Transcriptional regulation of CD4⁺ T_H cells that mediate tissue inflammation

Abstract

Acquired and genetic immunodeficiencies have revealed an indispensable role for CD4⁺ T cells in the induction of protective host immune responses against a myriad of microbial pathogens. Influenced by the cytokines present in the microenvironment, activated CD4⁺ T cells may differentiate into several highly-specialized helper subsets defined by the production of distinct signature cytokines tailored to combat diverse classes of pathogens. The process of specification and differentiation is controlled by networks of core, master, and accessory transcription factors, which ensure that CD4⁺ T helper (T_H) cell responses mounted against an invading microbe are of the correct specificity and type. However, aberrant activation or inactivation of transcription factors can result in sustained and elevated expression of immune-related genes, leading to chronic activation of CD4⁺ T_H cells and organ-specific autoimmunity. In this review, we provide an overview of the molecular basis of CD4⁺ T_H cell differentiation and examine how combinatorial expression of transcription factors, which promotes genetic plasticity of CD4⁺ T_H cells, can contribute to immunological dysfunction of CD4⁺ T_H responses. We also discuss recent studies which highlight the potential of exploiting the genetic plasticity of CD4⁺ T_H cells in the treatment of autoimmune and other immune-mediated disorders.

1 |. INTRODUCTION

After encountering a specific antigen, CD4⁺ T cells undergo clonal expansion and differentiate into functionally distinct T_H effector subsets, which orchestrate immune responses against diverse microbial pathogens (Fig. 1). Interferon-γ (IFN-γ)-producing T_H1 cells specialize in activating cell-mediated immune responses against intracellular bacteria. T_H2 cells target helminths through the production of interleukin (IL)-4, IL-5, and IL-13. Finally, T_H17 cell-derived cytokines IL-17A, IL-17F, IL-21, and IL-22 stimulate broad tissue responses that defend the host from extracellular bacterial and fungal infections (reviewed in Christie and Zhu,¹ Zhu and Paul,² and Zhu³). In addition, T follicular helper cells (T_FH), T_H9, T_H22, and T_GM-CSF may constitute unique T_H cell subsets or potentially represent differentiation states of the effector cells described above. Overactive CD4⁺ T_H responses carry the risk of initiating deleterious proinflammatory responses that can result in collateral tissue damage. Thus, the maintenance of immune homeostasis and prevention of immunopathology requires tight regulatory mechanisms of T_reg cells.

FIGURE 1. Transcriptional regulation of CD4⁺ T helper (T_H) cells.

When a naïve CD4⁺ T cell gets activated by an antigen presenting cell, it has a choice to develop into several different functional T_H cells. The decision-making process is influenced by the cytokines in the microenvironment, which activate signal transducers and activators of transcription (STAT) proteins whose primary function is to establish T_H lineage-specific enhancer landscape and induce the expression of master transcription factors. Master transcription factors establish and maintain T_H cell lineage identity. CD4⁺ T_H1 differentiation is under the control of the master transcription factor T-bet, which is induced by the IFN-γ-STAT1 and IL-12-STAT4 signaling pathways. The master transcription factor Gata3 is upregulated in response to IL-4-STAT6 and IL-2-STAT5 signaling, and it is responsible for coordinating the T_H2 differentiation program. T_H9 cells are an exception because their development is not controlled by a single lineage-specifying transcription factor, but rather by a complex network of transcription factors, which ensure Il9 gene expression and suppression of T_H2- and T_reg-cell-specific genes. Proinflammatory cytokines IL-6, IL-21, and IL-23 preferentially activate STAT3, which in conjunction with TGF-β-induced SMADs, induce the expression of RORγt, the master transcription factor of T_H17 cell-specific developmental program. T_FH cell differentiation is controlled by the master transcription factor Bcl6, whose expression is induced by IL-6-STAT3 and IL-21-STAT3 signaling. Differentiation of regulatory T (T_reg) cells is under the control of Foxp3, whose expression is induced and maintained in response to TGF-β-SMAD and IL-2-STAT5 signaling

In a traditionally accepted view of cell fate determination, CD4⁺ T_H differentiation involves several coordinated steps controlled at the transcriptional level: (i) specification, (ii) commitment, and (iii) terminal differentiation.⁴ Lineage specification of recently activated CD4⁺ T cells is initiated by TCR stimulation and cytokine signaling, two signals that lead to the activation of core transcription factors: NFAT-AP-1 or BATF-AP-1-IRF-4 and signal transducers and activators of transcription (STAT) proteins.¹ Initiation of T_H1 cell differentiation is contingent on IFN-γ-mediated STAT1 and IL-12-mediated STAT4 activation, while IL-2 and IL-4 signaling activate STAT5 and STAT6, respectively, to promote T 2 development.^5–11 Proinflammatory cytokines IL-6, IL-21, and IL-23 preferentially activate STAT3 and drive the transcriptional regulation of T_H17 lineage specification, whereas T_reg lineage commitment is dependent on IL-2-STAT5 signaling.^12,13

In addition to promoting targeted chromatin remodeling and establishing the T_H cell-specific enhancer landscape, core transcription factors also induce the expression of master transcription factors that control lineage commitment.¹⁴ Master transcription factors are necessary and sufficient to establish cell identity by coordinating and maintaining established cellular differentiation programs. T-bet, Gata3, RORγt, and Foxp3 are master transcription factors of T_H1,T_H 2, T_H 17 and T_reg differentiation programs, respectively.^15–20 Generally, master transcription factors facilitate lineage commitment by promoting the gene expression program of one T_H lineage while simultaneously repressing the developmental programs of opposing T_H lineages. Transcriptional regulation of T_H cell differentiation programs is further reinforced by a complex network of accessory transcription factors, which cooperate in the fine-tuning of feedforward or cross-inhibitory transcriptional circuits that modulate the duration, magnitude or specificity of CD4⁺ T_H responses.²

In mounting effective host immunity towards diverse microbial pathogens, transcriptional regulation of CD4⁺ T_H cell responses ensures the effective elimination of pathogens, while preventing robust CD4⁺ T cell activity from causing excessive self-damage. Here, we review the current understanding of molecular mechanisms that regulate CD4⁺ T_H cell differentiation and their functional plasticity in health and in the context of immune-mediated diseases.

