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Molecular control of CD4+ T cell lineage plasticity and integrity
International Immunopharmacology 28 (2015) 813–817
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Molecular control of CD4+ T cell lineage plasticity and integrity Wilfried Ellmeier Division of Immunobiology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria
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Article history: Received 27 February 2015 Accepted 28 March 2015 Available online 9 April 2015 Keywords: CD4+ cytotoxic T cells (CD4+ CTLs) CD4+ T cell lineage integrity Intestinal intraepithelial lymphocytes ThPOK Runx3 Histone deacetylases
a b s t r a c t CD4+ helper T cells and CD8+ cytotoxic T cells form the two major subsets of peripheral T lymphocytes. Helper T cells fulfill crucial roles in the activation and coordination of the immune response, while cytotoxic T cells kill virus-infected or tumor cells. Recent data suggest that the lineage identify of helper T cells is not fixed and that CD4+ T cells under certain physiological conditions can be reprogrammed to express CD8 lineage genes and to develop into intestinal intraepithelial CD4+ cytotoxic T lymphocytes that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, the analysis of mice with a conditional deletion of the transcription factor ThPOK or the histone deacetylases HDAC1 and HDAC2 indicated that CD8 lineage genes are actively repressed in CD4+ T cells in order to maintain the lineage integrity of helper T cells. In this review I summarize recent studies that indicate plasticity of CD4+ T cells towards a CTL program and that demonstrate that ThPOK and HDAC1–HDAC2 are part of a transcriptional regulatory circuit that counteracts the activity of the transcription factor Runx3 to maintain CD4+ T cell lineage integrity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction and background The two major subsets of peripheral T cells express either the CD4 or CD8 coreceptor molecules. T cells that express CD4 constitute the major histocompatibility complex (MHC) class II-restricted helper T cell population, while T cells that express CD8 (which on conventional T cells is formed by the CD8α and CD8β heterodimers) represent the MHC class I-restricted cytotoxic T cell lineage. CD4+ T cells can be subdivided into several T helper subsets and these subsets play key roles in the activation of other immune cells and coordination of the immune response, while cytotoxic T cells as part of their effector mechanism have the “license to kill” and thus eliminate virus infected cells or cancer cells [1]. The proper establishment of cell lineage-specific expression patterns and the stable repression of inappropriate genes and commitment to a particular lineage is crucial for the regulation of T cell development and the maintenance of T cell function. CD4+ and CD8+ T cells develop in the thymus from so-called double-positive (DP) thymocytes that express both CD4 and CD8. Studies performed during the last decades
Abbreviations: BTB, broad complex, Tram-track and bric-a-brac; CBFβ, core-binding factor β; CD4SP, CD4 single-positive; CD8SP, CD8 single-positive; CTL, cytotoxic T lymphocyte; DN, double-negative; DP, double-positive; HAT, histone acetyltransferase; HDAC, histone deacetylase; IEL, intraepithelial lymphocytes; IFN, interferon; LRF, leukemia/lymphoma related factor; MAZR, Myc-associated zinc finger-related factor; MHC, major histocompatibility complex; LN, lymph node; RA, retinoic acid; Rag, recombination activating gene; Runx, Runt-related transcription factor; ThPOK, T-helper inducing POZ/Krüppel like factor; TCR, T-cell receptor; TGFβ, transforming growth factor β; Th, T helper. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.intimp.2015.03.050 1567-5769/© 2015 Elsevier B.V. All rights reserved.
provided important insight in the mechanisms of CD4/CD8 cell fate choice and led to the identification of key transcription factors that regulate this process [2–5]. ThPOK, together with c-Myb, Tox and GATA-3, directs the development of the CD4+ T cell lineage [6–12], while Runx3, together with other factors such as MAZR [13], directs the differentiation of CD8+ T cells [14,15]. Thus, the identification of CD4+ and CD8+ T cell-lineage specific transcription factors and their distinct and different type of helper and cytotoxic effector functions suggested that mature CD4+ T cells and CD8+ T cells retain their lineage identity and integrity once they have been generated. Some subsets of CD4+ T cells have been described that display a cytotoxic activity and were designated as CD4+ cytotoxic T lymphocytes (CD4+ CTLs). This suggests some exceptions from the clear functional separation between helper and cytotoxic lineage T cells, although CD4+ CTLs were considered as being Th1-like [16–19]. However, as summarized below, recent data suggested that the lineage identity of helper T cells is not irreversible and that CD4+ T cells under certain physiological conditions have an unexpected plasticity and can be reprogrammed to express CD8 lineage genes and to develop into CD4+ CTLs that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, these studies also indicate that the potential for the induction of CD8 lineage genes and a CTL program has to be actively repressed in mature CD4+ T cells to maintain the lineage integrity of helper T cells. 2. ThPOK and LRF prevent the transdifferentiation of mature CD4+ T cells into CD8+ T cells A major breakthrough in the field of CD4/CD8 lineage differentiation was the identification that the BTB domain-containing transcription
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factor ThPOK (encoded by the Zbtb7b gene, referred here as the Thpok gene) is a key commitment factor for CD4+ T cell lineage development. Two studies independently showed that loss of ThPOK function during T cell development leads to the redirection of MHC class II-restricted cells into the CD8+ T cell lineage [6,7]. In contrast, enforced expression of ThPOK during T cell development is sufficient to direct MHC class I-restricted T cells into the CD4+ helper T cell lineage [6,7]. Similarly, enforced expression of ThPOK in peripheral CD8+ T cells induces CD4 lineage features, while CD8 lineage genes encoding for CD8α, Perforin, Granzyme B and Eomesodermin were down-regulated [20], suggesting that mature CD8+ T cells remain susceptibly to the activity of ThPOK. Soon after the discovery of ThPOK and its description as a key regulator of CD4+ T cell commitment, several studies were published that indicated that ThPOK function might be