Dynamic roles for IL-2-STAT5 signaling in effector and regulatory CD4⁺ T cell populations

Abstract

CD4⁺ T helper cells are responsible for orchestrating diverse, pathogen-specific immune responses through their differentiation into a number of subsets, including T helper 1 (T_H1), T_H2, T_H9, T follicular helper (T_FH), T follicular regulatory (T_FR), and regulatory T (T_REG) cells. The differentiation of each subset is guided by distinct regulatory requirements, including those derived from extracellular cytokine signals. Interleukin-2 (IL-2) has emerged as a critical immunomodulatory cytokine that both positively and negatively affects the differentiation of individual T helper cell subsets. IL-2 signals, in part, via activation of Signal Transducer and Activator of Transcription 5 (STAT5), which functions as a key regulator of CD4⁺ T cell gene programs. In this review, we discuss current understanding of the mechanisms that allow IL-2-STAT5 signaling to exert divergent effects across CD4⁺ T cell subsets and highlight specific roles for this pathway in the regulation of individual T helper cell differentiation programs.

Introduction

The cytokine interleukin-2 (IL-2) was first discovered in 1976 as a T cell growth factor (1). Subsequently, a large body of literature has identified IL-2 as a pivotal cytokine responsible for the formation and function of lymphocyte populations, including CD4⁺ T helper cells. IL-2 exhibits a broad range of functions across T helper cell populations and is an essential regulator of numerous signaling pathways, including those underlying cell survival, proliferation, differentiation, and effector functions. Thus, predictably, dysregulation of IL-2 signaling has been identified as a key factor in the genesis of autoimmune disorders and immunodeficiency. Here, we discuss basic aspects of the IL-2 signaling pathway and its pleiotropic effects on the differentiation of effector and regulatory CD4⁺ T cell populations.

Structure, binding affinity, and regulation of the IL-2 receptor

IL-2 signals through a heterotrimeric receptor composed of IL-2Rα, IL-2Rβ, and the gamma common (γc) chain subunits (2–8). Expression of individual subunits varies across immune cell populations, and expression levels are dependent upon both immune cell activation state and cytokine signals. In CD4⁺ T cells, while the γc chain is expressed constitutively, IL-2Rα and IL-2Rβ expression are upregulated following T cell receptor (TCR) stimulation and further induced by STAT5 activity downstream of IL-2 signaling (2, 9–12). In the presence of environmental IL-2, assembly of the trimeric form of IL-2R occurs sequentially, beginning with IL-2/IL-2Rα binding, followed by association with IL-2Rβ and subsequent γc chain recruitment. Formation of this heterotrimeric receptor results in high-affinity IL-2 signaling (12, 13). While the trimeric receptor combination predominates, weaker signals may be relayed through IL-2Rβ/γc, though downstream mechanisms are altered due to diminished IL-2-receptor binding affinity (14, 15).

Highlighting the importance of effective IL-2 signaling in immune function, mutations in IL-2R and downstream signaling molecules have been implicated in the generation of disrupted immune cell responses in humans. For example, mutations in the γc receptor subunit or JAK3 result in severe combined immunodeficiency (SCID) diseases (16–19). Conversely, patients with IL-2Rα or IL-2Rβ mutations develop autoimmune disease, due to disrupted immune tolerance, while paradoxically also exhibiting immunodeficiency (2, 20–23). Given its role in such disease states, the IL-2 signaling pathway is an attractive target for a number of immunotherapies, which focus on modulation of IL-2 responses to target individual T cell populations for the treatment of not only autoimmune disease, but also graft versus host disease (GVHD) and cancer. Different strategies to manipulate IL-2 signaling for therapeutic benefit will be briefly discussed below and have also been extensively reviewed elsewhere (2, 12, 24).