2 |. TRANSCRIPTIONAL REGULATION OF T_H 1 CELLS

2.1 |. Molecular basis of T_H1 polarization

The immune response activities of CD4⁺ T_H1 cells are largely mediated through the production of their signature cytokine, IFN-γ.²¹ The importance of IFN-γ in the immune system stems from its ability to enhance immunogenicity of tumor cells, directly inhibit viral replication, upregulate MHC Class I and MHC Class II protein expression, activate microbicidal mechanisms in macrophages, and recruit inflammatory cells to the site of inflammation. Thus, through IFN-γ production, T_H1 cells simultaneously regulate multiple facets of immune system activation and immunoregulation. Differentiation of CD4⁺ T cells into IFN-γ-producing T_H1 cells is under the control of T-bet, the master regulator of the T 1 differentiation program.¹⁵ Initially, T-bet is induced in response to TCR stimulation and IFN-γ-STAT1 signaling.^5,6,8 Because T-bet transactivates the Ifng gene, it establishes an IFN-γ-STAT1-T-bet feedforward loop that ensures more IFN-γ and T-bet expression. In this aspect, IFN-γ functions not only as an effector cytokine, but also as an autocrine T_H1-polarizing signal.⁸ Even though IFN-γ is a potent inducer of T-bet, it cannot drive T_H1 differentiation in the absence of IL-12.²² Following termination of TCR signaling and under the influence of IL-2, T-bet, and STAT5 induce the expression of Il12rb (encoding IL-12Rβ), enabling developing T_H1 cells to respond to IL-12. Full stabilization of the polarized T_H1 phenotype is then established by IL-12-induced STAT4 activation, which induces a second wave of sustained T-bet expression.^8,23 Thus, a two-step model of positive feedback regulation ensures that only CD4⁺ T cells which received proinflammatory signals that continue beyond initial antigen-recognition, go on to fully commit to the T_H 1 cell lineage.²⁴ T-bet-mediated transactivation of the Ifng gene is enhanced by accessory transcription factors, Runx3 and HLX, which interact with T-bet to promote heritable T_H 1 gene expression.^25,26 T-bet also controls the expression of genes encoding CXCR3 and chemokines responsible for the mobilization of leukocytes to the site of inflammation.²⁷ Accordingly, T-bet-deficient mice show increased susceptibility to infections with intracellular pathogens due to impaired T_H1 cell differentiation and diminished recruitment of effector cells to the site of challenge.²¹

In addition to promoting the expression of T_H1 cell-specific genes, T-bet reinforces the T_H1 cell differentiation program by concomitantly inhibiting alternative T_H cell differentiation pathways. T-bet accomplishes this either by suppressing the induction of other lineage specifying transcription factors or by interfering with their transcriptional activity.²⁸ For example, T-bet heterodimerizes with the T_FH cell specific master regulator Bcl6 and hijacks its transcriptional repressor activities for effective suppression of alternative helper T cell gene programs.²⁹ T-bet inhibits the T_H2 developmental program by binding directly to the T_H2 cell-specific master transcription factor, Gata3, and preventing it from transactivating T_H 2 cell-specific genes.³⁰ T-bet can also directly repress de novo expression of Gata3 by binding directly to the regulatory region in the Gata3 locus and promoting the deposition of repressive epigenetic marks.³¹ Additionally, T-bet-Runx3 transcriptional complexes silence Il4 gene expression and, thus, prevent expression of the T_H2 cell-polarizing cytokines during T_H1 differentiation.²⁵ Likewise, T-bet effectively inhibits commitment to the T_H17 cell lineage by blocking Runx1-mediated induction of the T_H17 cell-specific master transcription factor, RORγt.³² However, T-bet expression in already committed T_H17 cells results in their conversion into IFN-γ-producing T_H17 cells, which are strong drivers of T_H17 cell-mediated pathology (discussed in below sections).

Although in vitro studies have identified IL-12 and IFN-γ as central cytokine regulators of the T_H1 differentiation program, not all T_H1 cell responses require IL-12 and IFN-γ in vivo. For example, IL-12 is not required for the generation of T_H1 cells following infections with Mycobacterium avium, lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and mouse hepatitis virus (MHV) infections while IFN-γ-STAT1 signaling is not essential for the generation of T_H1 cell responses following Toxoplasma gondii infection.^33,34 These studies suggest that signals other than IL-12 and IFN-γ can instruct differentiation of T_H1 cells in vivo. In this context, it has been shown that microbial products can induce the expression of Delta-like ligands (DLLs) on antigen presenting cells, which upon binding to Notch3 on CD4⁺ T cells promote translocation of the intracellular Notch to the nucleus where it can directly activate the transcription of Tbx21 gene in an recombination-signal-binding protein for immunoglobulin-κ region (RBPJ)-dependent or NF-κB (p50/p65) dependent manner.^35,36

2.2 |. Transcriptional regulation of pathogenic T_H1 cells

Many of the immune-mediated diseases that were previously associated with T_H1 effector cells are, in fact, caused by IL-23-dependent T_H17 cells. In this context, the pathogenicity of T_H17 cells was shown to be tightly linked to the expression of the T_H1-specific master transcription factor, T-bet, and the acquisition of T_H1-specific effector functions. The confusion in the field was caused by the studies which showed that p40-deficiency results in resistance of mice to autoimmunity.³⁷ Since p40 encodes a shared subunit of IL-12 and IL-23, it was not until the generation of p19-deficient mice that it was convincingly established that IL-23 – and not IL-12 – drives autoimmune inflammation and generation of pathogenic T_H17 cells.^38,39 Nevertheless, there are a few diseases that develop due to strong T_H1 cell responses caused by dysregulated expression or transcriptional activity of T-bet (Fig. 2). For example, T-bet expression in T_H1 cells contributes to the pathogenesis of Crohn’s disease.^40–43 Several groups have detected higher T-bet expression and augmented IFN-γ production in the lamina propria of CD4⁺ T cells isolated from patients with Crohn’s disease, but not in those of patients with ulcerative colitis or healthy controls.^41–43 Higher T-bet expression is also detected in mouse models of chronic intestinal inflammation.⁴⁰ Additionally, T-bet-expressing T_H1 cells play a major role in the pathogenesis of autoimmune type 1 diabetes and chronic obstructive pulmonary disease (COPD).^44–46

FIGURE 2. Transcriptional regulation of CD4⁺ T helper (T_H) cells that mediate tissue inflammation.

CD4⁺ T_H cells are a heterogeneous population of effector cells with a significant degree of genetic plasticity that enables them to acquire effector functions and the cytokine profiles of the opposing T_H lineages. Generally, CD4⁺ T_H plasticity is associated with more aggressive CD4⁺ T_H responses and destructive inflammation-associated pathology. For example, T_H17 cells trans-differentiate into T_H1-like T_H17 cells in an IL-23-dependent manner and drive tissue pathology in multiple sclerosis, inflammatory bowel disease, diabetes, and autoimmune arthritis. Similarly, uncommitted T_reg cells, characterized by low Foxp3 and CD25 expression, can convert into IL-17A-producing effector cells and upregulate RANKL expression, which increases their osteoclastogenic activity. Such T_H17-like T_reg cells not only lost their suppressive functions, but also accelerated arthritis pathology. Future studies should be focused to elaborate and functionally characterize transcription factors that regulate the generation and functional plasticity of pathogenic CD4⁺ T_H effector cells

In contrast to other T_H cells, T_H1 cells appear to be terminally differentiated due to the dominant nature of the T-bet-STAT4 and Runx3 transcriptional regulatory network in maintaining the stability of the T_H 1 differentiation program.²¹ For this reason, effective modulation of T_H1-mediated autoimmunity represents a considerable clinical challenge as T_H1 cells may not be as malleable as other T_H subsets to manipulation. Pharmacological targeting of transcription factors is a daunting task since nuclear delivery of small molecule inhibitors is technically challenging to achieve. Furthermore, targeting the master regulator of a type 1 immune response may render patients immunocompromised and susceptible to infections. Careful analyses of immune responses against Toxoplasma gondii and Leishmania major have revealed T_H1 cells that co-express IFN-γ and IL-10 in the late stage of infection.^47,48 The appearance of IFN-γ and IL-10 co-producing T_H1 cells was induced by the parasites, and in the case of T. gondii, IL-10-producing T_H1 cells reduced immunopathology while in the case of L. major they promoted parasite persistence due to the immunosuppressive effects of IL-10 on the maintenance of T_H1 responses.^47,48 Therefore, induction of IL-10 production or exogenous administration of recombinant IL-10 could be the most effective strategy in the treatment of T_H1 mediated pathologies. In this context, a recent study reporting that the transcription factor Bhlhe40 acts a suppressor of the Il10 gene in T_H1 cells is of high significance.⁴⁹ CD4⁺ T cell-specific deletion of Bhlhe40 resulted in the reprograming of proinflammatory T_H1 cells into anti-inflammatory, IL-10-producing T_H1 cells.⁴⁹ Thus, understanding the molecular mechanisms that silence Bhlhe40 gene expression in T_H1 cells may offer new drug targets aimed at limiting T_H1-mediated inflammation.