continuously required in mature CD4+ T cells and that ThPOK maintains the lineage integrity of CD4+ T cells. One study showed that ThPOK is necessary for antagonizing Cd4 silencer activity in order to maintain CD4 expression during helper T cell differentiation [21]. Moreover, by using genetically modified mice that express a hypomorph Thpok allele, it was shown that ThPOK represses CD8 lineage genes such as those encoding for CD8α, CD8β, Runx3 and Eomesodermin in CD4+ T cells and that low ThPOK levels lead to the induction of a CD8 effector program [22,23]. These data demonstrated that one key function of ThPOK in mature CD4+ T cells is to repress CD8 lineage genes. Further insight into the role of ThPOK in the regulation of CD4 lineage function as well lineage integrity came from a study in which ThPOK was conditionally deleted in peripheral T cells (i.e. postthymic T cells) [24]. In these mice, due to the “late” peripheral deletion of ThPOK, CD4+ and CD8+ T cell development was normal and the impact of ThPOK deletion on CD4+ T cell function could be studied. Using this approach it was shown that ThPOK activity is essential for Th1-type T cells functions but not for Th17 differentiation. Furthermore, in agreement with studies using the hypomorph Thpok allele, ThPOKdeficient Th1 cells also up-regulated CD8α and CD8β and displayed increased Runx3 expression in comparison to wild-type Th1 cells. Moreover, ThPOK-deficient Th1 cells expressed also Eomesodermin as well as CD107a (a marker for cytotoxic cell degranulation [25,26]) in vitro and following Toxoplasma gondii infection in vivo. The upregulation of a CD8 effector program was dependent on Runx3, showing that ThPOK-mediated control of Runx3 expression levels is crucial for the repression of the CD8 effector program, although ThPOK might also act downstream of Runx3 to repress CD8 lineage genes that are otherwise induced by Runx3 [27]. Interestingly, although Runx factors are important regulators of Cd4 silencing, CD4 expression remained high in ThPOK-deficient Th1 cells despite high Runx3 expression. One possible explanation for this observation was that ThPOK-related factors might overcome the repressive activity of Runx3 on the Cd4 silencer. A candidate factor for such a compensatory function was LRF (encoded by the Zbtb7a gene), which is closely related to ThPOK and shown to be able to partially compensate for loss of ThPOK [28]. Indeed, CD4+ T cells from mice with a combined conditional peripheral deletion of ThPOK and LRF lost CD4 expression upon activation and Th1 differentiation and converted into CD4−CD8+ effector T cells. Thus, ThPOK and LRF are essential to repress the transdifferentiation of CD4+ T cells into CD8+ T cells. 3. Developmental plasticity of intestinal CD4+ T cells under physiological conditions
also CD8α (but not CD8β) and thus were within the subset of CD4+CD8αα+ intraepithelial lymphocytes (IELs). These cells had features similar to CD8+ cytotoxic T cells such as expression of Granzyme B and CD107a and, upon activation, to kill target cells in vitro. Fatetracking experiments revealed that these cells were derived from cells that previously expressed Thpok. Indeed, transfer of naïve CD4+ T cells (that carried a Thpok–GFP reporter allele to track ThPOK expression) into Rag-deficient mice led to a reprogramming of the transferred cells into MHC class II-restricted CD4+CD8αα+ T cells with a cytotoxic activity (CD4+ CTLs). While transferred CD4+ T cells that were isolated from the spleen or mesenteric LN of recipient mice still expressed ThPOK, intestinal intraepithelial CD4+ CTLs were observed in recipient mice that did not express Thpok. Of note, the reprogrammed CD4+ CTLs remained immunologically quiescent at steady state. However, in response to restimulation in the presence of IL15, these cells increased their inflammatory and cytolytic potential. Preceding this reprogramming of CD4+ T cells was the termination of ThPOK expression in antigenexperienced CD4+ effector T cells, which then led to the induction of CD8 lineage genes and a CTL program. The termination of ThPOK expression was under control of the Thpok silencer, since reprogramming of silencer-deficient CD4+ T cells into intestinal CD4+ CTLs was impaired. Similar, CD4+ T cells with a deletion of MAZR, which binds to the Thpok silencer and represses ThPOK during CD8 lineage differentiation [13], displayed a decreased reprogramming into CD4+ CTLs. Thus, the Thpok silencer is a crucial regulatory node in the differentiation of intestinal CD4 + T cells into intraepithelial CD4+ CTLs. Additional insight into the regulation of intestinal CD4+ T cell plasticity was provided in a study that addressed whether the post-thymic downregulation of ThPOK in intestinal CD4+ T cells is associated with changes in the expression of Runx3 [30]. Using reporter mice for monitoring either Runx3 or Thpok expression it was shown that the intestinal CD4+ T cells with a low expression of ThPOK had high expression levels of Runx3. It was further shown that Runx3 upregulation was the initiating event that led to the downmodulation of ThPOK, and the changes in the expression of both Runx3 and ThPOK were required for the induction of a CTL program in intestinal CD4+ T cells. The observation that Runx3 downregulated ThPOK in intestinal CD4+ T cells is different to ex vivo analyzed splenic ThPOK-deficient CD4+ T cells, where the deletion of ThPOK led to the induction of Runx3 [23,24]. Interestingly, the reprogramming of intestinal CD4+ T cells was is part induced by TGFβ and retinoic acid (RA) [30], indicating that intestinal environmental signals might regulate the expression of Runx3 and thus the differentiation of intestinal CD4+ T cells into intraepithelial CD4+CD8αα+ CTLs. This observation is also in agreement with a study showing that TGFβ induces CD8α expression on activated CD4+ T cells [31]. To determine the impact of the crossregulation between ThPOK and Runx3 for the function of intestinal CD4+ T cells, a T cell-mediated adoptive transfer colitis model was used to study the inflammatory potential of T cells that were deficient in either ThPOK or Runx3. Using this approach it was shown that forced loss of ThPOK in CD4+ T cells reduced inflammation despite normal Th17 differentiation, while forced loss of Runx3 resulted in a higher inflammation and enhanced Th17 differentiation [30]. Thus, the mutual expression of Runx3 and ThPOK might regulate intestinal CD4+ T cell immunity, perhaps by deviating pathogenic CD4+ T cells into CD4+ CTLs [32]. 4. HDAC1–HDAC2 maintain CD4+ T cell lineage integrity
