Immunol Res DOI 10.1007/s12026-014-8545-9 IMMUNOLOGY AT THE UNIVERSITY OF IOWA

From inception to output, Tcf1 and Lef1 safeguard development of T cells and innate immune cells Farrah C. Steinke • Hai-Hui Xue

Ó Springer Science+Business Media New York 2014

Hai-Hui Xue

Farrah C. Steinke

Abstract Transcription factors have recurring roles during T cell development and activation. Tcf1 and Lef1 are known to be essential for early stages of thymocyte maturation. Recent research has revealed several novel aspects of their functionality. Tcf1 is induced at the very earliest step of specifying hematopoietic progenitors to the T cell lineage as a key target gene downstream of Notch activation. In addition to promoting maturation of T-lineage-committed thymocytes, Tcf1 functions as a tumor suppressor in developing thymocytes, and this is mediated, paradoxically, by restraining Lef1 expression. After positive selection, Tcf1 and Lef1 act together to direct CD4?CD8? double positive thymocytes to a CD4? T cell fate. Although not required for CD8? T cell differentiation, Tcf1 and Lef1 cooperate with Runx factors to achieve stable silencing of the Cd4 gene in CD8? T cells. Tcf1 is also found to have versatile roles in innate immune cells, which partly mirror its functions in mature T helper cells. Discrepancy in requirements of Tcf1/Lef1 and b-catenin in T cells has been a long-standing enigma. We will review other protein factors interacting with Tcf1 and Lef1 and discuss their regulatory roles independent of b-catenin. Keywords

Tcf1  Lef1  T cell development  Innate lymphoid cells

Introduction T cell factor 1 (Tcf1) and lymphoid enhancer factor 1 (Lef1), encoded by Tcf7 and Lef1 genes, respectively, are members of the Tcf/Lef family transcription factors. Both Tcf1 and Lef1 are abundantly expressed in T-lineage lymphocytes and contain a highly conserved high mobility group (HMG) domain that binds directly to DNA [1–4]. It has been well established that Tcf1 and Lef1 are effector F. C. Steinke  H.-H. Xue Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA F. C. Steinke  H.-H. Xue Interdisciplinary Immunology Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA H.-H. Xue (&) 51 Newton Rd. BSB Rm. 3-772, Iowa City, IA 52242, USA e-mail: [email protected]

transcription factors of the canonical Wnt signaling pathway. In the absence of Wnt stimulation, Tcf1 and Lef1 are bound by the Groucho-related (Grg/Tle) corepressors, and the expression of Wnt target genes is therefore negatively regulated. A cytosolic b-catenin ‘‘destruction’’ complex is comprised of multiple components, including two scaffolding proteins, adenomatous polyposis coli (APC) and Axin, and two serine/threonine protein kinases, casein kinase 1 (CK1) and glycogen synthesis kinase 3b (GSK3b) [5]. These kinases phosphorylate a cluster of 4 Ser/Thr residues in the N-terminus of b-catenin, marking it for ubiquitination and proteasome-mediated degradation. Binding of Wnt ligands to their Frizzed (Fzd) receptors and low-density lipoprotein receptor-related protein 5 or 6 (Lrp5/6) co-receptors disrupts the ‘‘destruction’’ complex and inactivates CK1 and GSK3b kinases. As a result, unphosphorylated b-catenin is stabilized and accumulated. Upon its nuclear translocation, b-catenin displaces the Grg/ Tle corepressors, and the binding of b-catenin with Tcf1 or Lef1 leads to activation of Wnt target genes [5].

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The inception of T-lineage cells occurs when bone marrow-derived multipotent or lymphoid-biased precursors seed the thymus and give rise to early thymic progenitors (ETPs) with a CD44?c-KithiCD25- phenotype [6–9]. Signals from the thymic microenvironment especially Notch activation direct ETPs to fully commit to T cell lineage, reaching CD44-CD25? CD4-CD8- double negative (DN) 3 stage where the T cell receptor (TCR) b gene locus is rearranged. The DN3 thymocytes that complete the b-selection mature to CD4?CD8? double positive (DP) cells, which further rearrange their TCRa gene locus. The abTCRs are then tested for reactivity to self-antigens, and positively selected DP thymocytes make lineage choice decisions to become either CD4? helper T cells or CD8? cytotoxic T cells [10, 11]. Mature CD4? and CD8? single positive (SP) T cells resulting from thymic output then survey the periphery and adopt distinct differentiation steps upon activation, depending on the nature of pathogens. Several of these developmental and differentiation processes are critically regulated by Tcf1 and Lef1, such as thymocyte survival, differentiation of mature CD4? T cells to T helper 2 (Th2) and Th17, and formation and persistence of memory CD8? T cells. These aspects were previously reviewed in detail [1–4]. This review will focus on the substantial advances made during the past 3 years, on the novel regulatory roles of Tcf1 and Lef1 in the development of T cells as well as innate immune cells. A perplexing issue that has clouded the field for more than a decade is the discrepancy between targeting Tcf1/Lef1 and their cofactor b-catenin. Whereas deletion of Tcf1 alone or with Lef1 causes severe defects, ablating b-catenin alone or with its homolog c-catenin produces little impact on T-lineage cells. We will also review other proteins found to interact with Tcf/Lef factors thus far and discuss biological processes regulated by Tcf1 and Lef1 but independent of b-catenin.

