αβ T cells are divided into two subsets based on their expression of CD4 and CD8 surface molecules (hereafter referred to as coreceptors). CD4⁺ T cells, which recognize peptides bound to class II MHC (MHC-II), are traditionally referred to as “helper” cells (3, 4). Upon antigenic stimulation, they can adopt any of multiple specialized T helper (Th) fates defined by unique cytokine and transcription factor expression patterns. Conventional CD8⁺ T cells, which express both CD8α and CD8β molecules as CD8αβ dimers, recognize peptides bound to class I MHC (MHC-I). Contrasting with the polymorphism of helper cell differentiation, CD8⁺ T cells are heavily skewed towards cytotoxic effector differentiation and are responsible for eliminating infected or transformed cells.

Both CD4⁺ and CD8⁺ T cells develop from a common precursor through a differentiation process that has long served as a model for binary lineage decisions and is of interest from both an immunological and developmental standpoint. This common precursor, which expresses both CD4 and CD8 and is thus called ‘double positive’ (DP), itself originates from hematopoietic progenitors that have entered the thymus and initiated their development into T cells as CD4⁻CD8⁻ (‘double negative’, DN) thymocytes (5–8). The developmental sequence that leads these progenitors to become DP thymocytes includes multiple differentiation and proliferation events, which we will not discuss here. Critical for the CD4⁺-CD8⁺ differentiation decision is the rearrangement of the genes encoding TCRβ and TCRα. This allows the surface expression of TCRαβ complexes whose reactivity against pMHC expressed by the thymic stroma determines the death or survival of thymocytes (6, 9). Because of the broad allelic polymorphism among MHC molecules at the species level, most DP cells have little or no affinity for self-MHC ligands at the individual level; such cells die in the thymic cortex in a few days through death by neglect. At the opposite end, those thymocytes with high affinity for self-MHC, with the potential for causing auto-immune disease, are thought to be eliminated through active cell death (a process called negative selection); however, recent studies emphasize that a fraction of these cells are redirected towards regulatory or alternative functional fates (10, 11). As a result, only thymocytes with an intermediate affinity for self MHC peptide complexes survive, a process called positive selection, and become mature T cells.

In addition, the pMHC reactivity of positively selected thymocytes determines their choice of CD4⁺ vs. CD8⁺-lineage, so that MHC I-restricted DP cells become CD4⁻CD8⁺ ‘single positive’ (SP) thymocytes, whereas MHC II-restricted DP cells become CD4⁺CD8⁻ SP thymocytes (4, 8). Such matching is important because CD4 and CD8 coreceptors facilitate TCR recognition of the appropriate class of MHC molecules and subsequent initiation of intra-cellular signaling (12). Furthermore, there is evidence that the thymic choice of CD4 or CD8 coreceptor expression is accompanied by “pre-programming” for helper or cytotoxic functions, respectively (13–15). From this final differentiation stage, SP thymocytes egress to the peripheral immune system as naïve CD4⁺ or CD8⁺ T cells, prepared to respond to initial encounter with antigen. Although they can embrace multiple functional fates characterized by distinct gene expression patterns, MHC-I and MHC II-restricted T cells retain the coreceptor they committed to in the thymus. This review discusses the mechanisms enforcing this “lineage stability” that are emerging as essential for proper T cell function. Before addressing these issues, a brief introduction to the transcription factors that promote the emergence of CD4⁺ and CD8⁺ lineages in the thymus is in order.

Thpok and Runx3 enforce lineage commitment in the thymus

Setting the commitment circuitry

Work from several laboratories has led to a model of CD4⁺-CD8⁺ lineage differentiation where commitment to either lineage, biologically defined as the loss of the alternative developmental fate, is enforced by two transcription factors, Runx3 and Thpok, with mutually exclusive expression and opposite activities in thymocytes (16–18) (discussed below). However, other factors, which we will refer to as specification factors, are involved in initiating expression of Thpok and Runx3, as well as additional lineage specific genes. The CD4⁺ lineage specification factors, to which we will return later in this review, include E-box binding proteins (E-proteins) E2A and HEB, Gata3, and three HMG proteins, Tox, Tcf1 and Lef1, the latter two being highly related and carrying partly overlapping functions in developing T cells (19–24). Gata3 and Tcf1 bind the Thpok gene, suggesting a direct effect on its transcription (24–26). In addition to their role in CD4⁺-lineage specification, E-proteins, Gata3 and Tox are important for positive selection, whether MHC-I- or MHC II-induced (21, 24, 26, 27), and Gata3 represses Runx3 in thymocytes (28). The impact of Tcf1 on the differentiation of DP into SP thymocytes is even broader, as it contributes to Cd4 silencing in CD8⁺-lineage cells (24). The combined activity of these transcription factors lays the groundwork for Thpok expression and CD4⁺ lineage commitment, and an equivalent circuitry is thought to promote Runx3 expression and CD8⁺-lineage commitment (17, 29, 30).