IL-2 signaling pathways

IL-2 signaling is propagated through a number of signaling cascades, including the Janus Kinase (JAK)/STAT pathway (12, 13). Upon high-affinity IL-2 signaling, JAK1 and JAK3 associate with IL-2Rβ and γc, respectively, resulting in cross-phosphorylation and JAK activation (18, 25–30). Next, JAK1 and JAK3 phosphorylate specific tyrosine resides on IL-2Rβ, allowing for recruitment of members of the Signal Transducer and Activator of Transcription (STAT) family via their conserved SH2 domain (27, 31) (Fig. 1). While IL-2 has been shown to activate several STAT family members, including STAT1, STAT3, and STAT5, STAT5 is the predominant IL-2 signaling molecule (32, 33). Upon recruitment, two STAT5 isoforms are activated via JAK-mediated phosphorylation of the tyrosine 694 (STAT5A) and 699 (STAT5B) residues (34, 35) (Fig. 1). This results in STAT5 dimerization, nuclear translocation, and downstream transcriptional activities via direct DNA binding and co-factor recruitment (30). It is important to note that in addition to IL-2, signals from other cytokines, including IL-7, IL-9, and IL-15, are also propagated through STAT5. While this review focuses on the role of STAT5 downstream of signals from IL-2, these other STAT5-dependent pathways have been expertly discussed elsewhere (36–40).

Figure 1. Schematic depicting the key domains and phosphorylation sites of STAT5A and STAT5B.

IL-2 signaling through the high-affinity, trimeric IL-2R complex results in STAT5 activation via tyrosine phosphorylation at Y694 (STAT5A) and Y699 (STAT5B). Tyrosine-phosphorylated STAT5 dimerizes and translocates to the nucleus where it performs diverse functions to directly regulate the expression of individual target genes. STAT5 activity can also been modulated via phosphorylation of specific serine residues, detailed above. In addition to dimerization, STAT5 is also capable of forming tetramers mediated via STAT5 N-terminal domain interactions. Tetramerization requires specific isoleucine, tryptophan, lysine, phenylalanine, and leucine residues, as noted, as well as DNA binding at sequential GAS motifs (sequence provided above). Other domains involved in STAT5 function are also highlighted.

The expression and function of both STAT5 isoforms are critical for immune cell development and function, as their loss, similar to γc and JAK3, has been shown to result in SCID in mice (41). Curiously, in humans, individual mutations in STAT5A and STAT5B have highlighted their distinct roles in immune cell development and function, as STAT5B defects alone are sufficient to drive autoimmunity (42). Subsequent studies have since determined that these differential functions can be attributed to distinct gene targets for each isoform, discussed further below (43, 44).

While STAT5 is a critical downstream mediator of IL-2 signaling, studies utilizing expression of constitutively active STAT5 have demonstrated that its activity alone is insufficient to reproduce the full biological effects of IL-2 (45). Indeed, IL-2 has also been shown to signal via the Mitogen Activated Protein Kinase (MAPK) pathway, via extracellular signal-regulated kinase (ERK), as well as the phosphatidylinositol 3-kinase (PI3K) pathway (12, 14, 46). IL-2-dependent activation of PI3K results in subsequent activation of mammalian target of rapamycin (mTOR) and protein kinase B (AKT) (46). Collectively, these pathways have been implicated in regulating aspects of CD4⁺ T cell differentiation, effector function, metabolism, and survival.

Mechanisms by which STAT5 activity is regulated

STAT5 broadly influences the development of a number of immune cell populations through a diverse set of regulatory mechanisms. Such functional diversity is in turn mediated via mechanisms that control STAT5 activities. This includes alterations in STAT5 activation status mediated by post-translational modifications, including phosphorylation. First, as discussed briefly above, phosphorylation at Y694/9 (STAT5A/B) by JAK1/3 downstream of IL-2 signaling leads to its dimerization and subsequent transcriptional activity (35, 46). In addition to Y694/9, a number of serine residues have also been identified as phosphorylation sites within STAT5A and B, including S127/8 (STAT5A), S725 (STAT5A), S730 (STAT5B), and S780 (STAT5A) (Fig. 1) (46–49). While IL-2-dependent phosphorylation of these sites has been suggested to modulate STAT5 transcriptional activity, their precise functions and roles in T helper cell regulation are areas of active investigation (50, 51).

Given the requirement for STAT5 phosphorylation in propagating its function, predictably, dephosphorylation of STAT5 also regulates its activity by diminishing phosphorylation-dependent mechanisms. Enzymes identified as mediators of this process include the SH2 domain-containing protein (SHP-2), and low molecular weight protein tyrosine phosphatases (LMW-PTPs), which have both been shown to dephosphorylate STAT5 tyrosine residues (52, 53). Additionally, the dual-specificity phosphatase (DUSP) family member DUSP4 has been shown to dephosphorylate both tyrosine and serine residues to regulate STAT5 activity (54, 55).