Early stages of thymocyte development In the bone marrow (BM), specification and complete commitment of the common lymphoid progenitors to the B cell lineage depend on the sequential action of transcription factors including E2A, EBF, and Pax5 [12]. In contrast to this clearly defined road map for a B cell fate, many more transcriptional regulators exhibit essential roles in T cell lineage specification, and few of them are specific to T cells. For a long time, no single transcription factor has been experimentally demonstrated to be sufficient to confer a T cell fate. For example, the T cell-specific Gata3 is indispensable for generation of ETPs [13, 14]; however, forced expression of Gata3 in thymic progenitors rather blocks T cell development and instead activates a mast cell program [15].

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A Notch

B Fzd/Lrp

? ICN

βCat

CSL

Tcf1 Tcf7

DN1/ETP

preTCR

Fzd/Lrp5/6

?

βCat Tcf1

Gata3

Notch1, Dtx1

Bcl11b

Lef1, Id2, Sfpi1

DN2

Egr1,2,3

DN3

Fig. 1 Tcf1 specifies T cell fate, promotes b-selection, and guards early thymocytes from malignant transformation. At the DN1/ETP stage, Notch activation leads to nuclear translocation of intracellular domain of Notch1 (ICN), which binds the transcription factor CSL (CBF1, suppressor of hairless, and Lag-1) to directly induce Tcf7 expression. Tcf1 sustains its expression through auto-amplification and further activates the T cell transcriptional program including induction of Gata3 and Bcl11b to promote T-lineage specification and commitment (a). Upon maturation to the DN3 stage, Tcf1 promotes V(D)J recombination at the Tcrb locus through yet undefined mechanisms (not depicted) and facilitates b-selection, partly through induction of Egr genes (b). In addition, Tcf1 acts as a tumor suppressor, via direct suppression of Lef1, Id2, PU.1, Notch1, and Notch target genes. The requirement of Wnt signaling and b-catenin is less clear in these processes, but induced b-catenin stabilization has been shown to positively regulate Tcf7 in DN1 and negatively regulate Lef1 in DN3 cells. Arrows denote positive regulation, and lines ending with bars denote negative regulation

Tcf1 is known to be essential for T cell development. A recent breakthrough comes from the identification of Tcf1 as an important Notch target gene, with a –31.5 kb upstream regulatory region in the Tcf7 gene being occupied by Notch1 or CSL [16, 17]. Introduction of Tcf1 into BM progenitors is sufficient to activate a T cell transcriptional program including induction of Gata3, Bcl11b, and other TCR signaling components, even in the absence of Notch activation [16]. Intrathymic injection of Tcf1-expressing BM progenitors yields DP and SP thymocytes with TCRb expression [16]. Therefore, Tcf1 is a key mediator of Notch signaling in specification of BM progenitors to a T cell fate. To do this, Tcf1 can directly activate Gata3 transcription, as it does in Th2 differentiation [18]. Tcf1 can also positively regulate Bcl11b expression, by acting on an enhancer located ?850 kb downstream [19]. In addition, Tcf1 acts in a positive feedback loop to potentiate its own expression, which could be mediated through both proximal and distal Tcf/Lef binding motifs [16, 17]. We have also demonstrated that stabilization of b-catenin increases Tcf7 transcripts in DN1 but not DN3 thymocytes [20]. Collectively, Tcf1 employs multiple mechanisms to ensure T-lineage specification and commitment (Fig. 1a). Tcf1 continues to play essential roles in T-lineagecommitted DN3 thymocytes. Rearrangements at the Tcrb locus are much less efficient in Tcf1- and Lef1-deficient DN3 cells [21, 22]. How exactly Tcf1 and Lef1 participate in regulation of V(D)J recombination is not completely understood; yet important clues emerge from recent

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studies. ChIP-Seq of Tcf1 in total thymocytes reveals substantial overlap between Tcf1 and Rag1 binding sites across the genome, beyond the TCR gene loci [23]. DNAdependent kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is comprised of a catalytic subunit (DNA-PKcs), and Ku70 and Ku80 regulatory subunits. DNA-PK contributes to repair of DNA double-strand breaks (DSBs) and is involved in V(D)J recombination [24]. A recent study shows direct interaction of Lef1 with DNA-PKcs, and possibly with Ku80 as well [25]. These observations suggest that Tcf1 and Lef1 may be involved in both making DSBs and ensuing repair processes in DN3 thymocytes. Another notable observation is that although Tcf1-deficient DN3 cells can successfully pass b-selection and generate DP and SP thymocytes, double deficiency in Tcf1 and Lef1 further diminishes efficiency of b-selection and completely arrests T cell development at the DN stage. An essential role for Tcf1 in thymocyte survival has been well documented, which is mediated by positive regulation of Bcl-XL and Rorct [26, 27]. It is of note that pre-TCR signaling has been shown to stabilize b-catenin, and forced expression of stabilized b-catenin (via an Lck promoterdriven transgene) induces expression of the early growth response (Egr) genes to promote b-selection [28]. It remains an open question whether Tcf1 and Lef1 have additional roles beyond promoting thymocyte survival and b-selection to fully account for the complete DN block observed in the double null mutants.