Runx proteins: architects of CD8⁺-lineage differentiation

Runx proteins are characterized by an amino-terminal region of homology, the Runt domain, that binds specific DNA sequences (31). Members Runx1 and Runx3 are expressed in both thymocytes and mature T cells, and act as obligate heterodimers with the structurally unrelated CBFβ protein (31). Runx1 is expressed throughout thymocyte development and in CD4⁺ T cells, whereas Runx3 expression starts in differentiating CD8⁺ SP cells, after positive selection, and is maintained in mature CD8⁺ T cells (32–34). In the absence of Runx function (which, because of the functional redundancy between the two proteins, requires inactivation of both Runx1 and Runx3 or of CBFβ), MHC I-restricted thymocytes fail to become CD8⁺ T cells and are redirected into the CD4⁺ lineage, severing the link between MHC restriction and coreceptor expression (34, 35). Runx3 directly contributes to CD8⁺-lineage differentiation by direct binding to Cd4 and Cd8 loci, which it represses and activates, respectively (32, 36). Last, Runx1 and Runx3, together with Mazr, a zinc finger transcription factor related to Thpok, contribute to inhibit expression of Thpok, by binding a silencer located upstream of the Thpok promoter (35, 37–39).

Thpok: enforcer of CD4⁺-lineage commitment

The opposing transcription factor, Thpok, belongs to a large family characterized by a zinc finger-based DNA binding domain and an amino-terminal BTB motif that promotes dimerization and recruitment of additional transcriptional regulators (40, 41). While not expressed in DP cells, Thpok is upregulated in MHC II-selected thymocytes and its expression persists in both CD4⁺ SP thymocytes and mature CD4⁺ T cells (42, 43). Thpok is required for MHC II-restricted thymocytes to become CD4⁺ T cells, although not for their positive selection and maturation. That is, disruption of Thpok, or a mutation in one of its zinc finger domains, causes the redirection of MHC II-restricted thymocytes into the CD8⁺-lineage (26, 37, 42, 44, 45). Conversely, enforced expression of Thpok in DP thymocytes inhibits CD8⁺ T cell differentiation, and redirects MHC I-restricted cells towards the CD4⁺-lineage (42, 43). Thpok represses expression of Runx3 and Cd8 (37, 45–47) and binds to enhancers within the Cd8 genes (48, 49). In addition, Thpok antagonizes the activity of Runx proteins (37, 50), so that both Cd4 and Thpok are subject to Runx-mediated transcriptional repression in thymocytes lacking Thpok molecules (37, 50). Thpok molecules bind the Cd4 and Thpok silencers, suggesting a direct antagonism of DNA-bound Runx (37), including of Runx1 in CD4⁺-lineage cells, although there is also evidence that Thpok promotes expression of Cd4 and CD4⁺-lineage genes through indirect mechanisms (50, 51).

Although it is not yet fully understood how they are matched to MHC restriction (52, 53), the mutually exclusive and opposite activities of Thpok and Runx3 promote CD4⁺ and CD8⁺ commitment in thymocytes. Since both factors remain expressed in post-thymic T cells (16), the question arises of whether they contribute to maintain the post-thymic stability of CD4⁺ and CD8⁺ lineage.

Control of Cd4 expression in mature T cells: an epigenetic epic

Genetic elements controlling Cd4 expression

Conceptually, two non-mutually exclusive processes can be envisioned to maintain lineage stability: an active transcriptional circuitry, possibly involving Thpok and Runx3, or epigenetic DNA or chromatin modifications, established during CD4⁺-CD8⁺ lineage differentiation in the thymus and subsequently inherited through cell division. In fact, as we will see below, T cells have borrowed from these two mechanisms to maintain CD4⁺ and CD8⁺ lineages in a way that fits requirements for stable, MHC-matched coreceptor expression, and for functional plasticity, which tailors cell responses to specific stimuli and environments.

The first strike was for epigenetics and came from studies of Cd4 expression. At least four cis-regulatory elements contribute to control Cd4 expression in T cells (54–57): the promoter, near the transcriptional start site (TSS), the ‘proximal’ enhancer, E4p, located 13 kilobases upstream of the TSS, the silencer, S4, in the first Cd4 exon, and a recently identified presumptive enhancer located near the silencer (Fig. 1). Genetic analyses by homologous recombination have shown that Cd4 expression requires E4p in DP thymocytes but not in positively selected thymocytes and resting CD4⁺ T cells (58). However, neither E4p nor the promoter are involved in determining the CD4⁺-lineage specificity of Cd4 expression. Rather, this task falls to the silencer (55, 56), whose germline deletion causes ectopic expression of Cd4 in MHC I-restricted CD8⁺ T cells (59, 60). Silencing of Cd4 in the thymus requires binding of Runx molecules and of additional silencing factors (including Tcf1 and Lef1) (24, 32, 61).