Beyond phosphorylation, STAT5 activity is also modulated via differential oligomerization states. Following dimerization, STAT5 is capable of forming tetramers at specific tandemly linked gamma-activated sequence (GAS) DNA binding motifs (56–59). Tetramer formation is dependent upon conserved isoleucine 28 (I28), tryptophan 37 (W37), lysine 70 (K70), phenylalanine 81 (F81), and leucine 82 (L82) residues in the N-terminal domain of both STAT5A and STAT5B (57, 58, 60) (Fig. 1). Functionally, it has been established that mice incapable of forming STAT5 tetramers exhibit a reduced ability to generate a number of immune cell populations, including CD4⁺CD25⁺ regulatory T (T_REG) cells (57). Mechanistically, STAT5 tetramerization is essential for the direct regulation of a specific subset of genes activated in response to IL-2 signals, including the IL-2 receptor subunit Il2ra (CD25) and effector cytokines Ifng, and Tnf (57). The mechanisms underlying dimeric and tetrameric STAT5 function also appear to be distinct, as tetrameric STAT5 has been observed predominantly within intronic regions, while dimers are found proximal to the transcriptional start sites (TSS) of target gene loci (57). To date, much of the mechanism underlying STAT5 tetramer function, including the identity of recruited co-regulators in T helper cells, remains unclear. However, in B cells, tetrameric STAT5 has been shown to recruit the histone methyltransferase Ezh2, which works in concert with the polycomb repressive complex 2 (PRC2) to regulate histone modifications (61). While this role has not been directly demonstrated in CD4⁺ T cells, Ezh2 is known to regulate T_H1, T_H2, and T_REG programs, suggesting a potential mechanism by which STAT5 tetramerization may regulate gene expression patterns in these populations (62).

In addition to Ezh2, interactions between STAT5 and other transcriptional regulators are known to influence STAT5 transcriptional activity and downstream functions. In regulatory T cell populations, STAT5 has been shown to recruit the ten-eleven translocation enzymes Tet1 and Tet2, which function to demethylate target gene loci (63). Additionally, STAT5 has been shown to interact with a number of other co-regulators, including the transcriptional co-activators p300/CREB-binding protein, (p300/CBP) and nuclear receptor coactivator 1 (NcoA-1)/steroid receptor coactivator 1 (SRC-1), as well as co-repressors, such as silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT), Sac3 domain-containing protein (SHD1), and suppressor of cytokine signaling (SOCS) family member SOCS7 (64, 65). Ultimately, such interactions result in alterations to gene expression via changes in cytokine signaling and the accessibility of target gene loci. It is important to note that while much of the work defining STAT5 interaction with these factors was performed outside of the context of CD4⁺ T cells, it is possible that these interactions are conserved within T helper cell subsets. Thus, they may represent mechanisms underlying differential STAT5 activities across T helper gene programs.

Divergent roles for IL-2 signaling in CD4⁺ T helper cell differentiation and function

CD4⁺ T cells are essential to the adaptive immune response as they coordinate the pathogen-specific effector functions of an array of immune cell populations. Central to this process is the ability of naïve CD4⁺ T cells to differentiate into distinct subsets that perform individual functions during the course of immune responses to allergens, infection, and cancer. Key determinants of CD4⁺ T cell differentiation include signals from environmental cytokines, which drive the differentiation and function of individual subsets. Of these, IL-2 has been found to promote or repress the expression of specific T helper cell gene programs (12, 66, 67). The dynamic nature of this regulation is dependent upon diverse mechanisms including modulation of both cytokine and cytokine receptor expression, as well as positive and negative regulation of cell-type specific transcription factor expression and metabolic programs. The collective effects of IL-2-STAT5 signaling are summarized in Figure 2.

Figure 2. Functional regulation of individual T helper cell gene programs by IL-2-STAT5 signaling.

