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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Curr Opin Immunol. 2012 Feb 16;24(2):146–152. doi: 10.1016/j.coi.2012.01.015

The Role of STAT5 in Lymphocyte Development and Transformation

Lynn M Heltemes-Harris 1, Michael A Farrar 1,1
PMCID: PMC3319496  NIHMSID: NIHMS354354  PMID: 22342169

Abstract

STAT5 plays a critical role in B and T lymphocyte development. However, whether STAT5 primarily plays a role as a permissive factor, involved in lymphocyte survival, or an instructive factor, involved in lymphocyte differentiation, has been unclear. In addition, while STAT5 has been suggested to act as a transcriptional repressor, the mechanism by which it represses transcription was undefined. Recent reports have begun to shed new light on these roles for STAT5 in lymphocyte development, transcriptional repression, and leukemic transformation.

Introduction

The transcription factor STAT5 consists of two isoforms, STAT5a and STAT5b. These two proteins exhibit 95% sequence homology and appear to be largely redundant in the lymphoid system. STAT5 is a key target of multiple cytokine receptors that are known to play critical roles in lymphocyte development, including those for interleukin-2 (IL2) and -7 (IL7). The availability of mice expressing either STAT5 gain-of-function or loss-of-function mutants has provided strong evidence that STAT5 plays a key role downstream of these receptors in entraining the early stages of B and T cell development (Figure 1) [1]. However, the molecular mechanisms by which STAT5 affects lymphocyte development are still being elucidated.

Figure 1.

Figure 1

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Overview of lymphocyte development with an emphasis on the role of STAT5. The green arrows mark the different stages of development where STAT5 plays a positive role in development of different lymphocyte subsets. The red arrow marks where STAT5-dependent inhibition is required for normal development.

In this review we address the ongoing controversy of whether STAT5 acts primarily as a permissive factor that allows for survival of developing lymphocytes, or as an instructive factor that plays an active role in inducing the expression of factors required for lymphocyte differentiation. In addition, we describe recent advances in our understanding of how STAT5 can act as both a transcriptional activator and repressor. Finally, we review recent studies supporting the key role that STAT5 plays in lymphocyte transformation, focusing specifically on how it initiates B cell acute lymphoblastic leukemia.

T cell development

The cytokine receptors for IL7 and IL2 play important roles in distinct aspects of thymocyte development. Activation of the STAT5 signaling pathway downstream of these receptors has been shown to play a critical role in mediating the biological effects of IL2 and IL7. For example, IL7-dependent STAT5 activation has been shown to both initiate γδ T cell differentiation and promote double negative thymocyte survival [2,3]. These initial studies have been complemented by more recent reports that highlight both the instructive and the permissive roles of STAT5 in later stages of lymphocyte development. The first of these studies focused on the process of CD4 versus CD8 lineage commitment and how this is influenced by TCR signal strength and cytokine signals. Specifically, increased TCR signal strength during positive selection was shown to result in reduced IL7R expression, which leads to reduced CD8 expression. The end result of this process of “co-receptor tuning” is that thymocytes expressing TCRs that signal more strongly are preferentially selected into the CD4+ T cell lineage [4]. Until recently the mechanism that accounts for this remained unclear. However, studies from Singer and colleagues have demonstrated that co-receptor tuning is dependent on STAT5 signaling and that STAT5 acts in both a permissive manner, by promoting CD8+ T cell survival, and in an instructive manner, by inducing expression of Runx3, a transcription factor that is typically required for CD8 expression [5]. These studies also raised several points that are important to consider when studying the role of STAT5 in lymphocyte development. First, deletion of STAT5 may allow other STAT proteins to aberrantly substitute for STAT5. For example, Singer and colleagues found that STAT5 deletion allowed the IL7R to unexpectedly activate STAT6. STAT6, which is not normally activated by IL7, effectively substituted for STAT5 function in CD8 lineage commitment; only when both STAT5 and STAT6 were deleted could the actual biological role of STAT5 be revealed. Second, care must be taken when interpreting results of studies involving overexpression of pro-survival genes. In this study, overexpression of Bcl2 rescued CD8+ T cells in Il7r-/- mice; however, these CD8+ T cells did not express Runx3 and resembled an atypical CD8+ T cell lineage rather than conventional CD8+ thymocytes. Third, neither Bcl2 nor Runx3 expression alone was able to recapitulate the effects of STAT5 signaling. This result suggests that both the permissive/pro-survival and the instructive/differentiation functions of STAT5 are required to drive appropriate CD8+ T cell lineage commitment. Finally, continuous STAT5 signaling, as opposed to a transient STAT5 signal, was required to maintain CD8 expression. Thus, STAT5 plays a critical role as both a permissive and instructive factor required for CD8+ T cell differentiation.