3 |. TRANSCRIPTIONAL REGULATION OF T_H 2 CELLS

3.1 |. Molecular basis of T_H2 polarization

Shortly after activation in the lymph nodes, newly differentiated CD4⁺ T_H2 cells migrate to the site of parasite invasion, where they initiate protective anti-helminth immune responses primarily through the production of T_H2-associated cytokines: IL-4, IL-5, IL-9, and IL-13.T_H2 cell-derived cytokines have pleotropic effects, which include stimulation of immunoglobulin E secretion by B cells (through IL-4), recruitment of eosinophils and mast cells (through IL-5 and IL-9, respectively), and increased mucus production, smooth muscle cell contractility, and vascular permeability (through IL-4, IL-9, and IL-13). Collectively, these type 2 effector mechanisms culminate in the “weep and sweep” response leading to parasite expulsion.^50–52 Because extensive tissue damage may occur during infection or parasite expulsion, T_H2 cell-specific cytokines also promote tissue repair by inducing differentiation of wound-healing (M2) macrophages, which stimulate myofibroblast activation, angiogenesis, and extracellular matrix deposition.⁵³ However, the same T_H2 cell-specific effector responses, if inappropriately activated or insufficiently controlled during chronic exposure to innocuous allergens, can induce deleterious structural changes in affected tissues, characterized by pronounced collagen deposition and fibrosis. In this context, overzealous T_H2 cells have an established role as key instigators and drivers of immunopathology in asthma, allergy, and atopic dermatitis. Thus, dissecting the molecular basis of T_H2 polarization is important for understanding the pathogenesis of T_H2-mediated diseases and for finding new avenues for therapeutic targeting of T_H2 cells in asthma and allergy.⁵¹

CD4⁺ T_H2 differentiation is initiated following T cell receptor stimulation in response to IL-2 and IL-4 cytokines. Signals emanating from the TCR induce the assembly of core transcription factors BATF-AP-1-IRF4 at the AP-1-IRF4 composite elements (AICE) in the Il4 and Gata3 promoters, leading to the initiation of Il4 and Gata3 expression in CD4⁺ T_H precursors.^54–56 Importance of this transcriptional event is demonstrated by limited IL-4 production and T_H2 differentiation in mice that lack Irf4, c-Maf, and Junb expression.^54,55,57,58 Furthermore, IL-2 signaling activates STAT5 transcriptional activity, which induces Il4 and Il4ra gene expression, thus, increasing the responsiveness of developing T_H2 cells to autocrine and paracrine IL-4 stimulation.^10,11,59 The key function of IL-4 signaling is the activation of a core transcription factor, STAT6, which changes the genetic landscape of T_H2-specific loci and induces expression of the T_H2-specific master transcription factor, Gata3.^9,60,61 Studies demonstrating that ectopic expression of Gata3 is necessary and sufficient to trans-differentiate polarized T_H1 cells into IL-4-producing T_H2 cells and that Gata3-deficiency results in defective T_H2 cell commitment have placed Gata3 on the throne of the T_H 2 differentiation program.^20,62,63 Gata3 orchestrates diverse transcriptional events during T_H2 differentiation, including augmentation of IL-4 expression by binding to the Il4 enhancer.⁶⁴ Increased IL-4 signaling further enhances STAT6-mediated transcription of Gata3, thus generating a positive feedback loop that reinforces Gata3 expression in developing T_H2 cells. Once a critical level of Gata3 is achieved, Gata3 autoactivates its own expression and ensures sustained expression of Gata3 in T_H2 cells even when IL-4 signaling ceases.⁶⁵ Furthermore, Gata3 is solely responsible for transcription of Il5 and Il13 genes, as Gata3-deficient CD4⁺ T cells do not produce IL-5 and IL-13 cytokines.^62,66,67 Stability and maintenance of the T_H2 phenotype is dependent on Gata3-mediated suppression of the T_H1 lineage developmental program by inhibiting Stat4 and Il12rb gene expression, rendering T_H2 cells unresponsive to IL-12 signaling.⁶⁸ Additionally, Gata3 interacts with Runx3 and prevents Runx3-mediated upregulation of Eomesodermin and Ifng genes.⁶⁹ Although Gata3 executes pleotropic functions, it does not act alone during T_H2 differentiation. Accessory transcription factors such as Dec-2 and Tcf1 aid in establishing the genetic program of T_H2 cells by promoting expression of positive regulators of the T_H2 differentiation program (Gata3, Junb, Il2, and Il4) while transcription factors Tcf1, Gfi, and Ikaros inhibit induction of T_H1 and T_H17 cell-specific genes during T_H2 differentiation.^70–75

Although IL-4-STAT6 signaling pathway is essential for T_H2 polarization in vitro, CD4⁺ T_H2 responses can be generated in IL-4Rα-deficient and STAT6-deficient mice under certain inflammatory conditions.^76–80 Analogous to the differentiation of IL-12-STAT4 independent T_H1 cells, studies have demonstrated that the Notch pathway can provide signals for the differentiation of IL-4-STAT6 independent T_H2 cells. The induction of T_H2 cell differentiation depends on RBPJ and Notch1 or Notch2, which directly bind to the Gata3 promoter and a 3′ enhancer of Il4.^35,81,82 Mice that lack either RBPJ or Notch1 and Notch2 expression have impaired T_H2 responses to parasite antigens.⁸² Collectively, these studies demonstrate the importance of the Notch signaling pathway in promoting the generation of cytokine-independent T_H2 cells in vivo.

3.2 |. Transcriptional regulation of pathogenic T_H2 cells

Dysregulated CD4⁺ T_H2 cell responses are responsible for the pathogenesis of asthma, which affects 300 million people worldwide.⁸³ Specifically, CD62L^loCXCR3^lo memory T_H2 cells, which produce excessive amounts of IL-5, and amphiregulin-expressing ST2^hi memory T_H2 cells have recently been shown to drive severe pathology in mouse models of allergic airway inflammation by stimulating eosinophils to produce increased amounts of a key profibrotic factor osteopontin.^84,85 Although T_H2 cell-specific transcription factors generally suppress expression of T_H1-specific transcription factors, CD4⁺ T_H2 cells with intermediate levels of Gata3 express low amounts of T-bet and Eomesodermin, which appear to have important immunoregulatory functions. By binding to Gata3, T-bet sequesters Gata3 away from Gata3 DNA binding sites in the T_H 2 cytokine locus.³⁰ Thus, T-bet-deficient mice develop spontaneous airway changes and exhibit more severe IL-13-induced fibrosis in the lungs than wild-type mice during allergic airway inflammation due to increased expression of T_H 2 signature cytokines.^86,87 Additionally, low levels of Eomesodermin are sufficient to prevent Gata3 binding to the Il5 promoter and inhibit development of IL-5-hyperproducing pathogenic T_H2 cells.⁸⁵ Interestingly, CD62L^loCXCR3^lo memory T_H2 cells, that produce excessive levels of IL-5, have selectively silenced Eomes gene expression.⁸⁵ Pathogenicity of T_H2 cells is further enhanced by their functional plasticity and acquisition of effector functions normally associated with T_H17 cells.^88,89 Such T_H2/T_H17 cells, that co-express Gata3 and RORγt and co-produce T_H2 and T_H17 signature cytokines, are readily detected in the bronchoalveolar lavage samples of patients with severe forms of asthma that is refractory to steroid treatment.⁸⁹ Thus, the transcriptional state of pathogenic T_H2 cells in asthmatic patients is characterized by complete silencing of T_H1 cell-specific transcription factors, T-bet and Eomesodermin, which may or may not be accompanied by upregulation of the T_H17 cell-specific transcription factor, RORγt (Fig. 2).