IL-2-STAT5 signaling has been shown to modulate CD4⁺ T helper cell differentiation by both activating and repressing the expression of subset-specific gene programs. Notable genes regulated by STAT5, including those encoding cytokines, cytokine receptors, and key transcription factors are highlighted above. Starred genes are those induced/repressed in response to IL-2 signaling but, to our knowledge, STAT5 has not been found to directly associate with these target gene loci.

Effector CD4⁺ T helper cell subsets positively regulated by IL-2

T_H1 cells

T helper 1 (T_H1) cells coordinate immune responses to intracellular pathogens and promote anti-cancer immunity through production of effector molecules, including the inflammatory cytokine interferon gamma (IFN-γ) (68, 69). T_H1 differentiation is driven by signals from the cytokine IL-12, which are propagated through a heterodimeric receptor consisting of IL-12Rβ1 and IL-12Rβ2. This results in the activation of STAT4 and subsequent induction of the transcriptional activator T-bet (70–72). T-bet, the lineage-defining transcription factor for the T_H1 cell type, drives expression of IFN-γ, which further promotes T-bet expression via STAT1 activation in a feed-forward fashion (73–76). T_H1 differentiation also relies on expression of the transcriptional repressor Blimp-1, which promotes a terminal effector state and suppresses alternative T cell fates (77).

In addition to the above, IL-2 has well-characterized roles in the positive regulation of T_H1 differentiation. This has been demonstrated by studies illustrating that CD4⁺ T cells lacking either IL-2 or IL-2Rα display marked deficiencies in T_H1 development (12, 78, 79). Mechanistically, STAT5 activated downstream of IL-2 binds to gene-specific regulatory elements to directly induce the expression of IL-12Rβ2, Blimp-1, and IFN-γ to support both T_H1 cell differentiation and effector cytokine production, as well as cytotoxic function in a subset of cells (78, 80, 81). In this context, IL-2-STAT5 signals are amplified through a feed-forward mechanism, as STAT5 directly induces expression of IL-2Rα. Conversely, IL-2-activated STAT5 directly represses expression of Bcl-6, which drives the differentiation of the alternative T_FH cell gene program (66, 82–85).

IL-2 signaling has also been implicated in regulating T helper cell differentiation via modulation of subset-specific metabolic pathways. In T_H1 cells, IL-2 signals promote glycolytic metabolism by both governing the activity of the transcriptional regulators c-Myc and HIF-1α and by repressing the glycolytic pathway antagonist Bcl-6 (86). Increased glycolysis further supports T_H1 effector function by enhancing the production of IFN-γ (87, 88). Additionally, IL-2 signaling in T_H1 cells promotes activation of the serine-threonine kinase Akt and the metabolic regulator mTOR. These factors drive the expression of T-bet and Blimp-1, clonal expansion, nutrient uptake, cellular growth, and alterations to epigenetic marks and chromatin accessibility to support effector differentiation in T_H1 cells (89–91). Finally, IL-2 drives the accumulation of α-ketoglutarate (α-KG), a metabolite generated during glutaminolysis, which supports T_H1 differentiation via CCCTC-Binding Factor (CTCF)-mediated restructuring of the chromatin landscape (92). Thus, IL-2 signals drive T_H1 differentiation by supporting T_H1 gene expression patterns and metabolic function, while also repressing alterative gene programs.

T_H2 cells

T helper 2 (T_H2) cell populations are critical for immune responses to extracellular parasites and have also been implicated in the development of asthma and other allergic disorders (93). T_H2 cells secrete a number of effector cytokines, including IL-4, IL-5, IL-9, and IL-13, which exhibit various functions including the modulation of B cell immunoglobin class switching, activation of eosinophils and basophils, and macrophage polarization (94). Differentiation of T_H2 cells requires signals from IL-4, which are propagated via activation of STAT6 (95, 96). IL-4-STAT6 signaling drives expression of the T_H2 lineage-defining transcription factor GATA binding protein 3 (GATA3), which stabilizes the T_H2 program by directly inducing the expression of key T_H2 target genes, including those encoding the effector cytokines discussed above (97–102).