Late stages of thymocyte development Thymocytes that successfully pass b-selection next upregulate both CD4 and CD8 co-receptors to become double positive (DP). The DP thymocytes undergo negative and positive selection with self-peptides to eliminate nonresponsive or over-responsive ab TCRs and to preserve those with proper responsiveness. The role of Tcf1 in these selection processes is unclear at present. HY-TCR?CD8? T cells, which specifically recognize the HY male antigen, are negatively selected in male but positively selected in female mice. When the germline-targeted Tcf7-/- mice are crossed to the HY-TCR transgene, the HY-TCR?CD8? SP thymocytes are increased in males as well as females [29]. In addition, stabilization of b-catenin is reported to promote negative as well as positive selection in different models [29, 30]. These apparently conflicting observations await further investigation to reconcile. Positively selected DP thymocytes then make a final decision to become MHC-I-restricted CD8? or MHC-IIrestricted CD4? SP T cells. This process is known as lineage choice, which is instructed by intricate extracellular signals and intrinsic transcription factors. According to the

Post-select DP/CD4+8lo Mazr

CD8+

Runx3 Cd4

Tcf1/Lef1 Runx ? Cd4 silencer

Tox Gata3 Tcf1/Lef1

Thpok

See Figure 3

CD4+

Fig. 2 Novel roles of Tcf1 and Lef1 in CD4? versus CD8? lineage choice and Cd4 gene silencing during late stages of T cell development. Runx3 and Thpok appear to be a key convergence point for regulation of T cell lineage choice. Runx3 and Mazr negatively regulate Thpok expression and promote a CD8? T cell fate. Tcf1 and Lef1 represent an independent pathway along with Tox and Gata3 in positive regulation of Thpok, which instruct the bipotent precursors to CD4? T cell lineage. In mature CD8? T cells, Tcf1 and Lef1 exhibit a role switch and cooperate with Runx factors to achieve stable Cd4 gene silencing

kinetic signaling model, DP thymocytes first moderately down-modulate CD8 expression, generating intermediate CD4?CD8lo cells, which remain bipotent [10]. Whereas potent and persistent TCR signaling favors a CD4? T cell fate, interruption of TCR signals and response to cytokines such as IL-7 and IL-15 promotes differentiation to CD8? T cell lineage [31, 32]. Intrinsically, Myb, Gata3, Tox, and Thpok are essential for generation of CD4? T cells, and in contrast, CD8? T cells require Runx3, in particular the Runx3d isoform, which is transcribed from a distal promoter [33, 34]. In terms of genetic interaction, upregulation of Thpok in CD4?8lo intermediates is dependent on Gata3 and Tox [35, 36], and Thpok and Runx3d oppose each other’s transcription/activity to ensure respective lineage promoting effects [37–39] (Fig. 2). A recent study shows that Mazr promotes CD8? T cell differentiation by negatively regulating Thpok [40]. Due to multiple defects in early T cell development in germline-targeted Tcf7-/animals, it has been unknown whether Tcf1 and Lef1 are involved in regulation of lineage choice. We have recently conditionally targeted both Tcf1 and Lef1, and both factors can be completely abrogated in postselected TCRbhi DP thymocytes by CD4-Cre [41], without compromising early T cell development. Deletion of Tcf1 alone moderately reduces output of mature CD4? T cells, and targeting both Tcf1 and Lef1 results in a severe loss of CD4? T cells. In the absence of Tcf1 or both factors, MHC-II-selected T cells are redirected to the CD8? T cell lineage when tested in the context of MHC-I deficiency or an OT-II TCR transgene [41]. These observations indicate a CD4? to CD8? T cell fate change in the absence of Tcf1 or both factors. Upon examining connections of Tcf1 with other known factors regulating lineage choice, we found that Tcf1 deficiency results in diminished expression of Thpok with concomitant increase in Runx3d in DP and CD4?8lo thymocytes, without affecting that of Gata3, Tox,

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and Mazr ([41] and our unpublished observations). Complementing Tcf1 deficiency with a Thpok transgene partly restores CD4? T cell output and completely prevents CD4? to CD8? T cell lineage redirection; in contrast, deletion of Runx3 does not rectify these defects caused by loss of Tcf1. Additionally, in the Tcf1-deficient DP and CD4?8lo cells, Thpok transgene is sufficient to prevent the increase in Runx3 and Runx3d expression; however, ablating Runx3 does not restore the reduced Thpok expression. Collectively, Thpok, but not Runx3, is a primary target for Tcf1-mediated regulation of lineage choice (Fig. 2). Indeed, Tcf1 directly binds to a ‘‘general T lymphoid element’’ (GTE) enhancer, previously identified in an intron of the Thpok gene [38]. Mutation of the two conserved Tcf/Lef motifs in the Thpok GTE diminishes its enhancer activity in a reporter assay [41]. These new findings identify Tcf1 as a novel pathway, parallel to rather than upstream of Tox and Gata3 in regulating CD4? T cell differentiation. Prominently, all these factors converge on Thpok. It should be noted, however, the Thpok transgene is not effective in restoring CD4? T cell numbers caused by loss of Gata3 or both Tcf1 and Lef1 [35, 41]. It is therefore likely there are additional factor(s) that act downstream, regulated either jointly or separately by Gata3 and Tcf1/Lef1. On the other hand, TCR and cytokine signaling pathways have been the primary focus in lineage choice studies, and it remains to be determined whether Wnt-derived signals influence CD4? T cell fate decision by acting through Tcf1/Lef1. In fact, the Lck promoter-driven b-catenin transgene increases the frequency and numbers of CD8? SP thymocytes [30, 42]. In addition, IL-7-derived signals were previously shown to inhibit the expression of Tcf1 and Lef1 [43]. The interplay between Wnt and TCR/ cytokine signals in lineage choice merits in-depth studies. Although Tcf1 and Lef1 are not required for DP thymocytes to choose the CD8? T cell fate, CD8? T cells generated in the absence of Tcf1 or both factors exhibit an interesting phenotype, derepression of the CD4 co-receptor [41]. It has been well established that repression of the Cd4 gene in CD8? T cells is mediated by the interaction of Runx factors with a *430 bp Cd4 gene silencer located in its first intron [44]. The combination of Runx3 deficiency with Runx1 mutations, deletion of the Runx cofactor Cbfb, or mutation of both Runx motifs in the 50 -half of the Cd4 silencer each results in complete CD4 derepression in CD8? T cells [39, 45, 46]. ChIP-seq of Tcf1 and Runx3 in CD8? T cells revealed their co-localization at the Cd4 silencer [47, 41]. Importantly, both Tcf1 and Lef1 can be co-immunoprecipitated with Runx3 [48], and this interaction appears to be direct because it does not depend on the b-catenin-binding domain in the N-terminus of Tcf1 or the C-terminal ‘‘VWPRY’’ motif in Runx3 that binds the Grg/ Tle corepressors [41]. Deletion of either Runx3 or Tcf1