Uncontrolled persistence of T_H2 cell responses exacerbates the pathology of allergy and asthma. Since even low levels of T_H1 cell-specific transcription factors, T-bet and Eomesodermin, can reduce the pathogenicity of T_H2 cells, one could harness functional plasticity of T_H2 cells to reprogram them into nonpathogenic T_H1-like T_H2 cells. However, a major challenge to this specific therapeutic strategy is the lost sensitivity of T_H2 cells to signaling by the T_H1 inducing cytokine IL-12 due to Gata3-mediated downregulation of Stat4 and Il12rb.^68,90 Although challenging, functional reprograming of unfavorably differentiated T_H2 cells into T_H1-like T_H2 cells can be achieved by simultaneous TCR stimulation and concerted Type I and Type II IFN and IL-12 signaling.⁹⁰ In this context, stable T_H2/T_H1 cells can be maintained in vivo for months and co-existence of T_H2/T_H1 cell differentiation programs within CD4⁺ T_H cells has beneficial effects by providing a fine balance between protective immune responses while preventing immunopathology.⁹⁰ Administration of specific Toll-like receptor (TLR) ligands, such as TLR7 and TLR9 agonists, could be employed to repolarize asthma-inducing T_H2 cells into anti-inflammatory T_H1-like T_H2 cells.⁵¹ Furthermore, pharmacological targeting of T_H2 cell-specific transcription factors, such as Gata3 and STAT6, could have profound beneficial therapeutic effects by preventing differentiation of T_H2 cells with pathogenic effector functions.⁵¹

4 |. TRANSCRIPTIONAL REGULATION OF T_H 9 CELLS

4.1 |. Molecular basis of T_H9 polarization

Mouse studies have provided strong evidence for potent anti-helminth and anti-tumor activity of T_H9 cells, mediated primarily by its signature cytokine IL-9. IL-9 drives the expulsion of Trichuris muris,Nippostrongylus brasiliensis, and Trichinella spiralis by increasing the intestinal muscle contraction, mast cell activation, and epithelial cell mucus production.⁹¹ Adoptive transfer of T_H9 cells is sufficient to protect susceptible Il9-deficient mice from N. brasiliensis infection, demonstrating the importance of T_H9 cell-derived IL-9 in driving anti-helminth immune responses.⁹² Interestingly, T_H9 cells also exhibit effective anti-tumor activity against melanoma and lung adenocarcinoma.^93,94 The anti-tumor activity of T_H9 cells is dependent on IL-3, IL-9, and IL-21.⁹¹ T_H9 cell-derived IL-3 prolongs dendritic cell survival while IL-9 increases the anti-tumor activity of mast cells.^93,95 Additionally, exposure of T_H9 cells to IL-1β results in IRF-1-mediated augmentation of IL-21 secretion from T_H9 cells, which enhances IFN-γ production and the anti-tumor activity of NK cells and CD8⁺ T cells.⁹⁴

It is becoming increasingly clear that in addition to these beneficial activities, T_H9 cells can have detrimental functions in allergic, skin and intestinal inflammatory diseases (Fig. 2). Genetic studies have identified the link between the genes related to T_H9 cell function and increased incidence of asthma and allergies in humans.⁹¹ Mouse studies indicate that T_H9 cells are superior to T_H2 cells in stimulating cytokine production by type 2 innate lymphoid cells (ILC2), promoting mast cell and eosinophil activation and increasing bronchial hyperresponsiveness.⁹¹ Increased IL-9 production and the frequency of circulating T_H9 cells are also detected in patients with atopic dermatitis.⁹⁶ Neutralization of IL-9 abrogates the ability of T_H9 cells to induce atopic disease in mice, suggesting that T_H9 cells promote disease primarily through IL-9 production.^97,98 Likewise, there is a strong association between enhanced T_H9 responses and a more severe form of inflammatory bowel disease (IBD).^96,99,100 In mice, T_H9 cells are sufficient to cause severe colitis following the adoptive transfer while Il9 deficiency or neutralization of IL-9 effectively reduces intestinal pathology.⁹⁹ Functionally, it was demonstrated that T_H9 cell-derived IL-9 disrupts the epithelial cell barrier functions and impairs mucosal wound healing in vivo.⁹⁹ Thus, T_H9 cells have a strong colitogenic potential to drive intestinal inflammation.

A complex regulatory network of transcription factors controls T_H9 cell differentiation. None of the following transcription factors are T_H9-specific; however, their combinatorial effects culminate into a unique T_H9 gene expression program that shares very few similarities with T_H1, T_H2, T_H17, and T_reg cells. Differentiation of IL-9-secreting T_H cells is dependent on IL-2-STAT5, IL-4-STAT6, and TGF-β-signaling.⁹¹ IL-2 promotes T_H9 differentiation by activating STAT5, which binds directly to the Il9 promoter and blocks the binding of the T_FH-specific transcription factor Bcl6.¹⁰¹ Accordingly, loss of IL-2 or pharmacological inhibition of STAT5 transcriptional activity results in impaired T_H9 differentiation.^101,102 IL-4 and STAT6 promote T_H9 differentiation by blocking the generation of TGF-β-induced Foxp3⁺ T_reg cells and T-bet-expressing T_H1 cells.^103,104 Additionally, IL-4-STAT6 signaling induces the expression of transcription factors BATF and IRF4, both of which bind directly to the Il9 locus and coordinate the expression of STAT6-regulated genes in T_H9 cells.^97,105 Genetic deletion of STAT6, IRF4, or BATF results in impaired differentiation of T_H9 cells and increased expression of T 1 cell-associated genes.^97,103,105 Since IL-2-STAT5 and IL-4-STAT6 signaling pathways drive T_H2 cell differentiation, TGF-β is critically important for the suppression of T_H2 cell-specific genes in T_H 9 cells.^104,106 Furthermore, TGF-β-activated SMAD2/3/4 transcription factors can promote T_H9 cell differentiation by binding independently or in a complex with IRF4 to the Il9 locus. Another critical function of TGF-β is induction of the transcription factor PU.1.¹⁰⁷ PU.1 deficiency in CD4⁺ T cells results in markedly impaired IL-9 production, whereas ectopic expression of PU.1 in either T_H2 or T_H9 cells increases IL-9 production.¹⁰⁷ Mechanistically, PU.1 binds directly to the Il9 promoter and recruits the histone acetyltransferase GCN5 to induce Il9 promoter activity.¹⁰⁸ Recently, three independent studies have reported a novel function of the transcription factor Foxo1 in regulating the development of T_H 9 cells.^109–111 Activation of the PI3K/AKT pathway results in Foxo1 upregulation in CD4⁺ T cells, in which Foxo1 directly induces IRF4 and IL-9 expression while simultaneously inhibiting the expression of T_H17-cell specific genes.¹¹⁰

Since none of the aforementioned transcription factors are uniquely expressed in T_H9 cells, the differentiation of T_H9 cells is not under the control of a defined lineage-specifying master transcription factor, but rather an end-product of synergistic and antagonistic actions of multiple transcription factors, which ensure Il9 gene expression and concomitant suppression of alternative T_H fates.