Figure 1. Thymic imprint on the Cd4 locus.

Cd4 expression is regulated by a combination of at least 4 cis-regulatory elements: the promoter (P4) near the TSS, the proximal enhancer (E4p) located 13kb upstream of the TSS, the silencer (S4) situated in the first Cd4 intron and a recently identified enhancer (DHS+3) located 3’ of the silencer. In CD4⁺-lineage thymocytes (top). Thpok binds the silencer and promotes sustained Cd4 expression. Locus demethylation is achieved in an E4p-dependent manner, presumably involving recruitment of Tet enzymes. E-proteins E2A and HEB, which are required for CD4⁺-lineage specification and bind E4p may be involved in these processes. In CD8⁺-lineage cells, Runx3 binds the silencer, promoting Cd4 repression and silencing. The locus is hypermethylated in a silencer-dependent manner, involving DNA methyl transferases (Dnmt), conceivably recruited by Runx3. Black boxes indicate Cd4 exons, grey rectangles cis regulatory elements, black arrow: TSS, active in CD4⁺-lineage cells.

Methylation of the Cd4 locus: the thymic imprint

In contrast, and remarkably, the silencer is dispensable to maintain silencing in mature CD8⁺ T cells, even through cell division (59). This indicates that the absence of Cd4 expression in CD8⁺ T cells reflects truly epigenetic silencing. This spectacular findings stimulated a long search for the silencing mechanisms. Epigenetic gene silencing largely relies on two processes: methylation of DNA itself (at position 5 of cytosine [5 mC] typically within palindromic CpG dinucleotide pairs) (62, 63) and post-transcriptional modifications of histones, the core proteins permitting assemblage of eukaryotic chromatin into nucleosomes (64). While there is currently no reported evidence that histone modifications contribute to Cd4 silencing, DNA methylation has recently been shown to be a key player (65). CpG methylation, initiated by DNA methyl transferases Dnmt3a and Dnmt3b, and transmitted during cell division by Dnmt1, is thought to promote gene silencing either by direct inhibition of transcription factor binding or by recruiting methyl CpG-binding proteins associated with transcription repression complexes (66). Importantly, the symmetry of CpG motifs allows their methylation to be ‘copied’ by Dnmt1 to newly synthesized strands during DNA replication, thereby ensuring truly epigenetic inheritance of this mark. Indeed, the Cd4 locus is hypermethylated in CD8⁺ T cells and hypomethylated in CD4⁺ T cells (65). Hypermethylation of the Cd4 locus is established early at the DN3 stage and maintained through the DP stage to CD8 SP, in a silencer-dependent manner. However, in mature CD8⁺ T cells, hypermethylation is silencer independent and remains stable throughout cell division. Furthermore, Dnmt1 (redundantly with Dnmt3a and Dnmt3b) is necessary to maintain Cd4 silencing during cell division (65). Thus, DNA methylation is critical to Cd4 silencing in CD8⁺ T cells.

Reciprocally, hypomethylation of the Cd4 locus is important for its proper expression in CD4⁺ T cells (65). Because the locus is methylated in DP thymocytes and because DP thymocyte are non-dividing cells, hypomethylation involves removing methyl residues from CpG islands. While there is no known cytosine demethylase that would perform such function, a mechanisms involving the Tet family of DNA dioxygenases has emerged as a functional equivalent (62). These enzymes convert 5mC to 5-hydroxymethylcytosine (5hmC), leading to 5hmC excision and replacement with unmethylated cytosine. While future genetic studies will be needed to confirm the involvement of Tet enzymes in Cd4 demethylation, analyses of CpG hydroxymethyl modifications in thymocytes are consistent with this possibility (65).

The mechanisms that install and maintain hypomethylation are coming to light. Although E4p is not needed for Cd4 expression except in DP thymocytes, its deletion in thymocytes, but not in post-thymic cells, destabilizes Cd4 expression in proliferating post-thymic CD4⁺ T cells (58). This suggests that E4p activity in the thymus facilitates demethylation, potentially by Tet-dependent mechanisms, installing an ‘open’ epigenetic context that subsequently contributes to stable Cd4 expression. Of interest in this context is that E4p recruits the E-protein HEB (67), which, redundantly with the related factor E2A, promotes Cd4 expression in DP thymocytes; both factors also support CD4⁺-lineage specification (22, 23). This suggests that E-proteins could promote stable epigenetic opening of the Cd4 locus, and affect post-thymic Cd4 expression through their thymic functions. Additional experiments will be needed to evaluate this hypothesis and determine the mechanisms at play. In agreement with a role for the specification circuitry, the demethylation of Cd4 in post-selection Class II-restricted thymocytes appears initiated independently of Thpok expression (65), and MHC II-restricted Thpok-deficient T cells, which are CD4⁻CD8⁺ when resting but retain marks of CD4⁺-lineage specification, re-express CD4 upon activation (13)

Despite the importance of such Cd4 epigenetic opening, recent studies (discussed in the next section) indicate that the transcriptional circuitry is needed to maintain Cd4 expression in mature T cells (68). Thus, epigenetic mechanisms do not appear as powerful to maintain Cd4 expression in CD4⁺ T cells as they are to enforce its silencing in CD8⁺ T cells.