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alone results in variegated CD4 expression in CD8? T cells, and compound deletion of Runx3 with Tcf1 or with both Tcf1 and Lef1 exhibits much stronger CD4 derepression [41]. Our findings thus reveal an unexpected novel role for Tcf1 and Lef1 in cooperating with Runx factors to achieve stable Cd4 gene silencing (Fig. 2). Notably, the Cd4 silencer does contain a Tcf/Lef motif in its 30 -end, and deletion of the 30 -half of the silencer has been reported to cause CD4 derepression in CD8? T cells [49]. It would be interesting to examine whether Tcf1 and Lef1 are recruited to the Cd4 silencer by this cis-element or indirectly through interaction with Runx factors, and how they contribute to the epigenetic silencing of the Cd4 gene. These studies therefore demonstrate requirements of Tcf1 and Lef1 beyond the early stages of thymocyte maturation and also highlight the importance of genetic dissection in a developmental stage-specific manner.

Thymocyte transformation The proliferative capacity of developing thymocytes ensures sufficient production of a large pool of functionally competent T cells after vigorous selection processes. However, dysregulation of thymocyte proliferation and maturation can result in malignant transformation, causing T cell acute lymphoblastic leukemia (T-ALL) in humans [50]. Recent studies by us and others have revealed that Tcf1 unexpectedly functions as a tumor suppressor in developing thymocytes [51]. The thymic lymphomas that develop in Tcf7-/mice express cell-surface markers characteristic of DN and DP thymocytes and are clonal and highly metastatic [22, 52]. The Tcf7-/- lymphoma cells exhibit increased expression of Lef1, Id2, and some Notch target genes, and express truncated forms of Notch1 protein, which frequently contain mutations in the heterodimerization domain [22, 52]. As discussed above, usually Tcf1 and Lef1 exhibit partially redundant roles in promoting thymocyte maturation; it is unexpected to observe increased expression of Lef1 in premalignant and transformed Tcf7-/- early thymocytes. Detailed molecular dissection reveals that Tcf1 directly binds to a ‘‘–4.4 kb’’ regulatory element in the Lef1 gene to repress Lef1 expression when coupled with b-catenin activation. More importantly, conditional deletion of Lef1 in Tcf7-/- early thymocytes protects the cells from transformation in [70 % of animals [22]. It is also suggested that the short isoforms of Tcf1 may contribute to negative regulation of the *3.6 kb Lef1 promoter [52]. Collectively, these findings indicate that Tcf1 and Lef1 have opposing roles in early thymocytes, with Tcf1 directly restraining Lef1 expression (Fig. 1b). In addition to Lef1 suppression, Tcf1 may employ other mechanisms to protect thymocytes from transformation.

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Stabilization of b-catenin in DN3 thymocytes suppressed the expression of Id2, Notch1, and Notch target genes [20, 22]. Furthermore, deletion of Id2 in Tcf7-/- mice delays onset of thymic lymphomas [22]. An upstream regulatory element (URE) located at –14 kb of the PU.1 gene (encoded by Sfpi1) has been reported to function as a negative regulator of PU.1 in early thymocytes. The URE harbors a consensus Tcf/Lef binding motif, and mice lacking the URE in the germline develop aggressive thymic lymphomas with similar phenotypes as those occurred in Tcf7-/- mice [53]. It is of note that neither Tcf1-deficient nor URE-deleted T cell lymphomas express high levels of PU.1 ([53] and our unpublished observations), and it is suggested that transiently enhanced PU.1 expression in early thymocytes, due to loss of Tcf1-mediated restraint, contributes to initiation of transformation in a ‘‘hit-andrun’’ fashion. Therefore, loss of Tcf1 can be a critical event leading to tumorigenesis in thymocytes (Fig. 1b). Significantly, in human T-ALLs, an ETP subtype is associated with diminished TCF7 expression, and two of the 15 ETPALL cases harbor monoallelic TCF7 gene deletions [22]. These findings highlight an essential role for Tcf1 in guarding developing thymocytes from uncontrolled growth and transformation. Aberrant activation of the Wnt-b-catenin pathway has become a hallmark of various human cancers including hematological malignancy [54]. However, no activating mutations of this pathway have been causatively linked to human T-ALL. Several approaches are used to stabilize bcatenin in murine thymocytes and have yielded contradictory observations. In mice harboring a mutation in the Apc allele (Apcmin) or expressing an N-terminus-truncated form of b-catenin transgene (driven by the Lck promoter), no thymocyte transformation is found [55, 56]. In both models, stabilization of b-catenin in thymocytes diminishes thymocyte growth, enhances apoptosis, and promotes oncogene-induced senescence. Ablation of p53 in these models permits development of thymic lymphomas, presumably due to loss of p53-enforced apoptosis in thymocytes expressing activated b-catenin [55, 56]. These p53null lymphomas are not dependent on c-Myc or Notch activation. In key contrast, thymic lymphomas are independently observed in two other different b-catenin stabilization models. In one model, the exon 3 of Ctnnb1 gene, which encodes an N-terminal segment of b-catenin that contain all four Ser/Thr residues for GSK3b- and CK1-mediated phosphorylation, is floxed and excised by Lck-Cre or CD4Cre recombinase [57]. In the other model, a stabilized form of b-catenin harboring a Ser33 to Tyr mutation is inserted into the ubiquitously expressed Gt(ROSA)26Sor gene locus, and expression of the mutant b-catenin is activated via CD4Cre-mediated excision of a preceding floxed STOP cassette