Although STAT5 is not required for differentiation of conventional CD4+ thymocytes, it does play a key role in the development of CD4+Foxp3+ regulatory T cells (Tregs). Mice lacking STAT5 are strikingly devoid of CD4+Foxp3+ Tregs. In this case STAT5 functions as part of a signaling pathway emanating primarily from the IL2R. This observation is consistent with a newly proposed two-step model for Treg development [6,7]. In this model, TCR/CD28 dependent signals leads to the generation of CD4+ Treg progenitors that express high levels of the IL2R complex but do not express FOXP3. In the second step, IL2 stimulation, via a STAT5-dependent process, rapidly converts these Treg progenitors into CD4+Foxp3+ mature Tregs. The exact mechanism by which this occurs remains unclear. However, STAT5 has been shown by several groups to bind to the promoter and first intron of the Foxp3 gene and thus may play a role in initiating Foxp3 expression [8-10]. Once FOXP3 protein is made, it has been shown to participate in a positive feedback loop that promotes it’s own expression and likely limits the need for STAT5 to maintain Foxp3 expression [11]. In this model STAT5 likely plays a role as a switch that helps initiate Foxp3 transcription but is not required to maintain Foxp3 expression once the gene has been induced.

B Cell Development

Mice lacking the IL7 receptor (IL7R) have almost no pro B cells and lack more mature B cell subsets; furthermore, the few remaining B cells exhibit aberrant immunoglobulin gene rearrangement [12,13]. STAT5 plays an especially important role in this process as expression of a constitutively active form of STAT5b (Stat5b-CA) largely restores B cell development in Il7r-/- mice [14]. Subsequent studies using mice in which STAT5 could be selectively deleted in developing B cells confirmed the key role of STAT5 in B cell development [2,3]. This led to a series of studies suggesting that STAT5 plays a critical instructive role in B cell development. Specifically, STAT5 was shown to bind to VH gene promoters and thereby promote IgH gene rearrangement [15]. Consistent with this study, other reports observed prolonged contraction and pericentromeric localization of the IgH locus in Stat5b-CA mice; this latter finding supported a role for STAT5 in promoting accessibility of the IgH region to the recombination machinery [16]. Finally, IL7 and STAT5-dependent signals influence expression of Ebf1, although it is unclear whether this reflects a direct or indirect role for STAT5 in governing Ebf1 expression [17-19]. Consistent with this last report, we observed that Stat5b-CA mice exhibit modestly increased expression of both Ebf1 and Pax5 [20,21]; once again whether this reflects a direct or indirect effect of STAT5 on transcription of these genes remains unclear. Taken together these findings suggested that STAT5 plays an important instructive role in initiating B cell differentiation.

STAT5 plays an important permissive role in B cell development as well. It has long been appreciated that STAT5 can bind to regulatory regions of two pro-survival genes Bcl2 and Bcl2l1 and promote their transcription [22]. More recent work by Busslinger and colleagues using Rag1-Cre × Stat5FL/FL mice demonstrated that STAT5 is required for the expression of the pro-survival gene Mcl1 but not for Ebf1 or Pax5 in pro-B cells; moreover, they demonstrated that Rag1-Cre × Mcl1Fl/FL mice exhibit a similar phenotype to that seen in Rag1-Cre × Stat5FL/FL mice. Finally, this report demonstrated that expression of a pan hematopoietic Vav-Bcl2 transgene could partially restore pro-B cell development in Rag1-Cre × Stat5FL/FL mice [23]. In contrast, forced expression of Ebf1 did not rescue the developmental block observed in Rag1-Cre × Stat5FL/FL mice. Based on these findings Busslinger and colleagues concluded that STAT5 only plays a permissive role in B cell development by regulating cell survival and does not regulate B cell differentiation in an instructive manner as previously proposed.

The explanation for these contradictory findings is not completely clear, although several explanations are possible. First, as suggested by Sigvardsson and colleagues [24], the study by Busslinger and colleagues made use of a Cre construct driven by the Rag1 promoter, which may delete STAT5 too late. Other studies have shown that though Rag1-Cre is transcribed in CLPs, it is likely confined to the more mature Ly6D+ CLP stages that express much higher levels of Rag1 [25,26]. Thus, it is possible that in Rag1-Cre × Stat5FL/FL mice B cell progenitors may receive an IL-7/STAT5 signal prior to deletion of STAT5. In that scenario, early IL-7/STAT5 signaling may be sufficient to initiate Ebf1 expression allowing the cells to overcome the developmental block in B cell development. In such a model, STAT5 would function as a transient switch that initiates Ebf1 expression; EBF1 then establishes a positive feedback loop via induction of Pax5 that acts to maintain both Ebf1 and Pax5 in the absence of continued STAT5 signaling. In contrast, the pro-survival/permissive function of STAT5 clearly requires continuous STAT5 function throughout early B cell development (Fig. 2). A second potential explanation is that other STAT factors may substitute for STAT5 as was described for CD8+ T cell lineage commitment. Future studies will be required to fully define whether STAT5 acts solely as a pro-survival, permissive factor in B cell development or whether it has both permissive and instructive roles.