IL-2-STAT-5 signaling positively regulates T_H2 differentiation, as studies have shown that IL-2 neutralization disrupts expression of the T_H2 gene program, while conversely, expression of constitutively active STAT5 is sufficient to induce a subset of T_H2 genes (93, 103). Functionally, IL-2-STAT5 signaling promotes T_H2 differentiation in part by inducing expression of the IL-4 receptor subunit, IL-4Rα, thereby increasing T_H2 responsiveness to IL-4 signals (104). Additionally, STAT5 has been shown to bind to and modulate accessibility of the Il4 cytokine locus (105). STAT5 also regulates Il4 expression by promoting expression of the transcription factor cMAF and NLR Family Pyrin Domain Containing 3 (NLRP3), which are both direct inducers of IL-4 expression (93, 105–108). Beyond cytokine regulation, IL-2 signaling supports the T_H2 program by promoting the expression of Blimp-1, which represses key aspects of alternative T helper cell gene programs, including expression of the T_FH lineage-defining transcription factor Bcl-6 (77, 109, 110). Collectively, IL-2-STAT5 signaling promotes T_H2 differentiation by modulating both effector cytokine and key transcription factor expression.

T_H9 cells

IL-9 producing T helper 9 (T_H9) cells have been implicated in the clearance of extracellular pathogens including parasites, inflammatory allergic responses, and anti-tumor immunity (111–115). While the mechanisms underlying their formation are still being characterized, their differentiation is induced at least in part via signals received from the cytokines TGF-β1 and IL-4, which promote the expression of downstream T_H9-associated transcription factors PU.1 and IRF4 (115–117). T_H9 cells share developmental requirements with T_H2 cells, including a dependence on both IL-4-STAT6 signaling and the subsequent induction of GATA3.

A number of studies have identified IL-2 as a positive regulator of T_H9 effector function, as IL-9 production is disrupted upon the addition of IL-2- and/or IL-2Rα-neutralizing antibodies (118, 119). A recent study by Warren Leonard’s laboratory expanded upon this understanding to define the mechanisms underlying IL-2 function in T_H9 cells (120). Briefly, this study established that addition of IL-2 to T_H9 cell cultures augments expression of IRF4, which directly associates with the Il9 promoter to induce its expression. Further, IL-2-dependent STAT5 activation is required for mediating IL-9 production, as deletion of STAT5A, STAT5B, or both, resulted in a marked decrease in IL-9 expression (120). Mechanistically, in the presence of IL-2 signaling, both STAT5B and STAT6 were found to directly associate with the Il9 locus. Curiously, however, association of both STAT factors was reduced in the absence of IL-2 (120). As such, it has been suggested that IL-2 signals may also support IL-4-STAT6 signaling via positive regulation of IL-4 receptor expression, as previously observed in other T helper cell populations (104). Finally, opposition between STAT5 and Bcl-6 is also a key aspect of T_H9 differentiation (as with T_H1 and T_H2 populations), as STAT5 induces, while Bcl-6 represses, IL-9 production. These factors exhibit adjacent binding sites not only at the Il9 locus, but also within additional gene loci differentially expressed in T_H9 populations exposed to IL-2 versus the Bcl-6-promoting cytokine IL-21. These findings suggest that STAT5 and Bcl-6 may regulate the T_H9 program more broadly (120). Together, these data support roles for IL-2 in promoting T_H9 differentiation and function via cytokine receptor, transcription factor, and effector cytokine-mediated mechanisms.

Effector T helper cell subsets negatively regulated by IL-2

T_H17 cells

T_H17 cells secrete interleukin 17 (IL-17) and promote inflammatory responses that are key for the clearance of extracellular pathogens and mucosal immunity. Their differentiation is dependent upon combined signals from TGF-β and IL-6, which result in expression of the lineage-defining transcription factor RORγt and production of the cytokine IL-23, which further augments T_H17 differentiation (121–123). Both IL-6 and IL-23 signal via activation of STAT3, which is a key transcriptional activator of both RORγt and IL-17 expression (124, 125).

A number of studies have demonstrated that the IL-2-STAT5 pathway negatively regulates T_H17 differentiation and effector functions (124, 126, 127). For example, IL-2 signaling represses expression of the IL-6 receptor subunit IL-6Rα, thus diminishing the sensitivity of activated CD4⁺ T cells to signals received from IL-6 (78). IL-2-STAT5 signaling also results in reduced expression of the T_H17 lineage-defining transcription factor RORγt, which promotes expression of a number of T_H17 genes including the Il17a/f cytokine locus (124, 126). Mechanistically, STAT5 has also been shown to repress expression of IL-17 by competing with STAT3 for binding sites located within the Il17a/f locus (124, 126, 128).