4.2 |. Transcriptional regulation of pathogenic T_H9 cells

Since proinflammatory functions of T_H9 cells map to the expression of IL-9, transcription factors which transactivate Il9 gene directly support the development of pathogenic T_H9 cells. As expected, deficiency of IRF4, BATF, PU.1, or Foxo1 in CD4⁺ T cells results in ameliorated T_H9 dependent asthma, allergic inflammation, and colitis.^{97,99,105,107,110,111} Additionally, administration of neutralizing IL-9 antibody protected the recipient mice from T_H9-mediated pathologies, demonstrating that IL-9-targeted therapy may be particularly successful in limiting T_H9 driven inflammatory responses.

5 |. TRANSCRIPTIONAL REGULATION OF T_H 17 CELLS

5.1 |. Molecular basis of T_H17 polarization

Although T_H17 cells can be induced in multiple tissues under different inflammatory conditions, in the steady state, they are most commonly found at barrier sites such as intestine, lung, and skin. Here, T_H17 cells play an important role in providing protective barrier functions against fungal and bacterial pathogens and for maintaining host–commensal homeostasis. The prototypic barrier functions of T_H17 cells are mediated by the production of T_H17 cell signature cytokines (IL-17A, IL-17F, IL-21, and IL-22), which induce a broad range of tissue responses including the expression of anti-microbial peptides (lipocalin-2, Reg3β, Reg3γ, S100A8, S100A9), proinflammatory cytokines (IL-1β, IL-6,TNF-α, GM-CSF), CXC chemokines (CXCL8, CXCL1, CXCL2, CXCL10), and metalloproteases.¹¹² Accordingly, T_H17 cell-mediated effector mechanisms are indispensable in providing protection against Klebsiella pneumoniae, Staphylococcus aureus, Propionibacterium acnes, Bacteroides spp., Borrelia spp., Candida albicans, and Pneumocystis carinii.¹¹³ The broad distribution of IL-17R and IL-22R results in a massive tissue response to T_H17 cell-derived effector cytokines, which may explain the prominent ability of T_H17 cells to cause severe tissue inflammation.¹¹³ Indeed, T_H17 cells have emerged as an important mediator in inflammatory and autoimmune diseases. Strong clinical evidence points to the pathogenic functions of T_H17 cells in psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma.¹¹³ Systems biology approaches have been instrumental in identifying novel transcriptional networks that regulate the differentiation of protective and pathogenic T_H17 cells.^114,115

Because both T_H17 cells and T_reg cells require TGF-β for their development, the T_H17 cell transcriptional program is functionally coupled to the transcriptional program of regulatory (T_reg) cells, a feature unique to the T_H17 lineage.^116–120 When activated in the presence of TGF-β, CD4⁺ T cells transiently co-express two antagonizing transcription factors, Foxp3 and RORγt, which direct the differentiation program of regulatory T (T_reg) cells and T_H17 cells, respectively.^{16,17,121,122} From this intermediate Foxp3⁺RORγt⁺ stage, a CD4⁺ T cell has the potential to develop into either the T_reg or the T_H17 lineage. Their ultimate fate is determined by the relative abundance of TGF-β and IL-6 in the microenvironment. At high TGF-β concentrations, Foxp3 inhibits RORγt transcriptional activity, thereby tipping the balance away from the T_H17 transcriptional program towards T_reg cell differentiation. At low TGF-β concentrations, TGF-β synergizes with signals initiated by IL-6, which relieves RORγt from Foxp3-mediated suppression through STAT3 transcriptional activity.^121–123 Thus, IL-6-mediated activation of STAT3 and RORγt is critical for the initiation of the T_H17 lineage specification. Another important aspect of IL-6-STAT3 signaling is the induction of IL-21, which, in a STAT3-dependent manner, induces its own transcription and promotes the expression of the IL-23 receptor. IL-23, produced by activated antigen presenting cells, acts in synergy with IL-6, IL-21, and TGF-β to amplify RORγt-dependent terminal differentiation of T_H17 cells.^13,124 An extensive network of transcription factors, including IRF4-AP-1-BATF transcriptional complexes, IκBζ, Runx1, Ahr, and HIF-1α, is activated to stabilize the T_H17 cell transcriptional program by ensuring optimal induction of RORγt or by promoting RORγt transcriptional activity at T_H 17 cell-specific loci.^125–127

Neither IL-6 nor IL-23 alone efficiently generate T_H17 cells in the absence of TGF-β, demonstrating the importance of TGF-β signaling in murine T_H17 cell differentiation. However, a combination of IL-6, IL-23, and IL-1β effectively supports the development of TGF-β-independent T_H17 effector cells with enhanced pathogenic potential.¹²⁸ These findings point to the alternative modes of T_H17 cell differentiation in vivo, which can contribute to the heterogeneity of T_H17 cells and their differing abilities to cause autoimmune pathologies.¹²⁸

5.2 |. Transcriptional regulation of pathogenic T_H17 cells

CD4⁺ T cells activated in the presence of IL-6 and TGF-β differentiate into IL-10-producing T_H17 cells that lack pathogenic potential.¹²⁹ For T_H17 cells to become pathogenic, they must receive IL-23 signals or be differentiated in the presence of alternative cytokine combinations such IL-6, IL-1β, and IL-23.^115,128,130 IL-23R signaling results in the repression of anti-inflammatory IL-10 production and induction of genes within the “proinflammatory transcriptional module” of T_H17 cells.^115,129 Thus, transcription factors that control IL-23R expression will also affect the pathogenic potential of T_H17 cells. In addition to STAT3, the canonical Notch signaling mediator, RBPJ, was shown to directly enhance expression of the IL-23R and pathogenicity of T_H17 cells.¹³¹

Interestingly, propagation of committed T_H17 cells in the presence of IL-23 but without TGF-β induces the expression of the T_H1-specific master transcription factor T-bet and promotes the emergence of IFN-γ-producing T_H17 cells.^128,132 Indeed, under chronic inflammatory conditions during experimental autoimmune encephalomyelitis, inflammatory bowel disease, autoimmune arthritis, and diabetes, T_H17 cells trans-differentiate into T_H1-like T_H17 cells in an IL-23 dependent manner and drive tissue pathology (Fig. 2).^132–137 Transition of T_H17 cells into T_H1-like effector cells is critically dependent on the transcription factors T-bet, Runx1, and STAT4. Accordingly, T-bet, Runx1, or STAT4 deficiencies impair the development of T_H1-like T_H17 cells and protect the mice from autoimmunity.^135,138 However, not all T_H17-mediated diseases are driven by the functional plasticity of T_H17 cells. For example, T_H17 cells exhibit exceptional phenotypic stability in the lungs, where they contribute to allergen-induced airway hyper-responsiveness by promoting neutrophilic airway inflammation and enhancing airway smooth muscle contractions.^139–141 Similarly, psoriasis, a chronic inflammatory disease of the skin, is caused specifically by the T_H17 cell-derived cytokine, IL-22. In psoriasis, IL-23 stimulates T_H17 cells to secrete IL-22, which promotes dermal inflammation through STAT3 activation in keratinocytes.¹⁴² The pathology of dry eye disease, autoimmune uveitis, and scleritis is also driven by memory T_H17 cells.^143–146 Neutralization of IL-17A and IL-27/STAT1-mediated antagonism of the T_H17 cell phenotype have demonstrated significant therapeutic benefits in the treatment of eye inflammation.^143–145