Post-thymic control of Cd8 expression

Although the Cd8 locus is methylated (69, 70) and subject to histone modifications (71), and although there is evidence for epigenetic control of its expression (71, 72), whether there is a similar ‘imprint’ of thymic events on post-thymic Cd8 gene expression remains to be determined. In fact, the current evidence points to a greater control by the transcriptional circuitry: Runx3 is important to sustain expression of Cd8 in CD8⁺ T cells effectors (72), whereas, as we will see below, Thpok is critical for its repression in CD4⁺ T cells (47, 68, 73).

Thpok vs. Runx3 in mature T cells: the circuitry strikes back

Plasticity is critical for helper T cell responses

Would the tentative dichotomy in the control of coreceptor expression, predominantly epigenetic for Cd4 and dynamic for Cd8, also apply to the associated programming for helper vs. cytotoxic functions? Relevant to this question is the diversity of CD4⁺ T cell functional responses, corresponding to the variety of pathogens they fight (74–76). Th1 cells produce IFNγ in response to intra-cellular pathogens, Th2 cells fight extracellular parasites through production of IL-4, IL-5 and IL-13, whereas Th17 make IL-17 against extra-cellular bacteria and fungi. In addition, CD4⁺ T cells can post-thymically up-regulate Foxp3, adopting a regulatory fate and suppressing immune responses (77). Differentiation towards each of these fates is controlled by specific transcription factors, including T-bet, Gata3, and RORγt in Th1, Th2 and Th17 cells, respectively, in conjunction with cytokine-specific signal transducers of the Stat family. The current perspective is that each naïve CD4⁺ T cell emerging from the thymus, irrespective of its antigenic specificity, can potentially adopt any of these fates and preserves such potential by repressing expression of fate-specific transcription factors until antigen encounter (78). At that point, the cytokines generated by antigen presenting and innate immune cells initiate transcriptional program specific of each effector fate, and thereby match T cell functional responses to pathogenic stimuli.

Of particular interest for the present discussion is that differentiation into Th1 (CD4⁺) and cytotoxic (CD8⁺) T cells, which both target intra-cellular pathogens and produce IFNγ, relies on similar key factors in both cell types (76, 79, 80): Stat4 and Stat5, activated by IL-12 and IL-2, respectively, a T-box binding factor, typically T-bet in Th1 effectors and Eomes or T-bet in cytotoxic cells, and, last but not least, Runx3. Although Runx3 is CD8⁺-lineage specific in the thymus, it is expressed at similar levels in Th1 CD4⁺ effectors and cytotoxic CD8⁺ T cells, which raises puzzling issues. First, it implies that, even though Thpok represses Runx3 in the thymus, the circuitry in Th1 effectors must be plastic enough to accommodate Runx3, despite their Thpok expression. While Runx3 expression presumably involves cytokine signals that could be relayed by Stat5, as has been reported in thymocytes (29), how it is enabled despite concurrent Thpok expression has yet to be determined and will require a better understanding of the mechanisms of Thpok-mediated Runx3 repression. Second, Th1 and Th2 differentiation are mutually antagonistic; notably, Runx3 represses IL-4 expression and Th2 differentiation (79, 81). Thus, the question arises of whether preserving the Th2 potential of CD4⁺ T cells requires Thpok restraint of Runx3 expression. Last, because Th1 effectors express Cd4 and Thpok (47, 79, 80), they must counteract the repressive function of Runx3 on these genes, a task that has been suggested to fall on Thpok molecules (37, 50).

Thpok as a key enforcer of helper responses

A recent study shed light on the latter two points by evaluating Thpok functions through conditional deletion in naïve post-thymic CD4⁺ T cells, after their Thpok-dependent differentiation into CD4⁺ T cells in the thymus (68). These analyses showed that Thpok enforces Runx3 repression in naïve CD4⁺ T cells, and thereby a strict CD4⁺CD8⁻ coreceptor expression pattern. However, even after Thpok disruption and loss of Thpok protein, most MHC II-restricted naïve cells remain CD4⁺CD8⁻ and express little or no Runx3, suggesting that additional mechanisms, possibly epigenetic, contribute to control Cd4, Cd8, and Runx3 expression. Of note, the total number of MHC II-restricted cells was not affected by post-thymic Thpok disruption; thus, consistent with its dispensability for positive selection in the thymus, this factor is not essential for CD4⁺ T cell survival (13, 44, 47).