Although the role for IL-2-STAT5 in T_H17 differentiation seems relatively straightforward, a recent study determined that ablation of IL-2Rα or STAT5 reduced T_H17 generation in vivo (128). This may be explained by another study demonstrating that IL-2 signaling, while repressing the differentiation of T_H17 populations, may also support their expansion (129). Collectively, the current literature suggests that IL-2 may exert nuanced, stage-specific control over T_H17 differentiation, function, and proliferation.

T_FH cells

T follicular helper (T_FH) cells support humoral immunity by both interacting directly with B cells and producing cytokines, including IL-21, to promote B cell activation, germinal center (GC) formation, and high-affinity antibody production (130). T_FH cell differentiation from naïve CD4⁺ T cell progenitors is driven by a number of cytokines, including IL-6 and autocrine signals from IL-21, which are propagated via STAT3 activation to promote expression of the T_FH lineage-defining transcription factor Bcl-6. Bcl-6 broadly supports expression of the T_FH gene program by repressing expression of the T_FH antagonist Blimp-1, as well as other transcription factors that promote the differentiation of additional T helper cell subsets (77, 131–133).

The IL-2-STAT5 signaling axis is a well-established negative regulator of T_FH cell differentiation and function (82–85). In vivo, IL-2-deficient mice exhibit enhanced T_FH cell generation and GC formation, even in the absence of infection (134). Conversely, systemic administration of IL-2 during influenza infection results in deficient T_FH cell responses and GC formation (82). Similarly, the absence of regulatory T cells, which function to deplete environmental IL-2, has been shown to reduce the number of T_FH cells during influenza infection (135). These phenotypes have been attributed to repression of the T_FH gene program via STAT5-dependent mechanisms. First, IL-2-STAT5 signaling has been shown to repress expression of the IL-6R subunits IL-6Rα and gp130, leading to a reduction in IL-6 responsiveness and STAT3 activation (78, 83, 136). STAT5 also competes directly with STAT3 for binding at the Bcl6 promoter to repress its expression. In the absence of Bcl-6, the T_FH antagonist Blimp-1 is released from Bcl6-mediated repression and directly suppresses expression of T_FH target genes, including Cxcr5 (77, 83). In the absence of Cxcr5, T_FH cells are incapable of homing to the B cell follicle to interact with B cells and support GC formation. Finally, loss of Bcl-6 expression also permits both STAT5 binding and reduced DNA methylation within gene loci associated with non-T_FH gene programs, allowing for their expression (137). Thus, IL-2-STAT5 signals repress T_FH differentiation and activity by suppressing both T_FH-associated cytokine receptor and transcription factor expression.

IL-2 signaling and Regulatory T cell populations

Regulatory T cells (T_REG)

Regulatory T cell populations comprise a unique compartment of T helper cells that modulate immune responses, reduce inflammation, and prevent potential autoimmunity mediated by effector T cell (T_EFF) populations (138, 139). These cells are subdivided into thymically-derived nT_REG cells, and those induced in mature CD4⁺ T cells in the periphery (iT_REG) (140, 141). Differentiation of T_REG populations is dependent upon signals from TGF-β1 and expression of the lineage-defining transcription factor Foxp3, which performs both activating and repressive functions to support expression of the T_REG gene program (142). A hallmark of T_REG populations is their constitutive expression of IL-2Rα, which allows T_REGS, but not other T cell populations, to respond to low doses of IL-2 via constitutive high-affinity IL-2 signaling (143). Unlike effector T helper cell subsets, T_REG populations do not produce IL-2 and, indeed, one function of Foxp3 is to repress Il2 expression (2, 13, 144). Instead, T_REG cells depend upon paracrine IL-2 signals from effector T helper cells (13, 139).