Figure 2.

Figure 2

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Model for how STAT5 might regulate gene transcription in T and B cell development. In some instances, transient expression of STAT5 correlates with an instructive role in development. As illustrated for Ebf1 and Foxp3, STAT5 activation may initiate transcription but continued expression of genes is maintained by positive feedback loops that are STAT5-independent. Therefore, these genes would only require an initial threshold level of STAT5 signaling without a need for continuous STAT5 signaling. In contrast, continuous STAT5 signaling is required for permissive genes such as Mcl1, Bcl2, and Cyclin D2, although it can be required for instructive factors, such as Runx3, as well.

STAT5 as a transcriptional inhibitor

Most studies of STAT5 have focused on its ability to activate gene transcription. However, recent research has emphasized the role of STAT5 as an important repressor of gene transcription. This is perhaps most clearly demonstrated by the observations made by several groups that IL7 signaling prevents rearrangement at the immunoglobulin kappa light chain locus [23,27,28]. These studies have been extended to show that STAT5 is the critical IL7-dependent factor involved, as Rag1-Cre × Stat5FL/FL mice exhibit premature kappa light chain germline transcripts [23]. In these initial studies they show STAT5 bound to the intronic kappa enhancer (Eki) in a way that might prevent binding of a second transcription factor E2A, which is required for initiating kappa germline transcription. However, a recent report from Clark and colleagues demonstrated that STAT5 still acted as a repressor even when the STAT5 and E2A binding sites were physically separated [29]. Instead, they observed that STAT5 acted to recruit EZH2, a histone H3K27 methylase. This resulted in H3K27-tri-methylation of the kappa light chain locus, an epigenetic change associated with repressed chromatin (Fig. 3). These studies point toward a novel mechanism by which STAT5 can actively repress transcription [29].

Figure 3.

Figure 3

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STAT5 repressive mechanism at the light chain immunoglobulin locus. STAT5 homodimers translocate to the nucleus following cytokine activation. In the nucleus, STAT5 may bind as a tetramer to tandemly repeated STAT5 DNA-binding sites. These tetrameric complexes are capable of recruiting EZH2 and other members of the polycomb repressor family. The resulting complex generates repressive chromatin via H3K27 trimethylation (H3K27me3).

In addition to repressing kappa gene rearrangement, STAT5 also represses many other gene targets. One target of particular interest is the transcriptional repressor Bcl6 [30,31]. BCL6 represses pre-B cell proliferation, in part by inhibiting c-Myc transcription [31]. Thus, STAT5 repression of Bcl6 in large pre-B cells allows for appropriate expansion of this developing cell population. Upon downregulation of the IL7R and STAT5 inactivation, Bcl6 levels increase and cause pre-B cells to exit cell cycle. Importantly, BCL6 also exhibits a pro-survival role in later stages of B cell development. Intriguingly, this represents an important mechanism by which BCR-ABL+ leukemias escape death following treatment with imatinib [32]. Imatinib treatment blocks STAT5 activation which is normally important for survival of the transformed pre-B cell; paradoxically, however, this inhibition of STAT5 results in re-expression of Bcl6 which acts to protect leukemia cells from death. Thus strategies that inhibit both BCR-ABL and BCL6 are more effective at treating BCR-ABL+ leukemias [32]. Future studies of gene targets repressed by STAT5 are likely to provide additional important insights into STAT5 biology.

A major question that remained from these studies is why STAT5 acts as a transcriptional activator when bound to some STAT5 binding sites but a transcriptional repressor when bound to other sites. To address this question, Clark and colleagues carried out STAT5 ChIP-Seq studies in progenitor B cells. Subsequent analysis of these studies found that STAT5 appears to function as a transcriptional activator when to bound to sites that bind STAT5 as a STAT5 homodimer but functions as a transcriptional repressor when bound to sites that bind STAT5 as a larger tetrameric complex [29]. These results suggest that the nature of the STAT5 binding complex determines whether it interacts with transcriptional repressors such as EZH2. This is a very appealing model, although future studies will be required to address the mechanism by which this occurs in more detail.