IL-1β plays a unique and nonredundant role during differentiation of pathogenic T_H17 cells. IL-1R signaling is essential for T_H17 cell production of GM-CSF, which is the only cytokine proven to be critical for the encephalitogenicity of T_H17 cells in the experimental autoimmune encephalomyelitis.^147,148 IL-1β strongly induces the expression of the transcription factor Bhlhe40, which acts as a transcriptional activator of Csf2 (encoding GM-CSF) in T_H17 cells. Additionally, Bhlhe40 represses IL-10 production by T_H17 cells, demonstrating the importance of this IL-1β-induced transcription factor in regulating T_H 17 cell pathogenicity.^149,150 IL-1R expression is under strict negative control of a transcriptional repressor, Foxo1. Therefore, downregulation of Foxo1 by the miR-183-96-182 cluster is required to unleash the full pathogenic potential of T_H17 cells in response to IL-1 signaling.¹⁵¹

Given the strong association of T_H17 cells with multiple immunemediated diseases, it is not surprising that a substantial amount of research has been devoted to devising novel therapeutic strategies that can target T_H17 differentiation. Since T_H17 cells also have barrier protective functions, it is important that new therapies do not interfere with beneficial functions of T_H17 cells that safeguard the intestine from potential pathogens.¹¹² Although T_H17 cell functional plasticity has been linked to T_H17 cell pathogenicity, one could exploit this property of T_H17 cells as a therapeutic opportunity for the treatment of inflammatory diseases.¹⁵² Indeed, T_H17 cells can be redirected to adopt immunoregulatory functions and contribute to the resolution of intestinal inflammation in response to Ahr and TGF-β signaling.¹⁵² T_H17 cells can also deviate towards T_FH cell phenotype in Peyer’s patches where they support the generation of high-affinity antigen-specific IgA responses. In this way, T_H17 cell-derived T_FH cells contribute to the maintenance of the host/commensals equilibrium and constitute an effective arm of mucosal immune responses in the gut.¹⁵³ Understanding the precise molecular mechanisms that regulate T_H17 cell genetic and functional plasticity will broaden the repertoire of therapeutic approaches, which can harness T cell plasticity for the benefit of patients.¹⁵⁴

6 |. TRANSCRIPTIONAL REGULATION OF T_FH CELLS

6.1 |. Molecular basis of T_FH polarization

T_FH cells can be distinguished from all other T_H subsets by elevated IL-21 production and high expression of surface receptors that are critical for their development and function, including CXCR5, PD-1, ICOS, CD40L, BTLA, and CD84.¹⁵⁵ T_FH cells control humoral immunity by supporting the formation of germinal centers in which T_FH cells provide help to cognate B cells. Productive T_FH–B cell interactions result in the development of long-lived, antigen-specific memory B cells and plasma cells. Proper regulation of T_FH differentiation is necessary to enable generation of high-affinity neutralizing antibodies following vaccinations and infections; however, dysregulated T_FH responses can lead to pathogenic autoantibody production and autoimmunity. Aberrant activation of T_FH cells and increased autoantibody production are frequently observed in patients with systemic lupus erythematosus (SLE), Sjogren’s syndrome, rheumatoid arthritis, autoimmune thyroid diseases, type 1 diabetes, multiple sclerosis, neuromyelitis optica, Guillain-Barré syndrome, myasthenia gravis, and idiopathic thrombocytopenic purpura.¹⁵⁶ Therefore, investigations of transcriptional mechanisms that control generation of T_FH cells will provide us with a unique opportunity to identify molecules that could be targeted to attenuate T_FH responses for the treatment of immunological diseases caused by excessive autoantibody production.¹⁵⁵

Differentiation of T_FH cells is a multistage process spanning three spatiotemporal phases: initial priming, T-B border commitment phase, and germinal center phase (reviewed in Ma et al.,¹⁵⁵ Crotty,¹⁵⁷ Qi,¹⁵⁸, and King¹⁵⁹). The initial priming of naïve CD4⁺ T cells occurs in T cell zones of lymphoid organs after recognition of peptide-MHC Class II complexes on dendritic cells. Expression of high-affinity T cell-receptor or activation of CD4⁺ T cells in the presence of high doses of antigen favors the differentiation of T_FH cells.^157,160,161 The initiation of T_FH differentiation program is dependent on ICOS-ICOSL and IL-6-STAT3 signaling pathways, which induce Bcl6, c-maf and IL-21 expression in T_FH precursors.^162–165 IL-6 and T_FH cell-derived IL-21 ensure continuous STAT3 activation necessary for the maintenance of T_FH phenotype during different stages of differentiation.^163,165 These initial interactions between dendritic cells and T_FH precursors also produce phenotypic changes characterized by CXCR5 upregulation and CCR7 downregulation, which license T_FH precursors to move out of the T cell zone towards T cell–B cell border where the critical B cell dependent phase of T_FH differentiation occurs.¹⁶⁶ A continuous dialogue between T_FH cells and B cells, mediated by TCR-MHC Class II,ICOS-ICOSL, CD40-CD40L, and SAP-SLAM interactions, further stabilizes Bcl6 expression in the intermediate T_FH cells. After entry into the B cell follicle, T_FH cells complete their differentiation program in a B cell and ICOS-dependent manner.¹⁶² At this stage, germinal center T_FH cells acquire their most polarized state characterized by the highest level of Bcl6, CXCR5, PD-1, ICOS, and SAP expression.¹⁵⁷ Here,CXCR5^hiPD-1^hiBcl6^hi T_FH cells are needed to maintain germinal centers and facilitate differentiation of germinal center B cells into plasma cells and memory B cells.¹⁵⁷

Although Bcl6 is the master regulator of the T_FH cell differentiation program, the development of T_FH cells requires hierarchical and integrated functions of numerous transcription factors.^{155,167–169} STAT3-activating cytokines, IL-6 and IL-21, induce the expression of BATF, which subsequently binds to and transactivates Bcl6 and c-maf genes.^170,171 While primary function of c-maf is to induce Il21 expression, Bcl6 employs multiple mechanisms to imprint the T_FH phenotype. Specifically, Bcl6 suppresses expression and/or function of transcriptional regulators of alternative T_H lineages (T-bet, Gata3, and RORγt), Blimp-1, and the miR-17–92 cluster, which represses CXCR5 expression.^167–169