It is well-established that T_REG deficiency results in the development of severe autoimmune phenotypes. Similarly, early studies utilizing germline knockouts for IL-2, IL-2Rα, or IL2Rβ revealed that germline disruptions in the IL-2 signaling pathway result in an absence of T_REG generation and suppressive function, leading to the development of autoimmune disorders (139, 145–150). However, conditional deletion of IL-2Rα exclusively in T_REG populations results in a comparatively more severe phenotype, due to the aforementioned IL-2 requirements for specific effector populations as well (151). Functionally, IL-2 signaling is required for the early induction of Foxp3 via activation of STAT5, which directly binds to both promoter and enhancer elements to induce its expression (139, 146, 152–156). Thus, unsurprisingly, loss of STAT5A/B has been shown to result in a significant reduction in murine Foxp3⁺ T helper cells in vivo (157). In humans, this effect may be STAT5B specific, as STAT5B defects, in the presence of normal STAT5A expression, result in reduced Foxp3 expression and T_REG cell suppressive function, and are consequently sufficient to induce autoimmune disease (44).

Mechanistically, Foxp3 induction has been attributed to collaborative STAT5 and TGF-β-Smad3-dependent recruitment of the ten-eleven translocation (Tet) methylcytosine dioxygenases Tet1 and Tet2 to the Foxp3 locus, which results in decreased DNA methylation and stable Foxp3 expression (158). Further emphasizing the importance of STAT5 activity in T_REG development, a recent study found that inhibition of the cyclin dependent kinase (CDK)8 and its paralog CDK19 prevented repressive serine phosphorylation of STAT5 (Fig. 1). This resulted in enhanced STAT5 tyrosine phosphorylation and allowed for conversion of CD4⁺ T cells into Foxp3⁺T_REG cells (48). Furthermore, overexpression of a STAT5B mutant incapable of such phosphorylation promoted Foxp3⁺T_REG differentiation to a much higher degree than WT STAT5B. Together, these findings support the need for enhanced STAT5 activation signals for the differentiation of T_REG populations (48). Finally, much like T_H1 populations, IL-2 signaling has been found to support T_REG differentiation via STAT5 activation and Blimp-1 expression, as well as by supporting glycolytic metabolism, which is induced by IL-2-induced PI3K signaling in this population (159–163).

Expanding upon early global knockout studies, recent work has begun to define dichotomous roles for IL-2 signaling in early T_REG development versus the stability and suppressive capabilities of mature T_REG populations. First, in contrast to germline knockout studies, inducible deletion of IL-2Rα after thymic development or exclusively in mature pT_REG cells revealed that mature T_REG populations require IL-2 for long-term survival and homeostasis, but not to maintain Foxp3 expression (151, 160). Additionally, these cells exhibit reduced expression of the co-inhibitory receptor CTLA4, as well as disrupted metabolic and biosynthetic pathways, further supporting a role for IL-2 in T_REG differentiation via metabolic modulation (151, 160). While the precise roles of IL-2 during each stage of T_REG development are still being elucidated, these findings collectively suggest that IL-2 signaling is required for differentiation and functional maturation in early T_REG stages, and helps to maintain survival, homeostatic proliferation, and metabolic function in mature T_REG cell populations.

Due to their central role in regulating immune tolerance and their dependence upon IL-2 signaling, T_REG cells have been the target of a number of IL-2-based immunotherapeutic strategies to treat both autoimmune disorders and cancer (2, 164, 165). These include the use of IL-2 targeting antibodies, such as JES6–1, which selectively induce T_REG proliferation in efforts to treat a number of autoimmune diseases (166, 167). Conversely, antibodies that target IL-2 and/or modulate IL-2R subunit interactions have been used to limit T_REG responses and thus promote inflammatory environments that are beneficial in tumor immunotherapy approaches (166, 168, 169). Furthermore, the elevated expression of IL-2Rα on T_REG cells has been leveraged in therapies utilizing low doses of the IL-2 cytokine itself to preferentially enhance T_REG populations (170–173). Finally, efforts have been made to engineer altered versions of IL-2 or IL-2Rα that exhibit differential binding affinities and thus preferentially induce or limit T_REG responses in treatments of autoimmune disease and cancer, respectively (174–176).