In addition to these effects in naïve T cells, post-thymic Thpok is needed for the proper effector differentiation of CD4⁺ T cells, and specifically to restrain the expression of cytotoxic genes, including those encoding Runx3, Eomes, Granzymes A and B, Perforin, and CD8 (47, 68). In the absence of Thpok, this cytotoxic program takes over Th2 differentiation and strongly impairs IL-4 production, both in vitro and in vivo (68). This impact of Thpok disruption is consistent with the role of Runx3 in IFNγ production Th1 differentiation. However, and unexpectedly, Thpok is also needed for Th1 differentiation (68). Even though production of IFNγ per se does not depend on Thpok, this factor is necessary to prevent the cytotoxic ‘diversion’ of Th1 effectors. Transcriptome analyses show that Thpok disruption causes an almost complete Th1 to cytotoxic conversion of the effector response, accompanied by the acquisition of actual cytolytic functions. In addition to restraining expression of cytotoxic genes, Thpok is essential for sustained expression of helper genes, first and foremost Cd40lg, which is critical for CD4⁺ help to B cells and dendritic cells (82).

Given the role of Runx3 in inhibiting Il4 expression, is Runx3 repression underpinning the protective role of Thpok on Th2 differentiation? That is indeed the case, as disruption of both Thpok and Cbfβ fully restores Th2 differentiation and prevents cytotoxic gene expression (68). In contrast, since Th1 effectors normally express Runx3, Thpok-mediated inhibition of Runx3 transcription could not account for its role in Th1 differentiation. Nonetheless, Cbfβ disruption showed that both the expression of cytotoxic genes by Thpok-deficient ‘Th1’ effectors, and, to a partial extent, their repression of Cd40lg, are dependent on Runx3 (68). Thus, Thpok serves two Runx-related functions by which it preserves the potential diversity of CD4⁺ T cell responses (Fig. 2). The first is to restrain Runx3 expression, and thereby enable cytokine-activation of Th2 responses. The second, which operates in Th1 effector cells that normally express Runx3, constrains Runx3 functions and prevents its activation of the cytotoxic program (68).

Figure 2. Maintenance of lineage stability in thymocytes and mature T cells.

(top panel) In thymocytes, Thpok and Runx3 form a dual negative regulatory loop (1), in which Runx3 inhibits Thpok expression and drives CD8⁺ lineage gene expression, whereas Thpok inhibits CD8 and Runx3 expression, protecting the CD4⁺ lineage. The mutually exclusive expression of Thpok and Runx3 decides lineage differentiation. (bottom two panels) During CD4⁺ effector T cell differentiation, Thpok serves two functions to constrain Runx activity. First, it inhibits Runx3 and cytotoxic gene expression (2) and thereby protects helper functions and Th2 differentiation. Second, Thpok antagonizes the function of Runx proteins (3) (Runx1 and Runx3) and thereby prevent cytotoxic diversion of CD4⁺ T cell expressing Runx3, including Th1 effectors (bottom panel).

Unexpectedly, and in contrast with its role in the thymus, the post-thymic disruption of Thpok spares two genes characteristic of the CD4⁺ lineage, Cd4 itself and Thpok (assessed by a GFP-based reporter transgene) (47, 68). Functional redundancy with LRF, a zinc finger transcription factor closely related to Thpok that also inhibits Runx-mediated Cd4 repression (13, 68), underpins Thpok-independent Cd4 expression in CD4⁺ T cells. Indeed, the post-thymic disruption of both Thpok and LRF causes CD4⁺ T cells to fully convert into CD4⁻CD8⁺ cells (68). Because Cd4 hypomethylation contributes to its expression in CD4⁺ T cells (65), it will be interesting to determine if Thpok and LRF disruption results in re-methylation of the Cd4 locus, or allow its repression by Runx molecules while remaining unmethylated. In any case, these findings emphasize that active epigenetic marking of Cd4 is not sufficient for its sustained expression in CD4⁺ T cells, for which overlapping functions of Thpok or LRF are also needed.

Contrary to Cd4, sustained expression of the Thpok gene in MHC II-restricted T cells requires neither Thpok nor LRF molecules despite the inhibitory potential of Runx3 (68). Whether additional transcription factors, or lack of transcriptional repressors, support Thpok expression or whether it is maintained epigenetically remains to be determined.