The studies that analyzed CD4+ T cells with a hypomorphic Thpok allele or with a peripheral deletion of ThPOK indicated already that CD4+ T cells have the potential to acquire cytotoxic CD8+ T cell features. One intriguing example demonstrating in vivo plasticity of CD4+ T cells under physiological conditions was provided in a study that showed lineage conversion of intestinal CD4+ T cells towards a CTL phenotype [29]. The starting point of this study was the observation that a subset of intestinal CD4+ T cells did not express ThPOK. These cells expressed
Another genetic switch that represses a CD8 effector program in CD4 lineage T cells was identified during the analysis of mice with a deletion of the histone deacetylases (HDAC) 1 and HDAC2 in T cells [33]. An important regulatory level of the establishment and maintenance of cell lineage-specific expression patterns is formed by epigenetic and chromatin-mediated mechanism such as DNA methylation or histone acetylation and methylation. Modification of core histones by reversible
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lysine acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs), which are considered transcriptional coactivators and corepressors, respectively [34,35]. The HDAC family consists of 18 members grouped into 4 classes [36]. Many studies, using genetic approaches as well as by applying HDAC inhibitors, showed important roles for reversible lysine acetylation in the regulation of T cell development and function and several members of the HDAC family have been implicated in the regulation of T cells [37–40]. The generation of mice with a T cell-specific deletion of HDAC1 (using Cd4-Cre) showed that loss of HDAC1 leads to enhanced Th2 responses in vitro and in vivo leading to a more severe disease in a Th2-type allergic airway inflammation model [41]. Surprisingly, T cell development in the absence of HDAC1 was normal, most likely due to the observed compensatory up-regulation of HDAC2, which was also described in other cell lineages and biological processes [42–47]. In order to investigate whether the combined deletion of HDAC1 and HDAC2 leads to T cell developmental defects, mice with a T cell-specific deletion (using Cd4-Cre) of both HDAC1 and HDAC2 were generated [33]. Overall, the number of αβTCR+ T cells was reduced and the activation of HDAC1–HDAC2-deficient peripheral CD4+ T cells revealed that HDAC1–HDAC2-deficient CD4+ T cells undergo apoptosis if the cells are cultured for more that 3 days in vitro. These data show that HDAC1 and HDAC2 are essential for the efficient generation of the peripheral T cell pool and for the survival of proliferating CD4+ T cells. However, a surprising finding was that HDAC1–HDAC2-deficient CD4+ helper T cells acquired CD8 lineage effector features [33]. This was already observed under homeostatic conditions due to the appearance of a fraction of MHC class II-selected CD4+ helper T cells that expressed CD8+ T cell lineage genes encoding for CD8α, CD8β and Eomesodermin in peripheral lymphoid organs such as LN and spleen. Interestingly, the HDAC1–HDAC2-deficient CD4+CD8+ as well as CD4+CD8− T cells had normal expression of ThPOK protein ex vivo and up-regulated CD154 upon overnight activation, thus displaying characteristic and defining features of helper T cells. However, after short-term activation for 60 h, HDAC1–HDAC2-deficient CD4+CD8− lineage T cells acquired in addition to CD8α and CD8β expression also CD8 effector features, indicated by a strong further up-regulation of Eomesodermin, a high production of IFN-γ and increased expression of Runx3, T-bet, Granzyme B and Perforin. This was observed in Th0 and Th1-polarizing conditions, while the induction of a CD8 effector program was strongly impaired in Th2 conditions. Since Runx/CBFβ complexes are essential for the development of CD8+ T cells and their differentiation into cytotoxic effector T cells [14,48,49], the role of Runx factors in the induction of a CD8 effector program in the absence of HDAC1–HDAC2 was further explored by crossing conditional HDAC1–HDAC2 mice with conditional CBFβ mice, which leads to an inactivation of all Runx factors. This revealed that the expression of CD8 effector genes in activated HDAC1–HDAC2-deficient CD4+ T cells was dependent on Runx–CBFβ complexes. Moreover, the expression of CD8 effector genes correlated with the presence of Runx/CBFβ complexes and local histone hyperacetylation at the promoter regions of these genes [33]. Interestingly, the treatment of activated wild-type CD4+ T cells with class I HDAC inhibitors led to the induction of CD8α, Eomesodermin, T-bet and IFN-γ expression, indicating that HDAC1–HDAC2 are also required for the repression of CD8 lineage genes in mature CD4+ T cells and thus the maintenance of CD4+ T cell lineage integrity [33]. In addition, retroviral-mediated enforced expression of Runx3 in wild-type CD4+ T cells led to the induction of CD8α, Granzyme B and increased IFN-γ production and this was in part increased in synergy with HDAC inhibitors. Moreover, Runx3 expression together with HDAC inhibitor treatment led also to the induction of Eomesoderin in CD4+ T cells. Taken together, these observations revealed that HDAC1 and HDAC2 are essential for the maintenance of CD4+ T cell lineage integrity by repressing Runx–CBFβ complexes that otherwise induce CD8 lineage genes and a CTL-like program in CD4+ T cells.
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5. HDAC1–HDAC2, ThPOK/LRF and Runx factors form a transcriptional circuit that regulates CD4+ T cell lineage integrity The studies describing the conversion of intestinal CD4+ T cells into CD4+CD8αα+ CTLs demonstrate that this is a physiological process and the ThPOK and Runx3 expression patterns suggest that CD4+ CTLs represent a distinct Th subset different from Th1 cells [29,30,32]. Since ThPOK and HDAC1–HDAC2 are required to repress a Runx3-dependent CD8 lineage effector program in CD4+ T cells, it is conceivable that a transcriptional circuit containing HDAC1–HDAC2, ThPOK and Runx3 regulates the differentiation of CD4+ T cells into CD4+ CTLs (Fig. 1). At a molecular level one might envisage that HDAC1–HDAC2 as well as ThPOK (in part together with LRF) maintain the integrity of CD4+ T cells by keeping Runx3–CBFβ complexes below a certain threshold level to prevent the induction of a CD8 effector program in CD4+ T cells. It is likely that the strong induction of Runx3 expression in HDAC1–HDAC2-deficient or ThPOK-deficient CD4 lineage T cells is the initiating event in the induction of CD8 lineage genes and the acquisition of a CTL program. The induction of a