STAT5 and transformation

STAT5 is involved in both proliferation and survival of lymphoid cells. Therefore, it is perhaps not surprising that STAT5 activation has been correlated with lymphocyte transformation. For example, STAT5 is a known downstream target of the BCR-ABL fusion gene [33-35]. Consistent with this observation, Weber-Nordt and colleagues found that ~50% of human acute lymphoblastic leukemia (ALL) samples displayed elevated levels of activated STAT5, although only 3 fresh and 15 frozen primary samples were studied [36]. Using a much larger pool of 128 patients with acute lymphoblastic leukemia (ALL), we observed elevated levels of STAT5 phosphorylation (and hence activation) in >35% of these samples. This varied by subset with BCR-ABL-positive patients expressing the highest levels of STAT5 phosphorylation. Importantly, we observed that the level of STAT5 phosphorylation prior to therapy predicted the subsequent outcome to treatment of BCR-ABL-positive leukemia with high STAT5 phosphorylation correlating with poor overall survival [20]. These findings strongly suggest that STAT5 plays an important role in progenitor B cell transformation.

Studies of human patients with ALL indicate that STAT5 phosphorylation is increased in ALL and correlates with outcome. Such studies do not demonstrate that STAT5 is actually a major driver of transformation. However, work in mouse models of ALL clearly demonstrates the key role of STAT5 in transformation. For example, expression of a BCR-ABL cDNA in B cell progenitors leads to B-ALL in mouse models; transformation fails to occur in mice lacking STAT5 [37]. We were able to demonstrate a role for STAT5 in ALL utilizing a Stat5b-CA transgenic mouse model. Approximately 1-2% of Stat5b-CA mice develop a disease that resembles human ALL [38]. When these same mice were crossed to mice lacking the adaptor protein BLNK, the mice developed a highly penetrant form (80-90% penetrance) of the disease [38]. Interestingly, Blnk mutations have been noted in a small subset of human patients with B-ALL [39,40]. These studies suggest that STAT5 activation in concert with defective Blnk signaling plays an important role in initiating some forms of B-ALL.

More recent studies have indicated that STAT5 plays an important role in cooperating with defects in Ebf1 or Pax5 to initiate transformation. EBF1 and PAX5 are transcription factors critical for B cell development; surprisingly, deletions in Ebf1 and Pax5 were found in over 30% of patients with B-ALL [39]. Whether these mutations were important drivers of leukemia or merely passengers in the transformation process remained unclear. To address this issue, we generated Stat5b-CA × Pax5+/- and Stat5b-CA × Ebf1+/- mice; these mice rapidly developed fatal progenitor B-ALL. The role of STAT5 was not due solely to effects on survival as Ebf1+/- mice crossed to transgenic mice expressing the pro-survival factor Bcl2l1 failed to develop leukemia. Likewise, no difference in the rate of proliferation was seen between WT progenitor B cells and transformed B-ALL cells, suggesting that STAT5 did not initiate transformation by enhancing pro-B cell proliferation. However, transformation required the expression of the IL7R and correlated with increased expression of the TSLPR. This latter observation is interesting for three reasons: first, STAT5 can be activated downstream of TSLP signaling [41], second, elevated levels of TSLPR have been identified in human ALL [42,43], and third, gain-of-function mutations in the IL7Rα chain, one of the two receptor chains required for TSLP binding, have been found in human ALL [44]. The mechanism by which STAT5 initiates transformation is not entirely clear. However, it may involve synergistic effects of STAT5 activation coupled with defects in the EBF1/PAX5 signaling network. For example, one of the most highly expressed genes in Stat5b-CA × Ebf1+/- leukemia’s is RANKL; this is consistent with the suggestion that RANKL promotes pro-B cell expansion, as Rankl-/- mice have reduced numbers of progenitor B cells. However, although STAT5 induces RANKL expression in breast epithelial cells, it fails to do so in progenitor B cells; only in cells with reduced expression of EBF1 and PAX5, which both repress RANKL [45,46], is increased STAT5 activation capable of driving RANKL expression. Thus, cooperative effects between STAT5 activation and defects in the EBF1/PAX5 transcription factor network that orchestrates normal B cell development appear to be a potent combination that initiates progenitor B cell transformation

Summary and perspective

These studies have begun to shed light on the molecular mechanisms by which STAT5 affects lymphocyte development and leukemia. However, important questions remain to be answered. First, the mechanisms by which STAT5 initiates gene transcription remain poorly defined. Likewise, while we have gained some insights as to how STAT5 represses gene transcription, the precise mechanism by which STAT5 recruits EZH2 remain unclear. Second, although we now know that STAT5 can act as a transcription repressor, the important biological settings in which this occurs are just beginning to be identified. Third, whether STAT5 acts as an instructive factor in initiating B cell development remains unresolved. Finally, the mechanisms by which STAT5 drives progenitor B cell leukemia are just beginning to be understood. A better understanding of how STAT5 initiates leukemia and maintains the transformed state should suggest targets for future pharmaceutical intervention.

Highlights.

  • STAT5 plays a permissive role as a pro-survival factor in lymphocyte development

  • STAT5 regulates lymphocyte differentiation

  • STAT5 functions as both an activator and a repressor of transcription

  • STAT5 plays an important role in initiating acute lymphoblastic leukemia

Footnotes

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