The Thpok-Runx3 circuitry in CD8⁺ T cells

Aside from the epigenetic maintenance of Cd4 silencing, the mechanisms that post-thymically maintain CD8⁺-lineage differentiation are less well understood. Additional evidence for epigenetic repression of the helper program in CD8⁺ T cells comes from ectopic (retroviral) Thpok expression in post-thymic CD8⁺ T cells (46). While Thpok impairs the expression of CD8, cytotoxic and IFNγ genes, mirroring the effects of Thpok disruption in CD4⁺ T cells (47, 68), it only modestly upregulates markers of helper function (including Gata3 and IL-4), with little or no effect on Cd40lg and none on Cd4. Although they do not exclude that transcription factors critical for helper gene expression are missing in CD8⁺ T cells, these observations support the idea that epigenetic mechanisms similar to those at work at the Cd4 locus impose broad restraints on CD4⁺-helper differentiation in CD8⁺ T cells. Such epigenetic control could involve establishment of silencing marks (e.g. DNA methylation), or removal of active marks, during CD8⁺ T cell differentiation in the thymus. Of note, the repression of Thpok itself in CD8⁺ T cells is not as stringent as that of Cd4, as it is expressed upon antigen-induced activation (25, 83). Thpok amounts in activated CD8⁺ T cells are much lower than in CD4⁺ T cells, and have been reported to promote cytotoxic responses and memory CD8⁺ T cell differentiation (83), through mechanisms that remain to be defined.

Conversely, Runx complexes are not needed to maintain CD8 expression in activated cytotoxic effectors (72), and, similar to the Cd4 silencer itself, Runx3 was reported to be dispensable to maintain post-thymic Cd4 silencing (58). However, the post-thymic functions of Runx3 in CD8⁺ T cells remain to be clarified by in vivo stage-specific inactivation. This is in large part because Runx3 is not strictly required for CD8⁺ T cell development (owing to the functional overlap with Runx1) (32–34), so that studies of Runx3 functions in mature CD8⁺ T cells have mostly been conducted using genetic models that delete Runx3 in the thymus. These experiments have provided evidence that Runx3 is important for cytotoxic gene expression in CD8⁺ effectors, and notably suggested that it promotes expression of T-bet and Eomes (84). Of note, the disruption of both T-bet and Eomes causes the diversion of cytotoxic into IL-17 producing responses (85), and it remains to be determined whether the disruption of Runx activity would have similar consequences.

Where circuitry meets epigenetics

Thus, the Thpok-Runx3 based loop that decides CD4⁺-CD8⁺-lineage commitment in the thymus is also involved in maintaining CD4⁺ lineage integrity and helper responses after thymic egress (Fig. 2). The critical importance of these functions is highlighted by the fact that post-thymic deletion of Thpok in CD4⁺ T cells impairs long-term control of T. gondii, resulting in the death of infected animals (M.V. and R.B., unpublished observations). Whether post-thymic functions of Thpok are needed for differentiation into other effector fates (e.g. Th17 or T follicular helper) remains to be determined. Current evidence indicates that neither Thpok nor LRF are needed for Th17 differentiation nor to restrain the expression of IFNγ by Th17 cells, suggesting that the Th17 circuitry efficiently represses IFNγ(68). Contrasting with this broad impact of Thpok in CD4⁺ T cells, the available evidence, while limited, suggests that Runx3 contributes to the expression of cytotoxic genes in CD8⁺ T cells, but not to the repression of CD4⁺-lineage genes, even though it serves such functions in thymocytes or in Thpok-deficient CD4⁺ T cells.

Continued involvement of Thpok and Runx3 beyond the thymus also contrasts with stage-specific functions of some of CD4⁺-lineage specification factors mentioned earlier, including Tcf1 and Lef1, or Gata3. Post-thymic disruption of both Tcf1 and Lef1 compromises Tfh differentiation but does not affect overall CD4 expression or other helper functions (86). Similarly, disruption of Gata3 in mature CD4⁺ T cells impairs Th2 differentiation, but was not reported to affect CD4⁺ lineage integrity (87).

It is also important to emphasize that, as illustrated by a recent study of histone deacetylase (HDAC) functions in T cells (71), epigenetic and active transcriptional control collaborate to maintain appropriate gene expression. Acetylation of histone H3 on lysine residues 9 and 27, typically at gene promoters and enhancers, is associated with increased transcriptional activity (88). HDACs are part of large transcriptional repression complexes, some of which associate with members of the BTB zinc finger family (41, 89) and their recruitment dampens gene expression. Inactivation of both HDAC1 and HDAC2 in the thymus causes the conversion of CD4⁺ effectors into CD4⁺CD8⁺ cells expressing cytotoxic genes and Runx3, and producing IFNγ (71). Accordingly, activated CD4⁺ T cells from HDAC1 and HDAC2 deficient mice show increased H3K9 acetylation at the promoters of cytotoxic genes and at a Cd8 enhancer active in effector cells. Although these experiments assessed the consequences of thymic HDAC inactivation, treating post-thymic CD4⁺ T cells with an inhibitor of HDACs mimicked the effects of HDAC1 and HDAC2 deletion, indicating a persistent need for HDACs to repress CD8⁺-lineage genes (71).