T follicular regulatory (T_FR) cells

In addition to their modulation of pro-inflammatory T_EFF responses, Foxp3⁺T_REG cells have also been shown to suppress germinal center responses and B cell antibody production via differentiation into the T follicular regulatory (T_FR) cell type (159, 177–180). These cells can also arise directly from naïve CD4⁺ T cell precursors, in response to either self- or foreign antigen (181). T_FR cells exhibit both T_REG- and T_FH-associated traits, including expression of both Foxp3 and Bcl-6, as well as Cxcr5 (159, 177, 179–181). While IL-2 is needed for initial T_REG development as discussed above, it is perhaps unsurprising that strong signals from IL-2 have been shown to repress the differentiation of the Bcl-6-dependent T_FR population (159). Specifically, IL-2 signaling results in activation of STAT5, upregulation of Blimp-1, and subsequent repression of Bcl-6 (159). Thus, as IL-2 production wanes during the resolution of infection, loss of IL-2 signaling allows for the downregulation of IL-2Rα, upregulation of Bcl-6, and subsequent differentiation of the Foxp3⁺Treg population into T_FR cells (159, 182, 183). This is consistent with studies demonstrating that, while IL-2 is required for the initiation of Foxp3 in T_REG populations, its expression can be maintained in mature T_REG cells even in the absence of further IL-2 signals (151, 160). Thus, it would seem that this mechanism allows for concurrent Foxp3 and Bcl-6 expression in the T_FR compartment. This stage-specific, IL-2-dependent generation of T_FR populations thus functions to protect against autoimmunity, as humoral immune responses are directly suppressed during the resolution of infection (184).

Conclusions

IL-2 signaling has emerged as an essential and multi-functional regulator of an array of immune cell populations, including effector and regulatory CD4⁺ T cell subsets. As such, it is no surprise that this pathway has long been the target of therapeutic strategies to treat diseases ranging from cancer to autoimmunity. While the many, and sometimes counterintuitive, roles of IL-2 signaling have made this a challenging endeavor, these efforts have been aided by decades of work that have provided insight into the IL-2-dependent regulatory mechanisms that govern these critical immune responses. Continued research into the mechanisms that govern the IL-2-STAT5 signaling pathway and its subsequent effects on subset-specific cytokine pathways and transcription factors are thus an important avenue for further study moving forward. Such understanding will provide routes for more specific manipulation of IL-2 signals to augment T helper cell responses in immunotherapy approaches to treat human disease.

Abbreviations

α-KG

α-ketoglutarate

AKT

protein kinase B

CBP

Creb-binding protein

CCT

CCCTC-binding factor

CDK

Cyclin dependent kinase

CXCR5

C-X-C motif chemokine receptor 5

DNA

deoxyribonucleic acid

DUSP4

dual-specificity phosphatase family 4

ERK

extracellular signal-regulated kinase

EZH2

Enhancer of zeste homologue 2

F

phenylalanine

GAS

gamma-activated sequence

GATA3

GATA binding protein 3

GC

germinal center

HIF-1α

hypoxia inducible factor

I

isoleucine

IFN-γ

interferon gamma

IL

Interleukin

IRF4

interferon regulatory factor 4

Jak

Janus Kinase

K

lysine

L

leucine

LMW-PTP

low molecule weight protein tyrosine phosphatase

MAPK

mitogen activated protein kinase

mTOR

mammalian target of rapamycin

NcoA-1

nuclear receptor coactivator 1

NLRP3

NLR family pyrin domain containing 3

PI3K

phosphatidylinositol 3-kinase

PRC2

polycomb repressive complex 2

RORγt

RAR-related orphan receptor gamma

S

Serine

SCID

Severe combined immunodeficiency

SHD1

Sac3-domain containing protein

SHP-2

SH2 domain containing protein

SMRT

silencing mediator for retinoic acid receptor and thyroid hormone receptor

SOCS7

suppressor of cytokines signaling 7

SRC-1

steroid receptor coactivator 1

STAT

signal transducer and activator of transcription

TCR

T cell receptor

T_EFF

T effector

T_FH

T follicular helper

T_FR

T follicular regulatory

TGF-β1

transforming growth factor beta 1

T_H1

T helper 1

T_H2

T helper 2

T_H9

T helper 9

T_REG

Regulatory T cells

TSS

Transcriptional start site

W

Tryptophan

Y

Tyrosine