CD8 effector program is regulated in part via recruitment of Runx–CBFβ complexes to target genes such as Cd8, GzmB, Prf1 and Ifng as shown in HDAC1–HDAC2-deficient CD4+ T cells [33]. Since environmental cues such as TGFβ and RA induce the reprogramming into intestinal CD4+ CTLs [30], it is possible that these signals might (transiently) modulate the activity of HDAC1– HDAC2 and/or ThPOK and thus allow the induction of a CTL program in CD4+ T cells. Further, activated HDAC1–HDAC2-deficient or ThPOKdeficient CD4+ T cells upregulated Eomesodermin in a Runx–CBFβ-dependent manner, and HDAC1–HDAC2-deficient CD4+ T cells also upregulated T-bet. Since Eomesodermin and T-bet are important for the induction of the cytotoxic program [48–51], it is likely that Runx3, Eomesodermin and T-bet together regulate the induction of the CD8 effector program in HDAC1–HDAC2-deficient or ThPOK-deficient CD4+ T cells. HDACs are considered as general chromatin-modifying regulators of gene expression, however many non-histone targets of HATs and HDACs are currently emerging [52,53]. Lysine acetylation of proteins has been shown to affect protein–protein and protein–DNA interactions, protein stability and intracellular localization [34,52,54]. This raises the important question whether the induction of a CTL program in HDAC1–HDAC2-deficient CD4+ T cells is a consequence of chromatin-mediated or of non-chromatin-mediated effects, or a combination of both. The transcriptome analysis of HDAC1–HDAC2-deficient CD4+ T cells and the observation that HDAC1–HDAC2 are recruited to the Runx3 locus strongly suggest that HDAC1–HDAC2 directly modulate the expression levels of the Runx3 gene in CD4+ T cells [33]. However, it has been shown that the stability and function of Runx factors can be controlled by reversible lysine acetylation [55,56], suggesting that Runx3 expression might be also regulated at the post-translational level. Further studies are required to investigate whether posttranslational modifications and potentially Runx3 stability/function in T cells are altered in the absence of HDAC1 and HDAC2. There is a striking similarity between the phenotypes of HDAC1– HDAC2-deficient CD4+ T cells and ThPOK- (or ThPOK/LRF)-deficient as CD4+ T cells with respect to the induction of a CD8 effector program. This raises the question whether there is a direct link between HDAC1– HDAC1 and ThPOK. Of note, ex vivo HDAC1–HDAC2-deficient CD4+CD8+ T cells displayed Th-POK protein expression levels similar to wild-type CD4+ T cells, despite slightly reduced Thpok mRNA expression. Thus, CD8α and CD8β coreceptor expression is induced in HDAC1– HDAC2-deficient CD4+ T cells under homeostatic conditions despite wild-type ThPOK levels. This is an unexpected finding, since ThPOK has been shown to repress the Cd8a and Cd8b1 genes via binding to the Cd8 gene loci [57]. Similar to Runx factors, ThPOK is also modified by reversible lysine acetylation and acetylation stabilizes Th-POK protein [58]. Thus, HDAC1–HDAC2 might be part of a regulatory complex that controls Th-POK stability and perhaps also ThPOK
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Fig. 1. A regulatory network that maintains CD4+ T cell lineage integrity. A regulatory circuit that includes HDAC1–HDAC2 and ThPOK (and in part the ThPOK-related factor LRF) represses CD8 lineage genes by keeping Runx3 expression below a certain threshold to prevent the induction of a CTL program in CD4+ T cells. As a consequence, CD4+ T cell lineage integrity is maintained. Since Thpok expression is decreased in activated HDAC1–HDAC2-deficient CD4+ T cells, it is likely that loss of ThPOK activity contributes to the induction of CD8 lineage genes in the absence of HDAC1–HDAC2. The down-regulation of ThPOK might occur via HDAC1–HDAC2-mediated and/or Runx3-mediated cross-regulation. However, whether the down-regulation of ThPOK is essential for the induction of CD8 lineage genes in the absence of HDAC1–HDAC2 is not known. In intestinal CD4+ T cells, the upregulation of Runx3 might precede the downmodulation of ThPOK, which then leads to the development of CD4+CD8αα+ CTLs. The reprograming of intestinal intraepithelial CD4+ T cells is in part regulated by microenvironmental signals such as TGFβ and RA. Whether these signals directly regulate Runx3 expression or Runx3 activity is not known. It is tempting to speculate that TGFβ and RA, inflammatory conditions or other factors (transiently) modulate the expression and/or activities of HDAC1–HDAC2 or ThPOK and thus facilitate the differentiation of CD4+ CTLs. Dotted lines indicate potential additional regulatory crosstalks. See text for more details.
function, and this might explain why CD8α and CD8β are induced despite wild-type ThPOK protein expression levels. However, upon activation there was a strong down-regulation of Thpok gene expression in HDAC1–HDAC2-deficient CD4+ T cells in comparison to activated wild-type CD4+ T cells, which might be also be caused by the increased expression of Runx3 that in turn might lead to the downregulation of ThPOK [30,59]. Thus, it is likely that the reduced Thpok expression might enhance and/or strongly contribute to the induction of a CD8 effector program upon activation of CD4+ T cells in the absence of HDAC1 and HDAC2.
6. Concluding remarks The observed reprogramming of CD4+ T cells into intestinal intraepithelial CD4+CD8αα+ CTLs clearly indicates that this occurs under physiological conditions influenced by the local microenvironment. Thus, it is conceivable that other (i.e. non-intestinal) CD4+ CTLs that are arising in humans and mice under inflammatory conditions, e.g. during viral infections or in association with autoimmunity [16–19], might be regulated by a similar underlying molecular process. Therefore, it is tempting to speculate that a particular local inflammatory microenvironment generated during an adaptive immune response in response to viral infections might (transiently) modulate HDAC1– HDAC2-activity, repress ThPOK and/or induce Runx3 expression and thus promotes the induction of a CTL program in CD4+ T cells. Further studies are required to investigate the regulatory hierarchy and interactions among HDAC1–HDAC2, Th-POK and Runx factors to better understand how CD4+ T cell lineage integrity is maintained, how the expression and the function of these factors is influenced by microenvironment, and how this regulatory network contributes to the generation of CD4+ CTLs and thus the fine tuning of the adaptive T cell response. Insight into these processes will also help to better understand the role of CD4+ CTLs in health and disease.