[58]. Formation of T cell lymphomas in these two models depends on c-Myc and Rag activity but does not involve Notch activation, and all these features are distinctive from Tcf7-/- lymphomas. A recent study has further revealed that stabilized b-catenin causes genomic instability, with frequent translocation of Myc to the Tcra locus [23, 58]. Genome-wide mapping of Tcf1 binding locations in whole thymocytes shows an impressive [80 % overlapping of Tcf1 binding sites with Rag2-occupied and H3K4me3marked genomic locations [23]. Coupled with a direct interaction of Lef1 and the DNA-PK complex [25], Tcf1 and Lef1 may contribute to sensing and repair of DSBs. In line with this notion, b-catenin stabilization causes increased DNA damage at the Tcra and Myc loci, impairs repair of Rag-mediated DSBs, and enhances survival of damaged thymocyte, all of which could have contributed to malignant transformation [23]. Another form of genomic instability due to b-catenin stabilization is loss of Pten expression, and deletion of one Pten allele accelerates onset of thymic lymphoma in the b-catenin-stabilized mice [58]. It is of interest to note that unphosphorylated b-catenin is increased in thymic lymphomas derived from Pten-deficient hematopoietic progenitors, and ablating one allele of the b-catenin gene substantially decreases/delays the occurrence of thymocyte malignancy caused by loss of Pten [59]. In spite of these findings, it remains to be determined whether cooperation of Wnt/b-catenin activation with Myc and/or Pten represents a Notch-independent pathway in human T-ALL etiology. The reason(s) for these directly contradictory effects, i.e., senescence vs. transformation, in different bcatenin stabilization models is not immediately evident. In all these models, the impact of b-catenin stabilization on thymocyte development is clearly different as well, ranging from no apparent alterations to severe blocks at DN or DP stages. It appears that at least the level and timing of bcatenin activation may be accountable for these discrepancies. As observed in hematopoietic stem cells, moderate activation of b-catenin is beneficial, whereas strong activation has detrimental effects [60].

CD41 helper T cell differentiation and function CD4? T cells differentiate into distinct helper lineages upon activation, depending on the cytokine milieu elicited by various types of infections. A role for Tcf1/b-catenin in repressing Th1 and promoting Th2 differentiation was previously reviewed [1]. Tcf1 has also been implicated in negative regulation of Th17 differentiation [61, 62], and this role may involve different mechanisms (Fig. 3). Tcf1 directly binds to an intronic region of the Il17a gene in activated or Th17-polarized CD4? T cells [61, 63] and thereby dampens IL-17A production. Predisposition of

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Fig. 3 Comparison of requirements of Tcf1 and Lef1 in subsets of T helper and innate immune cells. Whereas molecular details on a role in Th1 and NK/ILC1 cells are less known, Tcf1 promotes differentiation of Th2 and ILC2 by positive regulation of Gata3. In Th17, Tcd17, and NKp46? as well as NCR- ILC3 cells, a common role of Tcf1 is to restrain production of IL-17A. The precise function of Tcf1 in Treg cells remains to be elucidated. Bcl2l1 encodes Bcl-XL. ‘‘?’’ and ‘‘-’’denote positive and negative regulation by Tcf1, respectively

Tcf1-deficient T cells to the Th17 lineage may also stem from alterations in epigenetic modification during thymic development [62]. The Il17a gene locus is associated with increased histone acetylation in Tcf7-/- thymocytes as well as naı¨ve T cells, which could not be reversed even when Tcf1 expression is reintroduced into Tcf7-/- mature CD4? T cells [62]. Consequently, Tcf7-/- mice are more susceptible to induction of experimental autoimmune encephalomyelitis (EAE) [61, 62]. Although Tcf1 does not bind to the Rorct gene or affect Rorct expression, a recent study showed direct binding of Lef1 to the Rorct gene locus in polarized Th17 cells [63]. Albeit the biological importance of this binding remains unknown, this observation suggests an interesting possibility that Tcf1 and Lef1 may act through distinct target genes in activated CD4? T cells. In line with the role of Tcf1, activation of Wnt-b-catenin pathway represses Th17 differentiation as tested by inclusion of Wnt ligands or a GSK3b inhibitor in Th17 polarization assays [64]. Conversely, inhibition of Wnt signaling with secreted Frizzled-related protein 1 (sFRP1), Dickkopf1 (Dkk1), or Wnt inhibitory factor 1 (Wif1) enhances Th17 production. sFRP1, in particular, sensitizes CD4? T cells to TGF-b stimulation, leading to increased Smad2/3 phosphorylation. Previous studies suggested a positive regulatory role for b-catenin in regulatory T cells (Tregs). Stabilization of bcatenin by inhibitors of GSK3b has been reported to potentiate in vitro suppression activity of Tregs [65]. Systematic administration of GSK3b inhibitors extends survival of allograft islet transplantation [65] and ameliorates EAE [66]. In addition, forced expression of a mutant form