The mechanistic bases for these effects of HDAC1 and HDAC2 remain to be elucidated. The parallel with Thpok functions suggests that Thpok-mediated repression of cytotoxic genes could involve its recruitment of Class I HDACs 1 and 2, similar to its homolog Bcl6 (89). However, it was reported that Thpok binds Class II HDACs 4, 5 and 10, which it recruits to the Cd8 locus, but not HDACs 1 and 2 (48); additionally, it is possible that Thpok and HDACs serve independently to repress CD8⁺-lineage expression. Thus, a comprehensive picture of Thpok association with HDACs has not yet emerged. Further highlighting the challenges of integrating such studies into a unified conceptual framework, HDACs act on non-histone acetyl-lysine substrates. A comparison of mass spectrometry and mRNA expression analyses showed that HDAC1 and HDAC2 tinactivation affected almost 200 genes at the protein but not at the mRNA level. Thus, it is possible that re-expression of CD8⁺-lineage genes in HDAC-deficient CD4⁺ T cells is mediated by deacetylation of non-histone proteins. Although such substrates could include Thpok and Runx3, both of which have shown to be acetylated and thereby protected from ubiquitin-mediated degradation (90, 91), the impact of such modifications in vivo remains to be assessed.

Crossing lineage boundaries: risks and benefits

Cytotoxic CD4⁺ T cells

Although Thpok preserves the functional diversity of helper effector responses by restraining the cytotoxic program, there is evidence that CD4⁺ T cells can acquire cytotoxic functions, especially during chronic infections (reviewed in Refs.(92, 93). These CD4⁺ T cells are distinct from Th1 cells, with upregulated Eomes as well as cytolytic activity mediated by both perforin and Fas. Such cells could function to eliminate pathogen-infected cells that express MHC-II during infection (94), or tumor cells (95), or conceivably to dampen antigen presentation to avoid excessive immune responses.

It remains to be determined whether acquisition of cytotoxic gene expression implies the cessation of Thpok expression, or antagonism of its functions. A potential scenario has emerged from studies of a specific subset of intra-epithelial lymphocytes (IELs), defined by the co-expression of CD4 and CD8α but not CD8β(49, 73). These cells thus differ from the larger IEL subset expressing CD8α but neither CD4 nor CD8β(96).Most such CD4⁺CD8α⁺ IELs cells fail to express Thpok, and adoptive transfer experiments, together with elegant fate mapping strategies, suggest that at least some of them derive from bona fide MHC II-restricted CD4⁺ T cells that have terminated Thpok expression, driven by TGFβ and retinoic acid signaling in the intestinal milieu (73). While such post-thymic Thpok silencing was shown to depend on Mazr and the Thpok silencer, it was only partly dependent on Runx3 expression (73). The latter observation suggests mechanisms distinct from the Runx-mediated Thpok repression operating during thymic selection. Consistent with this idea, the double disruption of Thpok and Lrf, despite promoting Runx3 expression, does not impair Thpok gene activity in CD4⁺ effectors, unlike its effect on Cd4 (68).

Determining whether post-thymic Thpok silencing underpins the differentiation of “conventional” cytotoxic CD4⁺ effectors during chronic infections is an important goal for future research. Such cells do not express CD8α, and their Thpok expression status has not been reported. Conversely, neither the antigenic specificity of CD4⁺CD8α⁺ IELs nor their functions have been definitely identified. Whereas there is evidence that at least some CD4⁻CD8α⁺ IELS derive from self-reactive thymocytes escaping deletion by negative selection in the thymus (10, 11) whether the same is true of CD4⁺CD8α⁺ IELs remains to be determined. Functionally, they could serve as a first line of defense against viruses trophic for Class II expressing cells in the gut, or carry regulatory functions against “pathogenic” inflammatory Th17 or Th1 cells (49, 73).

Switching coreceptor expression

Additional studies in non human primates (including African Green and Patas monkeys) have reported a shift in Class-II restricted T cells from their normal CD4⁺CD8⁻ to a CD4⁻CD8^lo phenotype (97, 98). Such conversion is only observed in memory cells (naïve cells remain CD4⁺CD8⁻), and is accompanied by impaired expression of helper-specific genes at the single-cell level. However, at the organism level, the cessation of CD4 expression is thought to prevent viral dissemination after SIV infection, and therefore to be advantageous by preserving the MHC II-restricted repertoire. The transcriptional control of this conversion, and whether it involves Thpok or LRF, remain to be determined.

Despite their potential importance, such changes in coreceptor expression are the exception rather than the rule, as T cells take great care of maintaining coreceptor expression matched to their MHC specificity. This is highlighted by the strong epigenetic Cd4 silencing in CD8⁺ T cells. However, there is evidence that the epigenetic silencing of Cd4 is not as stringent in human than in mouse CD8⁺ T cells, despite conservation of the Cd4 silencer in the human genome (99). Indeed, naïve human CD8⁺ cells, from either adults or neonates, can re-induce CD4 expression upon long-term activation (100, 101), and this has been proposed to make them susceptible to HIV infection. However, such “leakiness” of CD4 expression was limited, as most CD8⁺ T cells remained CD4⁻ in such experiments, and it is not known if “leaky” cells acquired helper functions together with CD4 expression.