Acknowledgments Work in my laboratory has been supported by the Vienna Science and Technology Fund (WWTF) through project LS09-031 and by the Austrian Science Fund FWF (I00698, P19930, P23641, P26193, W1212). I thank Drs. Nicole Boucheron and Shinya Sakaguchi for critical reading of the manuscript. References [1] A.C. Carpenter, R. Bosselut, Decision checkpoints in the thymus, Nat. Immunol. 11 (2010) 666–673. [2] A. Singer, S. Adoro, J.H. Park, Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice, Nat. Rev. Immunol. 8 (2008) 788–801. [3] X. He, K. Park, D.J. Kappes, The role of ThPOK in control of CD4/CD8 lineage commitment, Annu. Rev. Immunol. 28 (2010) 295–320. [4] I. Taniuchi, W. Ellmeier, Transcriptional and epigenetic regulation of CD4/CD8 lineage choice, Adv. Immunol. 110 (2011) 71–110. [5] W. Ellmeier, L. Haust, R. Tschismarov, Transcriptional control of CD4 and CD8 coreceptor expression during T cell development, Cell. Mol. Life Sci. 70 (2013) 4537–4553. [6] X. He, X. He, V.P. Dave, Y. Zhang, X. Hua, E. Nicolas, et al., The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment, Nature 433 (2005) 826–833. [7] G. Sun, X. Liu, P. Mercado, S.R. Jenkinson, M. Kypriotou, L. Feigenbaum, et al., The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection, Nat. Immunol. 6 (2005) 373–381. [8] L. Wang, K.F. Wildt, J. Zhu, X. Zhang, L. Feigenbaum, L. Tessarollo, et al., Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells, Nat. Immunol. 9 (2008) 1122–1130. [9] S. Goyama, Y. Yamaguchi, Y. Imai, M. Kawazu, M. Nakagawa, T. Asai, et al., The transcriptionally active form of AML1 is required for hematopoietic rescue of the AML1-deficient embryonic para-aortic splanchnopleural (P-Sp) region, Blood 104 (2004) 3558–3564. [10] G. Hernandez-Hoyos, M.K. Anderson, C. Wang, E.V. Rothenberg, J. Alberola-Ila, GATA-3 expression is controlled by TCR signals and regulates CD4/CD8 differentiation, Immunity 19 (2003) 83–94. [11] T.P. Bender, C.S. Kremer, M. Kraus, T. Buch, K. Rajewsky, Critical functions for c-Myb at three checkpoints during thymocyte development, Nat. Immunol. 5 (2004) 721–729.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Molecular control of CD4+ T cell lineage plasticity and integrity Wilfried Ellmeier Division of Immunobiology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria
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Article history: Received 27 February 2015 Accepted 28 March 2015 Available online 9 April 2015 Keywords: CD4+ cytotoxic T cells (CD4+ CTLs) CD4+ T cell lineage integrity Intestinal intraepithelial lymphocytes ThPOK Runx3 Histone deacetylases
a b s t r a c t CD4+ helper T cells and CD8+ cytotoxic T cells form the two major subsets of peripheral T lymphocytes. Helper T cells fulfill crucial roles in the activation and coordination of the immune response, while cytotoxic T cells kill virus-infected or tumor cells. Recent data suggest that the lineage identify of helper T cells is not fixed and that CD4+ T cells under certain physiological conditions can be reprogrammed to express CD8 lineage genes and to develop into intestinal intraepithelial CD4+ cytotoxic T lymphocytes that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, the analysis of mice with a conditional deletion of the transcription factor ThPOK or the histone deacetylases HDAC1 and HDAC2 indicated that CD8 lineage genes are actively repressed in CD4+ T cells in order to maintain the lineage integrity of helper T cells. In this review I summarize recent studies that indicate plasticity of CD4+ T cells towards a CTL program and that demonstrate that ThPOK and HDAC1–HDAC2 are part of a transcriptional regulatory circuit that counteracts the activity of the transcription factor Runx3 to maintain CD4+ T cell lineage integrity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction and background The two major subsets of peripheral T cells express either the CD4 or CD8 coreceptor molecules. T cells that express CD4 constitute the major histocompatibility complex (MHC) class II-restricted helper T cell population, while T cells that express CD8 (which on conventional T cells is formed by the CD8α and CD8β heterodimers) represent the MHC class I-restricted cytotoxic T cell lineage. CD4+ T cells can be subdivided into several T helper subsets and these subsets play key roles in the activation of other immune cells and coordination of the immune response, while cytotoxic T cells as part of their effector mechanism have the “license to kill” and thus eliminate virus infected cells or cancer cells [1]. The proper establishment of cell lineage-specific expression patterns and the stable repression of inappropriate genes and commitment to a particular lineage is crucial for the regulation of T cell development and the maintenance of T cell function. CD4+ and CD8+ T cells develop in the thymus from so-called double-positive (DP) thymocytes that express both CD4 and CD8. Studies performed during the last decades
Abbreviations: BTB, broad complex, Tram-track and bric-a-brac; CBFβ, core-binding factor β; CD4SP, CD4 single-positive; CD8SP, CD8 single-positive; CTL, cytotoxic T lymphocyte; DN, double-negative; DP, double-positive; HAT, histone acetyltransferase; HDAC, histone deacetylase; IEL, intraepithelial lymphocytes; IFN, interferon; LRF, leukemia/lymphoma related factor; MAZR, Myc-associated zinc finger-related factor; MHC, major histocompatibility complex; LN, lymph node; RA, retinoic acid; Rag, recombination activating gene; Runx, Runt-related transcription factor; ThPOK, T-helper inducing POZ/Krüppel like factor; TCR, T-cell receptor; TGFβ, transforming growth factor β; Th, T helper. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.intimp.2015.03.050 1567-5769/© 2015 Elsevier B.V. All rights reserved.