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of stabilized b-catenin extends the survival of Tregs without affecting their in vitro suppression activity, and consequently, fewer b-catenin-expressing Tregs are required for protection against naı¨ve CD4? T cell-induced colitis [67]. A more recent study, however, presents a completely opposite view, e.g., activation of Wnt-b-catenin pathway negatively regulates Treg activity [68]. Using an in vitro assay, stabilizing b-catenin in Tregs with GSK3b inhibitors, recombinant Wnt3a protein, or through Apc gene mutation (Apcmin/?) each compromises Treg suppression activity, and this effect is abrogated in Tcf1deficient Tregs [68]. Conversely, inclusion of a Wnt antagonist or inhibition of Wnt production potentiates Treg-mediated suppression. Short-term treatment of Tregs with a GSK3b inhibitor appears to have a long-lasting effect, and the treated Tregs exhibit diminished capacity to protect from autoimmune colitis in vivo. Furthermore, systematic administration of a GSK3b inhibitor exacerbates proteoglycan-induced arthritis. These discrepancies are enigmatic and thus caution for case-by-case evaluation on how to harness Treg activity through manipulation of Wnt-b-catenin pathway. On the mechanistic side, forced expression of stabilized b-catenin in Tregs induces Bcl-XL but reduces c-Myc and Bax expression [67]. The recent report demonstrates direct interaction between Tcf1 and Foxp3 proteins and considerable overlap between b-catenin and Foxp3 binding peaks across the Treg genome by ChIP-seq [68]. Among the Foxp3 target genes, the Il2 promoter is co-occupied by Tcf1, b-catenin, and Foxp3, and in addition, stabilization of bcatenin is able to override Foxp3-mediated repression of IL2 production [68]. Further analysis of Tcf1/b-catenin and Foxp3 targets under different experimental conditions may help reconcile these apparently conflicting observations.

Innate immune cells Innate immune cells provide the initial protection against infectious microorganisms. In the spotlight during the past few years is the expanding family of lymphocytes with innate cell characteristics, i.e., innate lymphoid cells (ILCs), which are important for multiple forms of protective immunity at the acute phase of infections [69, 70]. The ILC family exhibits remarkable functional diversity and produces distinct groups of cytokines that are reminiscent of T helper cells. Based on recent ILC grouping proposals [71], three functionally distinct ILCs are recognized. ILC1 contains the prototypical NK cells and other IFN-c-producing ILCs, resembling Th1 cells. Like Th2, ILC2 produces IL-5 and IL-13. ILC3 comprises the classical lymphoid inducer (LTi) cells and LTi-like cells with or without the expression of natural cytotoxicity receptors

University of Iowa Immunology 2014 Fig. 4 Summary of proteins that directly interact with Tcf/ Lef factors. If mapped, the domains in Tcf/Lef factors responsible for the interaction are illustrated. bCat-BD, bcatenin-binding domain; CAD, context-dependent activation domain; HMG, high mobility group DNA-binding domain

(NCRs), with the capacity to secrete Th17-type cytokines, IL-17 and/or IL-22. In terms of ontogeny, development of ILC subsets is guided by transcriptional programs that are analogous to those directing T cell development and/or Th differentiation [69]. Th1 differentiation depends on a T-box transcription factor, T-bet [72]. T-bet and its homolog Eomes are recently shown to regulate critical checkpoints in NK cell development [73]. In particular, Eomes is essential for expression of a diverse repertoire of Ly49 receptors in NK cells [73]. Whereas deficiency in Tcf1 exhibits a moderate effect, the combination of Lef1 null mutation with hypomorphic Tcf1 expression greatly diminishes NK cell numbers [74, 75]. Tcf1 deficiency also perturbs balanced expression of the Ly49 receptor repertoire [74, 75]. We have demonstrated positive regulation of Eomes by Tcf1 in cytotoxic CD8? T cells [76]. Genetic interaction between Tcf1/Lef1 and T-box factors in NK/ILC1 cells might be an interesting subject to pursue (Fig. 3). Similar to the essential role of Gata3 in Th2 cells [77], development of ILC2 depends on Gata3 [78]. In both early thymocytes and Th2 cells, Tcf1 acts upstream of Gata3 [16, 18]. It is therefore reassuring that the Tcf1-Gata3 axis is also