6.2 |. Transcriptional regulation of pathogenic T_FH cells

Resolution of the germinal center response after pathogen clearance is essential for the prevention of autoimmunity. Aberrant expression of T_FH cell-associated cytokines and co-stimulatory molecules, particularly IL-21 and ICOS, has been directly linked to the development of lupus.¹⁷² Antibody-mediated neutralization of IL-21 or Il21r gene deficiency in different strains of lupus-prone mice significantly ameliorated autoimmune pathology, suggesting that IL-21-targeted therapies have a great therapeutic potential in terminating T_FH-driven inflammatory processes.¹⁷² In mice, homozygosity for the “san” allele of Roquin, which encodes a post-transcriptional negative regulator of Icos, results in increased accumulation of T_FH cells, spontaneous germinal center formation and lupus pathology.¹⁷³ The pathogenic accumulation of T_FH cells in Roquin^san/san mice is caused by excessive IFN-γ signaling, which promoted Bcl6 overexpression and hyperproliferation of T_FH cells, suggesting that elevated IFN-γ production during infections carries a risk of triggering germinal center-driven autoimmunity.¹⁷³

CD4⁺ T cells isolated from the salivary glands of Sjogren’s syndrome patients express bona fide T_FH phenotype, characterized by high expression of BCL6, CXCR5, ICOS, and IL-21.¹⁷⁴ Similarly, increased frequency of PD-1^hiCXCR5⁺ T_FH-like cells was detected in the circulation of multiple sclerosis patients during the relapse of disease.¹⁷⁵ Using the experimental autoimmune encephalomyelitis model of multiple sclerosis, the authors have shown that CXCR5⁺ T_FH-like cells and B cells infiltrate the CNS parenchyma where they form organized ectopic lymphoid structures. In these structures, T_FH-like cells are juxtaposed with mature IgD⁺ B cells and CD138⁺ plasma cells, suggesting that ectopic lymphoid structures may be the precise location of continuous T_FH-like cell–B cell interactions driving a germinal center-like response and chronic inflammation.¹⁷⁵ In contrast, CD4⁺ T cells isolated from the synovium of rheumatoid arthritis patients expressed some, but not all, T_FH-specific markers (PD-1^hiICOS⁺SAP⁺MAF⁺ IL-21⁺CXCR5⁻BCL6⁻).¹⁷⁶ These pathogenic PD-1^hiCXCR5⁻ CD4⁺ T cells provided B cell help in an IL-21 and SLAMF5-dependent manner.¹⁷⁶ Although they have demonstrated helper functions normally associated with T_FH cells, peripherally-expanded pathogenic T_FH-like cells were negative for BCL6 expression,¹⁷⁶ suggesting that they may not be susceptible to the negative regulation by the IL-2-STAT5-Blimp1 pathway like bona fide T_FH cells.^177,178 In this case, antibody-mediated blockade of PD-1, SLAMF5, and IL-21 may be the only therapeutic strategy for targeting tissue-specific T-B cell interactions.¹⁷⁶

7 |. TRANSCRIPTIONAL REGULATION OF T_reg CELLS

7.1 |. Molecular basis of T_reg polarization

The “central tolerance” purging mechanisms of autoreactive T cells are imperfect, and a certain fraction of self-reactive T cells will escape thymic negative selection. The presence of self-reactive T cells in the circulation is a constant threat to the host. Aberrant activation of self-reactive T cells will inevitably result in fatal autoimmunity unless such a response is suppressed by regulatory T (T_reg) cells.¹⁷⁹ Like-wise, activated CD4⁺ T_H cell responses may provoke an overexuberant, deleterious inflammatory cascade, unless the magnitude or duration of such a response is curtailed by T_reg cells. Therefore, the primary function of CD4⁺ T_reg cells is to maintain immunological homeostasis by suppressing excessive and destructive immune responses against innocuous, self and foreign antigens. T_reg cells can be grouped into thymic (natural) T_reg cells or peripheral (induced) T_reg cells, depending on where they originate. Thymic selection of T_reg cells requires strong TCR (upon recognition of MHC Class II-restricted self-peptides), CD28 co-stimulation and IL-2-STAT5 signaling.¹² Gut-associated lymphoid tissue is a preferential site for the differentiation of peripheral T cells, where CD103⁺ reg dendritic cells induce T_reg cell differentiation via a mechanism that is dependent on TGF-β and retinoic acid signaling.^180,181

Development of thymic and peripheral T_reg cells remains under the control of the master transcription factor, Foxp3, which plays a critical role in the establishment and maintenance of T_reg lineage identity and suppressor functions.^18,182–185 Ectopic expression of Foxp3 in CD4⁺ T cells is sufficient to endow them with T_reg-suppressive functions, and conversely, genetic deletion of Foxp3 results in the loss of T_reg cells and fatal autoimmunity.^16,18,182 Because proper functioning of the immune system requires balanced activation of CD4⁺ T_H effector responses, Foxp3 expression must be stringently regulated. A network of transcription factors, including Smad3, NFAT, c-Rel, Foxo1, Foxo3, Runx1, Stat5, CREB, and the Nr4a family of transcription factors, binds to regulatory regions in the Foxp3 gene locus; ensuring the timely, stable, and heritable cell-restricted expression of Foxp3 in T_reg cells (reviewed in Lee and Lee¹⁸⁶). Rather than generating a T_reg-specific chromatin landscape, Foxp3 exploits a preformed network of accessible enhancers established by TCR signaling for promoting T_reg cell differentiation.¹⁸⁷ Only a small fraction of genes (~6%) in the entire Foxp3-dependent gene expression program is directly regulated by Foxp3, while the expression of the remaining genes is controlled indirectly through interactions with other transcription factors.¹⁸³ Thus, Foxp3 forms transcriptional complexes with Eos, Runx1, and NFAT to activate (Il2ra, Ctla4) or repress (Il2, Ifng) target gene expression.^188–190

Clearly, maintaining T_reg cell identity is critically important for immune homeostasis. Because the TCR repertoire of thymic T_reg cells is heavily biased toward autoreactivity,¹⁹¹ conversion of T_reg cells into inflammatory effector cells could have dire consequences. Several transcriptional mechanisms are put in place to ensure that T_reg cells are phenotypically and functionally stable under inflammatory conditions. In addition to regulating the induction of Foxp3 gene expression,Smad2/3 and Foxo1-mediated control of T_reg cell specification includes active suppression of T-bet and IFN-γ.^192–194 The transcription factor, musclin, promotes the unidirectional development of peripheral T_reg cells by suppressing the T_H 2 transcriptional program,¹⁹⁵ while the transcriptional repressor Bach2 stabilizes T_reg lineage identity by antagonizing differentiation programs of multiple T_H effector subsets.¹⁹⁶