provided important insight in the mechanisms of CD4/CD8 cell fate choice and led to the identification of key transcription factors that regulate this process [2–5]. ThPOK, together with c-Myb, Tox and GATA-3, directs the development of the CD4+ T cell lineage [6–12], while Runx3, together with other factors such as MAZR [13], directs the differentiation of CD8+ T cells [14,15]. Thus, the identification of CD4+ and CD8+ T cell-lineage specific transcription factors and their distinct and different type of helper and cytotoxic effector functions suggested that mature CD4+ T cells and CD8+ T cells retain their lineage identity and integrity once they have been generated. Some subsets of CD4+ T cells have been described that display a cytotoxic activity and were designated as CD4+ cytotoxic T lymphocytes (CD4+ CTLs). This suggests some exceptions from the clear functional separation between helper and cytotoxic lineage T cells, although CD4+ CTLs were considered as being Th1-like [16–19]. However, as summarized below, recent data suggested that the lineage identity of helper T cells is not irreversible and that CD4+ T cells under certain physiological conditions have an unexpected plasticity and can be reprogrammed to express CD8 lineage genes and to develop into CD4+ CTLs that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, these studies also indicate that the potential for the induction of CD8 lineage genes and a CTL program has to be actively repressed in mature CD4+ T cells to maintain the lineage integrity of helper T cells. 2. ThPOK and LRF prevent the transdifferentiation of mature CD4+ T cells into CD8+ T cells A major breakthrough in the field of CD4/CD8 lineage differentiation was the identification that the BTB domain-containing transcription
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factor ThPOK (encoded by the Zbtb7b gene, referred here as the Thpok gene) is a key commitment factor for CD4+ T cell lineage development. Two studies independently showed that loss of ThPOK function during T cell development leads to the redirection of MHC class II-restricted cells into the CD8+ T cell lineage [6,7]. In contrast, enforced expression of ThPOK during T cell development is sufficient to direct MHC class I-restricted T cells into the CD4+ helper T cell lineage [6,7]. Similarly, enforced expression of ThPOK in peripheral CD8+ T cells induces CD4 lineage features, while CD8 lineage genes encoding for CD8α, Perforin, Granzyme B and Eomesodermin were down-regulated [20], suggesting that mature CD8+ T cells remain susceptibly to the activity of ThPOK. Soon after the discovery of ThPOK and its description as a key regulator of CD4+ T cell commitment, several studies were published that indicated that ThPOK function might be continuously required in mature CD4+ T cells and that ThPOK maintains the lineage integrity of CD4+ T cells. One study showed that ThPOK is necessary for antagonizing Cd4 silencer activity in order to maintain CD4 expression during helper T cell differentiation [21]. Moreover, by using genetically modified mice that express a hypomorph Thpok allele, it was shown that ThPOK represses CD8 lineage genes such as those encoding for CD8α, CD8β, Runx3 and Eomesodermin in CD4+ T cells and that low ThPOK levels lead to the induction of a CD8 effector program [22,23]. These data demonstrated that one key function of ThPOK in mature CD4+ T cells is to repress CD8 lineage genes. Further insight into the role of ThPOK in the regulation of CD4 lineage function as well lineage integrity came from a study in which ThPOK was conditionally deleted in peripheral T cells (i.e. postthymic T cells) [24]. In these mice, due to the “late” peripheral deletion of ThPOK, CD4+ and CD8+ T cell development was normal and the impact of ThPOK deletion on CD4+ T cell function could be studied. Using this approach it was shown that ThPOK activity is essential for Th1-type T cells functions but not for Th17 differentiation. Furthermore, in agreement with studies using the hypomorph Thpok allele, ThPOKdeficient Th1 cells also up-regulated CD8α and CD8β and displayed increased Runx3 expression in comparison to wild-type Th1 cells. Moreover, ThPOK-deficient Th1 cells expressed also Eomesodermin as well as CD107a (a marker for cytotoxic cell degranulation [25,26]) in vitro and following Toxoplasma gondii infection in vivo. The upregulation of a CD8 effector program was dependent on Runx3, showing that ThPOK-mediated control of Runx3 expression levels is crucial for the repression of the CD8 effector program, although ThPOK might also act downstream of Runx3 to repress CD8 lineage genes that are otherwise induced by Runx3 [27]. Interestingly, although Runx factors are important regulators of Cd4 silencing, CD4 expression remained high in ThPOK-deficient Th1 cells despite high Runx3 expression. One possible explanation for this observation was that ThPOK-related factors might overcome the repressive activity of Runx3 on the Cd4 silencer. A candidate factor for such a compensatory function was LRF (encoded by the Zbtb7a gene), which is closely related to ThPOK and shown to be able to partially compensate for loss of ThPOK [28]. Indeed, CD4+ T cells from mice with a combined conditional peripheral deletion of ThPOK and LRF lost CD4 expression upon activation and Th1 differentiation and converted into CD4−CD8+ effector T cells. Thus, ThPOK and LRF are essential to repress the transdifferentiation of CD4+ T cells into CD8+ T cells. 3. Developmental plasticity of intestinal CD4+ T cells under physiological conditions
also CD8α (but not CD8β) and thus were within the subset of CD4+CD8αα+ intraepithelial lymphocytes (IELs). These cells had features similar to CD8+ cytotoxic T cells such as expression of Granzyme B and CD107a and, upon activation, to kill target cells in vitro. Fatetracking experiments revealed that these cells were derived from cells that previously expressed Thpok. Indeed, transfer of naïve CD4+ T cells (that carried a Thpok–GFP reporter allele to track ThPOK expression) into Rag-deficient mice led to a reprogramming of the transferred cells into MHC class II-restricted CD4+CD8αα+ T cells with a cytotoxic activity (CD4+ CTLs). While transferred CD4+ T cells that were isolated from the spleen or mesenteric LN of recipient mice still expressed ThPOK, intestinal intraepithelial CD4+ CTLs were observed in recipient mice that did not express Thpok. Of note, the reprogrammed CD4+ CTLs remained immunologically quiescent at steady state. However, in response to restimulation in the presence of IL15, these cells increased their inflammatory and cytolytic potential. Preceding this reprogramming of CD4+ T cells was the termination of ThPOK expression in antigenexperienced CD4+ effector T cells, which then led to the induction of CD8 lineage genes and a CTL program. The termination of ThPOK expression was under control of the Thpok silencer, since reprogramming of silencer-deficient CD4+ T cells into intestinal CD4+ CTLs was impaired. Similar, CD4+ T cells with a deletion of MAZR, which binds to the Thpok silencer and represses ThPOK during CD8 lineage differentiation [13], displayed a decreased reprogramming into CD4+ CTLs. Thus, the Thpok silencer is a crucial regulatory node in the differentiation of intestinal CD4 + T cells into intraepithelial CD4+ CTLs. Additional insight into the regulation of