operational in ILC2 cells, where Tcf1 binds to the Gata3 gene locus, and loss of Tcf1 reduces Gata3 expression [79]. Indeed, the numbers of mature ILC2 are severely reduced in the lung, gut, and bone marrow of Tcf7-/- mice [79, 80]. The few remaining Tcf1-deficient ILC2 cells are impaired in IL-5 and IL-13 production, resulting in diminished inflammatory responses such as eosinophil infiltration in the lung [79, 80] and clearance of helminth infection [79]. As discussed above, Notch signals directly upregulate Tcf1 expression during early thymocyte development [16, 17]. Notch also promotes generation of ILC2 [79, 81], and significantly, forced expression of Tcf1 is sufficient to drive ILC2 production even when Notch signals are inhibited [79]. Therefore, the ‘‘Notch-Tcf1Gata3’’ pathway in ILC2 appears to represent an express bypass to generate protective innate immune cells from hematopoietic progenitors. It is noteworthy that Tcf1 has Gata3-independent roles in ILC2 as well, such as direct positive regulation of IL-7Ra [79] (Fig. 3). Generation of all ILC3 subsets strictly depends on the orphan nuclear receptor Rorct [82, 83], reminiscent of Th17 differentiation [84]. Among the ILC3 subsets, the NCR-expressing NKp46? ILC3 cells are more specifically diminished in Tcf7-/- mice [63, 80]. Accordingly, the

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Tcf7-/- mice exhibit more severe colon mucosal damage and increased bacterial dissemination following C. rodentium infection [80]. The impact of Tcf1 deficiency on IL-22 production ex vivo varies depending on the phenotypic ILC3 subsets isolated and stimulation conditions; however, loss of Tcf1 causes uniformly increased IL-17 expression in ILC3 including the NKp46? and NCR- subsets [63, 80]. The latter effect is in concordance with a role for Tcf1 in restraining Th17 responses. Another classical innate immune cell type in the lymphoid lineage is cd T cells, which express cd TCRs with limited diversity but can rapidly produce cytokines to promote pathogen clearance in epithelium and mucosaassociated lymphoid tissues [85]. cd and ab T cells both originate from common progenitors in the thymus [85], and development of cd T cells requires a cd-specific transcription factor, Sox13, which contains an HMG DNAbinding domain, like Tcf1 and Lef1 [86]. Forced expression of Sox13 promotes cd T cell differentiation at the expense of ab T cells, and conversely, inactivation Sox13 greatly diminishes cd T cell output. Tcf1 and Lef1 are expressed in cd T cells; however, deficiency in both factors does not affect cd T development in fetal thymic organ culture [21], and Tcf7-/- mice have normal numbers of cd T cells in the thymus and spleen although they lack cd T cells in intraepithelial lymphocytes (IELs) in the intestine [87]. One mechanism of Sox13 function in cd T cells is to sequester Tcf1 by direct physical interaction and hence oppose Tcf1 activity. On the other hand, Tcf1-deficient DP or CD8? SP thymocytes express rearranged Vc2 transcript, suggesting that Tcf1 has an essential role in conferring lineage fidelity for ab T cells. If Tcf1 activity has to be repressed, why is the expression of Tcf1 necessary in cd T cells at all? An answer to this question comes from a recent study of Tcd17 cells, a subset of Vc2 TCR? cd T cells that express Rorct and produce IL-17 upon activation [88]. Inactivation of either Sox4 or Sox13 completely abrogates the generation of Tcd17, without affecting Eomes? IFN-c-producing Vc2? cd T cells [63]. Deletion of Tcf1, however, skews almost all Vc2? cells to Tcd17 and even skews IFN-cproducing Vc1.1? cells toward Rorct expression and IL-17 production [63]. Therefore, one critical role of Tcf1 in cd T cells appears to be ensuring generation of balanced innate effector subsets so as to preserve proper responses to different types of pathogens. Sox13 may oppose Tcf1-mediated repression of Rorct and/or Il17a genes to facilitate development of Tcd17 cells, by direct sequestration of Tcf1 protein or competitive binding to target regulatory sequences. Consistent with this notion, Sox13, Sox4, and Tcf1 exhibit co-occupancy at the Rorct and Blk gene loci in Vc2? but not Vc2- cd T cells [63]. It remains to be determined whether Tcf1 is recruited to these genes via

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direct (binding to DNA cis-elements) or indirect (binding to Sox13) mechanisms. In contrast to lack of interaction of Sox13 with the Il17a gene, Tcf1 and Lef1 bind to this gene in Vc2? as well as Vc2- cells, in addition to in vitro polarized Th17 cells [61, 63]. Collectively, restraining IL17 production by Tcf1/Lef1 factors is a conserved event in Th17, cd T cells, and NKp46? ILCs. Innate memory-like CD8? T cells represent another subset of ‘‘innate’’ T cells arising during thymocyte maturation [89] and share characteristics with conventional memory CD8? T cells such as higher expression of Eomes, CD44 and CD122, and rapid IFN-c production upon TCR stimulation. Generation of the innate memory-like CD8? T cells does not require encounter with antigens but depends on IL-4 produced from thymocytes expressing the PLZF transcription factor. The innate CD8? T cells and PLZFexpressing thymocytes are more readily detectable in naı¨ve Balb/c than in C57BL/6 mice; however, deficiency in Klf2, Id3, CBP, or Itk (a Tek family tyrosine kinase) causes accumulation of the innate CD8? T cells in C57BL/6 mice [89]. Interestingly, a similar increase in innate CD8? T cells is also observed in the Lck promoter-driven b-catenin transgenic mice on C57BL/6 background [90]. The accumulation of innate CD8? T cells does not intrinsically depend on b-catenin, but is rather secondary to expansion of IL-4-producing PLZF? NKT and CD4? thymocytes caused by forced expression of stabilized b-catenin. The Tcf1-b-catenin pathway thus indirectly promotes generation of the innate memory-like CD8? T cells, and its genetic interaction with other known factors such as Klf2 remains to be elucidated.