Nevertheless, a certain degree of phenotypic and functional heterogeneity within the T_reg population must be permitted for proper immunoregulation of qualitatively different types of inflammation induced by distinct T_H1, T_H2, and T_H17 effector subsets. For example, a single gene deletion of either T-bet or Gata3 specifically in T_reg cells does not affect T_reg cell functionality and immune homeostasis in the steady state; however, combined deletion of T-bet and Gata3 leads to the loss of Foxp3 expression, T_reg cell instability and spontaneous autoimmune lymphoproliferative disease.¹⁹⁷ Several elegant studies have demonstrated that during polarized T_H1-type inflammatory responses, T_reg cells selectively upregulate T-bet expression, which is necessary for efficient T_reg cell trafficking, proliferation, and survival.^19,198 Following selective depletion of T-bet⁻ T_reg cells, the remaining T-bet-expressing T_reg cells were fully sufficient to control T_H 1 and CD8⁺ T cell responses while specific elimination of T-bet-expressing T_reg cells resulted in severe T_H1 autoimmunity.¹⁹ Similarly, in an autoimmune model of diabetes, CXCR3⁺ T-bet-expressing T_reg cells played a crucial role in controlling autoimmune inflammation in the pancreas in a manner that could not be compensated by T-bet⁻ T_reg cells.¹⁹⁹ These studies demonstrate that T_reg cells may employ certain components of the T-bet/T_H1-specific transcriptional machinery to fine-tune their functional properties under type 1 response inflammatory conditions.¹⁹ Similarly, T_reg cell expression of IRF4, a transcription factor essential for T_H2 effector cell differentiation, is required for T_reg-mediated suppression of T_H 2-type inflammatory responses,²⁰⁰ while the control of pathogenic T_H17 cells is dependent upon T_reg cell-restricted expression of STAT3, a transcription factor that controls T_H17 effector cell differentiation.²⁰¹ Furthermore, individual intestinal symbionts induce a distinct population of RORγt-expressing T_reg cells, which contribute substantially to the regulation of intestinal T_H2- and T_H1/T_H17 mediated inflammatory responses.^202,203 Development and immune suppressive functions of RORγt-expressing T_reg cells are dependent on the expression of another transcription factor c-maf, which integrates TGF-β receptor signals with pathobiont-induced cytokine dependent STAT3 activation to promote the differentiation of RORγt⁺ T_reg cells.²⁰⁴ From these studies, a model has emerged in which selective expression of T_H1, T_H2-, and T_H17-specific transcriptional regulators is necessary for functional and phenotypical specialization of T_reg cells that are better equipped to restrain specific types of CD4⁺ T cell responses.²⁰⁵

7.2 |. Transcriptional regulation of pathogenic T_reg cells

Recent studies have challenged the stability of the T_reg cell lineage, demonstrating that T_reg cells could be fully converted into inflammatory T_H effector cells and become major drivers of immunopathology (Fig. 2). For example, CD25^loFoxp3⁺ T_reg cells isolated from the pancreas of diabetic mice have an activated memory phenotype, express T_H1-like effector functions, and upon adoptive transfer into näive recipient mice cause a rapid onset of autoimmune type 1 diabetes, demonstrating that pathogenic ex-T_reg cells could be generated under certain inflammatory conditions.²⁰⁶ In the context of food allergic responses, augmented IL-4R-STAT6 signaling is sufficient to promote the reprogramming of T_reg cells towards a T_H2-cell-like lineage. Consistent with this observation, peripheral T_reg cells isolated from children with food allergies demonstrate increased expression of T_H2-specific transcription factors (Gata3 and IRF4) and T_H2 cellassociated cytokines.²⁰⁷ T_reg conversion into T_H2-cell-like lineage was critical for the development of food allergy and could be reversed by the blockade of IL-4/IL-4Rα signaling pathway in T_reg cells.²⁰⁷ Additionally, in rheumatoid arthritis patients, CD4⁺ T cells isolated from the inflamed joints of patients are characterized by co-expression of Foxp3, RORγt, and IL-17A.²⁰⁸ Fate-mapping studies have revealed that a substantial proportion of arthritogenic CD4⁺ T_H17 cells originated from CD25^loFoxp3⁺ T_reg cells.²⁰⁸ A local IL-6-rich inflammatory milieu, created by joint synovial fibroblasts, shifts the balance, not only by promoting de novo T_H17 differentiation, but also by converting existing Foxp3⁺ T_reg cells into IL-17A-producing effector cells. Interestingly, the T_reg-derived T_H17 cells express high levels of RANKL, a central cytokine for osteoclast activation and differentiation, and are more potent osteoclastogenic T cells than de novo differentiated T_H17 cells.²⁰⁸ Thus, CD25^loFoxp3⁺ T_reg cells fail to suppress collagen induced arthritis in mice and accelerate arthritis pathology more efficiently, probably due to their higher degree of self-reactivity. Collectively, these studies demonstrate that under specific inflammatory conditions, Foxp3⁺ T_reg cells can give rise to pathogenic T_H1, T_H2, and T_H17 cells.

While expression of T_H1-, T_H2-, and T_H17-specific transcriptional regulators promotes functional specialization of T_reg cells that improves their immunoregulatory abilities, in other contexts, expression of the same transcriptional regulators can transform T_reg cells into aggressive effector T_H cell subsets. This paradox has raised the question of why certain Foxp3⁺ T_reg cells cannot maintain their lineage identity under inflammatory conditions. Genetic fate-mapping approaches have revealed that there is a degree of heterogeneity within T_reg cell population with respect to the level of Foxp3 and CD25 (IL-2Rα subunit) expression.^206,208,209 Fully committed T_reg cells, defined by high expression of Foxp3 and CD25, will remain stable and retain their suppressive functions in inflammatory environments, whereas uncommitted T_reg cells, which express low or transient expression of Foxp3 and CD25, will acquire effector functions and become pathogenic.^206,208,209 The availability of IL-2 and IL-2/STAT5 signaling is critically important for the maintenance of Foxp3 expression.²¹⁰ Inevitably, immune system activation will result in limited IL-2 availability due to IL-2 consumption by CD4⁺ T_H effector cells. This turned out to be the key destabilizing factor of T_reg cells in a strong T_H1-polarizing environment following Toxoplasma gondii infection.²¹¹ Importantly, the collapse of T_reg cells can be rescued by IL-2 treatment, which stabilizes Foxp3 and CD25 expression in T_reg cells.^211,212 Thus, therapeutic approaches aimed at inducing or transferring T_reg cells during the onset of autoimmune disease should consider administration of biologics that will shift the cytokine balance in favor of T_reg cells. For instance, administration of IL-2 with concomitant blockade of IL-6 signaling might allow for the stabilization of Foxp3⁺ T_reg cells in tissue during an ongoing pathogenic T_H 17 cell response.²⁰⁸

8 |. CONCLUDING REMARKS

Originally, CD4⁺ T_H cell differentiation was viewed as a linear and irreversible event whereupon each T_H cell subset expressed its lineage specific transcription factors and unique signature cytokines. This functional specialization was deemed to be a necessary adaptation of the immune response to ensure that the appropriate effector mechanisms of each CD4⁺ T_H subset are tailored to respond to a specific challenge. However, with the emergence of more sophisticated technologies, it became apparent that CD4⁺ T_H cells are a heterogeneous population of effector cells with a significant degree of genetic plasticity that enables them to acquire effector functions and the cytokine profiles of the opposing T_H lineages. Generally, CD4⁺ T_H plasticity is associated with more aggressive CD4⁺ T_H responses and destructive inflammation-associated pathology. In this perspective, CD4⁺ T_H17 cells have been notoriously detrimental; responsible for causing and sustaining tissue damage in various models of organ-specific autoimmunity. Despite the indisputable role of T_H17 cells in driving chronic inflammation, a complete knowledge of what makes T_H17 cells so destructive is lacking. There is a substantial gap in our understanding of the origin of pathogenic T_H17 cells. Are all T_H17 cells capable of acquiring pathogenic effector functions if they receive appropriate signals in a specific tissue microenvironment, and is this phenotype reversible? Are there two different functional subsets of T_H17 cells – protective T_H17 cells at mucosal sites and pathogenic T_H17 cells as key drivers of autoimmunity? With these questions in mind, it will be necessary to further elaborate and functionally characterize transcription factors that regulate the generation and functional plasticity of pathogenic CD4⁺ T_H effector cells. Furthermore, ongoing developments in understanding the transcriptional regulation of CD4⁺ T_H responses will foster new translational opportunities for therapeutic interventions in the treatment of immune-mediated diseases.