intestinal CD4+ T cell plasticity was provided in a study that addressed whether the post-thymic downregulation of ThPOK in intestinal CD4+ T cells is associated with changes in the expression of Runx3 [30]. Using reporter mice for monitoring either Runx3 or Thpok expression it was shown that the intestinal CD4+ T cells with a low expression of ThPOK had high expression levels of Runx3. It was further shown that Runx3 upregulation was the initiating event that led to the downmodulation of ThPOK, and the changes in the expression of both Runx3 and ThPOK were required for the induction of a CTL program in intestinal CD4+ T cells. The observation that Runx3 downregulated ThPOK in intestinal CD4+ T cells is different to ex vivo analyzed splenic ThPOK-deficient CD4+ T cells, where the deletion of ThPOK led to the induction of Runx3 [23,24]. Interestingly, the reprogramming of intestinal CD4+ T cells was is part induced by TGFβ and retinoic acid (RA) [30], indicating that intestinal environmental signals might regulate the expression of Runx3 and thus the differentiation of intestinal CD4+ T cells into intraepithelial CD4+CD8αα+ CTLs. This observation is also in agreement with a study showing that TGFβ induces CD8α expression on activated CD4+ T cells [31]. To determine the impact of the crossregulation between ThPOK and Runx3 for the function of intestinal CD4+ T cells, a T cell-mediated adoptive transfer colitis model was used to study the inflammatory potential of T cells that were deficient in either ThPOK or Runx3. Using this approach it was shown that forced loss of ThPOK in CD4+ T cells reduced inflammation despite normal Th17 differentiation, while forced loss of Runx3 resulted in a higher inflammation and enhanced Th17 differentiation [30]. Thus, the mutual expression of Runx3 and ThPOK might regulate intestinal CD4+ T cell immunity, perhaps by deviating pathogenic CD4+ T cells into CD4+ CTLs [32]. 4. HDAC1–HDAC2 maintain CD4+ T cell lineage integrity
The studies that analyzed CD4+ T cells with a hypomorphic Thpok allele or with a peripheral deletion of ThPOK indicated already that CD4+ T cells have the potential to acquire cytotoxic CD8+ T cell features. One intriguing example demonstrating in vivo plasticity of CD4+ T cells under physiological conditions was provided in a study that showed lineage conversion of intestinal CD4+ T cells towards a CTL phenotype [29]. The starting point of this study was the observation that a subset of intestinal CD4+ T cells did not express ThPOK. These cells expressed
Another genetic switch that represses a CD8 effector program in CD4 lineage T cells was identified during the analysis of mice with a deletion of the histone deacetylases (HDAC) 1 and HDAC2 in T cells [33]. An important regulatory level of the establishment and maintenance of cell lineage-specific expression patterns is formed by epigenetic and chromatin-mediated mechanism such as DNA methylation or histone acetylation and methylation. Modification of core histones by reversible
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lysine acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs), which are considered transcriptional coactivators and corepressors, respectively [34,35]. The HDAC family consists of 18 members grouped into 4 classes [36]. Many studies, using genetic approaches as well as by applying HDAC inhibitors, showed important roles for reversible lysine acetylation in the regulation of T cell development and function and several members of the HDAC family have been implicated in the regulation of T cells [37–40]. The generation of mice with a T cell-specific deletion of HDAC1 (using Cd4-Cre) showed that loss of HDAC1 leads to enhanced Th2 responses in vitro and in vivo leading to a more severe disease in a Th2-type allergic airway inflammation model [41]. Surprisingly, T cell development in the absence of HDAC1 was normal, most likely due to the observed compensatory up-regulation of HDAC2, which was also described in other cell lineages and biological processes [42–47]. In order to investigate whether the combined deletion of HDAC1 and HDAC2 leads to T cell developmental defects, mice with a T cell-specific deletion (using Cd4-Cre) of both HDAC1 and HDAC2 were generated [33]. Overall, the number of αβTCR+ T cells was reduced and the activation of HDAC1–HDAC2-deficient peripheral CD4+ T cells revealed that HDAC1–HDAC2-deficient CD4+ T cells undergo apoptosis if the cells are cultured for more that 3 days in vitro. These data show that HDAC1 and HDAC2 are essential for the efficient generation of the peripheral T cell pool and for the survival of proliferating CD4+ T cells. However, a surprising finding was that HDAC1–HDAC2-deficient CD4+ helper T cells acquired CD8 lineage effector features [33]. This was already observed under homeostatic conditions due to the appearance of a fraction of MHC class II-selected CD4+ helper T cells that expressed CD8+ T cell lineage genes encoding for CD8α, CD8β and Eomesodermin in peripheral lymphoid organs such as LN and spleen. Interestingly, the HDAC1–HDAC2-deficient CD4+CD8+ as well as CD4+CD8− T cells had normal expression of ThPOK protein ex vivo and up-regulated CD154 upon overnight activation, thus displaying characteristic and defining features of helper T cells. However, after short-term activation for 60 h, HDAC1–HDAC2-deficient CD4+CD8− lineage T cells acquired in addition to CD8α and CD8β expression also CD8 effector features, indicated by a strong further up-regulation of Eomesodermin, a high production of IFN-γ and increased expression of Runx3, T-bet, Granzyme B and Perforin. This was observed in Th0 and Th1-polarizing conditions, while the induction of a CD8 effector program was strongly impaired in Th2 conditions. Since Runx/CBFβ complexes are essential for the development of CD8+ T cells and their differentiation into cytotoxic effector T cells [14,48,49], the role of Runx factors in the induction of a CD8 effector program in the absence of HDAC1–HDAC2 was further explored by crossing conditional HDAC1–HDAC2 mice with conditional CBFβ mice, which leads to an inactivation of all Runx factors. This revealed that the expression of CD8 effector genes in activated HDAC1–HDAC2-deficient CD4+ T cells was dependent on Runx–CBFβ complexes. Moreover, the expression of CD8 effector genes correlated with the presence of Runx/CBFβ complexes and local histone hyperacetylation at the promoter regions of these genes [33]. Interestingly, the treatment of activated wild-type CD4+ T cells with class I HDAC inhibitors led to the induction of CD8α, Eomesodermin, T-bet and IFN-γ expression, indicating that HDAC1–HDAC2 are also required for the repression of CD8 lineage genes in mature CD4+ T cells and thus the maintenance of CD4+ T cell lineage integrity [33]. In addition, retroviral-mediated enforced expression of Runx3 in wild-type CD4+ T cells led to the induction of CD8α, Granzyme B and increased IFN-γ production and this was in part increased in synergy with HDAC inhibitors. Moreover, Runx3 expression together with HDAC inhibitor treatment led also to the induction of Eomesoderin in CD4+ T cells. Taken together, these observations revealed that HDAC1 and HDAC2 are essential for the maintenance of CD4+ T cell lineage integrity by repressing Runx–CBFβ complexes that otherwise induce CD8 lineage genes and a CTL-like program in CD4+ T cells.