Cofactors for Tcf/Lef, beyond b-catenin It has been a long-standing issue that inactivation of bcatenin, alone or in combination with its homolog c-catenin, does not phenocopy deletion of Tcf1 or both Tcf1 and Lef1 in T cells and other hematopoietic cells. Several possible explanations have been proposed (reviewed in [1]). Accumulating evidence suggests that Tcf1 and Lef1 do not solely act downstream of the Wnt-b-catenin pathway and can employ additional cofactors to regulate their target genes in b-catenin-dependent and/or b-cateninindependent manners (Fig. 4). One prominent group of Tcf/Lef-interacting proteins is transcription factors. As discussed above, Runx3 can interact with all four Tcf/Lef family members, and this interaction attenuates Tcf4-bcatenin signaling during intestinal tumorigenesis [48]. We find that interaction of Tcf1/Lef1 with Runx3 contributes to stable silencing of the Cd4 gene in CD8? T cells [41]. Tcf1 also interacts with Foxp3, and this interaction may oppose the regulatory roles of Foxp3, such as repression of Il2

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transcription in Tregs [68]. Direct interaction between Lef1 and Gata3 is found in a Th2 cell line, and forced expression of Lef1 in Th2 cells diminishes Th2 cytokine production [91]. Sox13 sequesters Tcf1 by direct interaction in cd T cells [86]. In the context of Xtwn gene regulation in Xenopus, Lef1 is found to interact with TGF-b-regulated Smad2 and Smad3 as well as the common Smad, Smad4 [92, 93], serving as a convergence point for TGF-b and Wnt signaling pathways to activate Tcf/Lef target genes. Although inhibiting the Wnt pathway appears to sensitize TGF-b signaling in Th17 cells [64], it remains to be determined whether the interaction between Lef1 and Smad factors is operational in Th17 or Tregs. Cooperation of Tcf/Lef with AP-1 family transcription factors was initially identified through interaction of Tcf4 and c-Jun in promoting intestinal tumorigenesis [94]. Tcf1 and Lef1 do not interact with c-Jun, but can strongly bind ATF2 and CREB5 (members of the ATF subfamily of AP-1 factors), respectively [95]. Coexpression of the ATF subfamily members promotes the expression of Tcf1/Lef1 target genes in hematopoietic tumor cells, independent of bcatenin [95]. In addition to these transcription factors, ALY (Ally of AML1/Runx1 and Lef1) and IGF1R (insulin-like growth factor-1 receptor) can function as coactivators for Lef1 through direct protein–protein interaction [96, 97]. CREBbinding protein (CBP) and p300 are broadly utilized transcriptional coactivators and have histone acetyltransferase activity. In Drosophila, CBP was initially found to bind directly to Tcf and acetylate a lysine residue in the Nterminus of Tcf, compromising the interaction between Tcf and b-catenin [98]. In vertebrates, CBP is frequently recruited by b-catenin and contributes to positive regulation of Wnt target genes [99]. However, repressive effects by CBP/p300 are observed in human colorectal cancer cell line, which is at least partly mediated by direct interaction between Tcf4 and p300 [100]. Whether and how these cofactors modulate Tcf1 and Lef1 activities in developing and mature T cells would be of interest to investigate. As detailed above, Lef1 interacts with DNA-PKcs and possibly Ku80 in the DNA-PK complex [25]. Tcf4 is also shown to interact with Ku70 and Ku80 as well as poly(ADPribose) polymerase-1 (PARP1), which is also involved in DNA repair [101, 102]. In conclusion, remarkable progress has been made during the past few years regarding the roles of Tcf1 and Lef1 in T cells and innate immune cells. The observation that forced expression of Tcf1 in hematopoietic progenitors activates a T cell transcriptional program may have a farreaching impact on directing pluripotent cells to differentiation into functionally competent T cells. In most cases, Tcf1 and Lef1 have partially redundant functions; the finding that Tcf1 restrains Lef1 expression in early

thymocytes suggests that these factors have distinct individuality in terms of target selection and biological output, which should not be overlooked in the future works. Specific targeting of Tcf1 and Lef1 at a late stage of T cell development revealed their novel roles in lineage choice and Cd4 gene silencing. These effects are not evident in germline-deleted Tcf7-/- mice, which have been used in almost all relevant studies thus far. The defects in early stages of T cell or ILC development in these Tcf7-/- mice may have strongly impacted on and/or blurred its function at late developmental stages or in mature immune cells populating the periphery. It might be worthwhile to revisit some key issues to discern the actual requirements for Tcf1 and Lef1 in a stage-specific manner. Besides b-catenin, Tcf/Lef factors can interact with a number of other proteins including transcription factors, depending upon the cell context. Precisely which cofactor(s) contribute to regulation of T cells and ILCs is of great interest. In-depth understanding of these key issues will equip us with new knowledge, with which we can specifically boost innate and cellular immunity to control infections, autoimmune diseases, and cancers. Acknowledgments F.C.S. is a recipient of T32 predoctoral training grant (AI007485, NIAID, NIH). H.-H.X. is supported by grants from the American Cancer Society (RSG-11-161-01-MPC) and the NIH (AI105351). The authors declare no competing financial interests.

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