Why would CD4 re-expression be so detrimental that CD8⁺ T cells have developed redundant mechanisms to avoid it? It can be envisioned to have distinct consequences based on the cell’s reactivity to MHC-II. For most CD8⁺ T cells, carrying TCRs unable to cross-react with MHC-II peptide complexes, the main impact of CD4 re-expression would be on TCR signaling. Both CD4 and CD8 bind the tyrosine kinase Lck, which initiates intra-cellular TCR signaling upon pMHC engagement (2, 102). However, Lck binds with greater affinity to CD4 than to CD8; as a result, in cells expressing both CD4 and CD8, CD4 molecules trap a large fraction of Lck away from CD8, and therefore diminish the contribution of CD8 to MHC I-induced TCR signaling (103). Additional and deeper consequences are predicted in CD8⁺ T cells carrying TCRs cross-reacting with MHC-II peptide complexes, in which Cd4 reexpression could increase MHC II-induced TCR signaling. Recent studies suggest that a substantial fraction of thymocytes carry TCRs that can recognize both MHC-I and MHC-II peptide complexes, and therefore carry potential for cross-reactivity (11, 104). While many such cross-reactive cells die by negative selection in the thymus, some escape deletion, as shown by the existence of MHC-II-selected CD4⁺ T cells that can bind MHC-I peptide complexes (104). Acquisition of MHC II-reactivity by CD8⁺ T cells has the potential to allow their inappropriate activation by MHC-II-expressing APCs, and therefore to lead to auto-immunity.

Conclusions

Several conclusions arise from the studies summarized in this review. First, the current evidence favors asymmetric control of Cd4 and Cd8 expression in post-thymic cells. The transcriptional circuitry remains a key controller of Cd8 expression, in contrast with Cd4, for which both silencing in CD8⁺ T cells and stable expression in CD4⁺ T cells have a strong epigenetic component, involving cytosine methylation.

Second, the core components of the thymic commitment circuitry, Thpok and Runx proteins, remain involved in maintaining post-thymic T cell lineage integrity (Fig. 2). The current evidence suggests that Runx3 in CD8⁺ T cells promotes expression of the cytotoxic program but not repression of helper or CD4⁺-lineage genes. This contrasts with the role of Thpok (in part redundantly with LRF) as both a repressor of the cytotoxic program and a ‘protector’ of Th1 and Th2 helper differentiation. Although it remains to be determined if Thpok is also needed for additional helper fates (including Th17 and Tfh), the emerging picture is that post-thymic Thpok maintains the ability of naïve CD4⁺ T cells to adopt any of the helper fates upon activation. The latter appears as a key requirement for a functional adaptive immune system, since there is no a priori link between antigenic specificity and functional responses (determined by the type of pathogen involved). In contrast, the persistent expression of Runx3 in CD8⁺ T cells strongly biases their responses towards cytotoxicity, suggesting that only strong cytokine-induced signals can overcome such a bias and direct CD8⁺ T cells towards alternative fates, similar to those adopted by effector CD4⁺ T cells including IL-17 production (reviewed in Ref. (105). Whether Runx3 similarly inhibits adoption of such alternative functional fates remains to be assessed.

Recent studies have highlighted the similarities between T cell effector programs and those elicited in innate lymphoid cells (ILCs) (76, 106). A major difference between these two cell types is that the function of ILCs does not require antigen recognition by a clonotypic receptor. As a result, no benefit would be expected from maintaining ILCs in a naïve status with multi-functional potential, and they acquire effector-specific transcriptional programs, e.g. promoting IFNγ production for ILC1 or cytotoxic gene expression for NK cells, during their development (106, 107). In this perspective, which remains to be experimentally validated, functions similar to those of Thpok in CD4⁺ T cells are not needed once ILCs have completed their development.

The critical role of Thpok in CD4⁺ T cell effector differentiation raises the question of whether it could be targeted to inhibit or redirect effector differentiation for therapeutic purposes. A correlate of this question is whether Thpok also controls CD4⁺ lineage integrity in human cells. Thpok and LRF amino-acid sequences, as well as those of Runx proteins, are highly conserved among mammalian species, and there is evidence for preferential CD4⁺-lineage Thpok expression in human thymocytes and T cells, although possibly not as pronounced as in mice (108, 109). However, there are also differences in the permissivity of Cd4 silencing and expression of CD40L (110) in human CD8⁺ T cells, as well as in the lineage plasticity in non-human primate T cells described above. Determining the role of Thpok in human T cell responses should be facilitated by the emergence of tools for genome editing, and would be a first step to explore the potential of Thpok as a target for therapeutic manipulation of immune responses for human disease.