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5. HDAC1–HDAC2, ThPOK/LRF and Runx factors form a transcriptional circuit that regulates CD4+ T cell lineage integrity The studies describing the conversion of intestinal CD4+ T cells into CD4+CD8αα+ CTLs demonstrate that this is a physiological process and the ThPOK and Runx3 expression patterns suggest that CD4+ CTLs represent a distinct Th subset different from Th1 cells [29,30,32]. Since ThPOK and HDAC1–HDAC2 are required to repress a Runx3-dependent CD8 lineage effector program in CD4+ T cells, it is conceivable that a transcriptional circuit containing HDAC1–HDAC2, ThPOK and Runx3 regulates the differentiation of CD4+ T cells into CD4+ CTLs (Fig. 1). At a molecular level one might envisage that HDAC1–HDAC2 as well as ThPOK (in part together with LRF) maintain the integrity of CD4+ T cells by keeping Runx3–CBFβ complexes below a certain threshold level to prevent the induction of a CD8 effector program in CD4+ T cells. It is likely that the strong induction of Runx3 expression in HDAC1–HDAC2-deficient or ThPOK-deficient CD4 lineage T cells is the initiating event in the induction of CD8 lineage genes and the acquisition of a CTL program. The induction of a CD8 effector program is regulated in part via recruitment of Runx–CBFβ complexes to target genes such as Cd8, GzmB, Prf1 and Ifng as shown in HDAC1–HDAC2-deficient CD4+ T cells [33]. Since environmental cues such as TGFβ and RA induce the reprogramming into intestinal CD4+ CTLs [30], it is possible that these signals might (transiently) modulate the activity of HDAC1– HDAC2 and/or ThPOK and thus allow the induction of a CTL program in CD4+ T cells. Further, activated HDAC1–HDAC2-deficient or ThPOKdeficient CD4+ T cells upregulated Eomesodermin in a Runx–CBFβ-dependent manner, and HDAC1–HDAC2-deficient CD4+ T cells also upregulated T-bet. Since Eomesodermin and T-bet are important for the induction of the cytotoxic program [48–51], it is likely that Runx3, Eomesodermin and T-bet together regulate the induction of the CD8 effector program in HDAC1–HDAC2-deficient or ThPOK-deficient CD4+ T cells. HDACs are considered as general chromatin-modifying regulators of gene expression, however many non-histone targets of HATs and HDACs are currently emerging [52,53]. Lysine acetylation of proteins has been shown to affect protein–protein and protein–DNA interactions, protein stability and intracellular localization [34,52,54]. This raises the important question whether the induction of a CTL program in HDAC1–HDAC2-deficient CD4+ T cells is a consequence of chromatin-mediated or of non-chromatin-mediated effects, or a combination of both. The transcriptome analysis of HDAC1–HDAC2-deficient CD4+ T cells and the observation that HDAC1–HDAC2 are recruited to the Runx3 locus strongly suggest that HDAC1–HDAC2 directly modulate the expression levels of the Runx3 gene in CD4+ T cells [33]. However, it has been shown that the stability and function of Runx factors can be controlled by reversible lysine acetylation [55,56], suggesting that Runx3 expression might be also regulated at the post-translational level. Further studies are required to investigate whether posttranslational modifications and potentially Runx3 stability/function in T cells are altered in the absence of HDAC1 and HDAC2. There is a striking similarity between the phenotypes of HDAC1– HDAC2-deficient CD4+ T cells and ThPOK- (or ThPOK/LRF)-deficient as CD4+ T cells with respect to the induction of a CD8 effector program. This raises the question whether there is a direct link between HDAC1– HDAC1 and ThPOK. Of note, ex vivo HDAC1–HDAC2-deficient CD4+CD8+ T cells displayed Th-POK protein expression levels similar to wild-type CD4+ T cells, despite slightly reduced Thpok mRNA expression. Thus, CD8α and CD8β coreceptor expression is induced in HDAC1– HDAC2-deficient CD4+ T cells under homeostatic conditions despite wild-type ThPOK levels. This is an unexpected finding, since ThPOK has been shown to repress the Cd8a and Cd8b1 genes via binding to the Cd8 gene loci [57]. Similar to Runx factors, ThPOK is also modified by reversible lysine acetylation and acetylation stabilizes Th-POK protein [58]. Thus, HDAC1–HDAC2 might be part of a regulatory complex that controls Th-POK stability and perhaps also ThPOK
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Fig. 1. A regulatory network that maintains CD4+ T cell lineage integrity. A regulatory circuit that includes HDAC1–HDAC2 and ThPOK (and in part the ThPOK-related factor LRF) represses CD8 lineage genes by keeping Runx3 expression below a certain threshold to prevent the induction of a CTL program in CD4+ T cells. As a consequence, CD4+ T cell lineage integrity is maintained. Since Thpok expression is decreased in activated HDAC1–HDAC2-deficient CD4+ T cells, it is likely that loss of ThPOK activity contributes to the induction of CD8 lineage genes in the absence of HDAC1–HDAC2. The down-regulation of ThPOK might occur via HDAC1–HDAC2-mediated and/or Runx3-mediated cross-regulation. However, whether the down-regulation of ThPOK is essential for the induction of CD8 lineage genes in the absence of HDAC1–HDAC2 is not known. In intestinal CD4+ T cells, the upregulation of Runx3 might precede the downmodulation of ThPOK, which then leads to the development of CD4+CD8αα+ CTLs. The reprograming of intestinal intraepithelial CD4+ T cells is in part regulated by microenvironmental signals such as TGFβ and RA. Whether these signals directly regulate Runx3 expression or Runx3 activity is not known. It is tempting to speculate that TGFβ and RA, inflammatory conditions or other factors (transiently) modulate the expression and/or activities of HDAC1–HDAC2 or ThPOK and thus facilitate the differentiation of CD4+ CTLs. Dotted lines indicate potential additional regulatory crosstalks. See text for more details.
function, and this might explain why CD8α and CD8β are induced despite wild-type ThPOK protein expression levels. However, upon activation there was a strong down-regulation of Thpok gene expression in HDAC1–HDAC2-deficient CD4+ T cells in comparison to activated wild-type CD4+ T cells, which might be also be caused by the increased expression of Runx3 that in turn might lead to the downregulation of ThPOK [30,59]. Thus, it is likely that the reduced Thpok expression might enhance and/or strongly contribute to the induction of a CD8 effector program upon activation of CD4+ T cells in the absence of HDAC1 and HDAC2.
6. Concluding remarks The observed reprogramming of CD4+ T cells into intestinal intraepithelial CD4+CD8αα+ CTLs clearly indicates that this occurs under physiological conditions influenced by the local microenvironment. Thus, it is conceivable that other (i.e. non-intestinal) CD4+ CTLs that are arising in humans and mice under inflammatory conditions, e.g. during viral infections or in association with autoimmunity [16–19], might be regulated by a similar underlying molecular process. Therefore, it is tempting to speculate that a particular local inflammatory microenvironment generated during an adaptive immune response in response to viral infections might (transiently) modulate HDAC1– HDAC2-activity, repress ThPOK and/or induce Runx3 expression and thus promotes the induction of a CTL program in CD4+ T cells. Further studies are required to investigate the regulatory hierarchy and interactions among HDAC1–HDAC2, Th-POK and Runx factors to better understand how CD4+ T cell lineage integrity is maintained, how the expression and the function of these factors is influenced by microenvironment, and how this regulatory network contributes to the generation of CD4+ CTLs and thus the fine tuning of the adaptive T cell response. Insight into these processes will also help to better understand the role of CD4+ CTLs in health and disease.
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