The increased IL-17A production by CD4⁺ T cells from T cell receptor (TCR) transgenic DO11.10 mice was first observed by Infante-Duarte and colleagues in 2000 [1]. The transgenic TCR recognizes ovalbumin (OVA) peptide 322-339 in association with I-A^d. When stimulated with OVA_322-339 in the presence of cell lysates of Borrelia burgdorferi, these CD4⁺ Th cells preferentially produce IL-17A along with tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytokine profile produced by these CD4⁺ Th cells is distinctive from those by Th1 or Th2 cells. Interleukin 6 (IL-6) was shown to have a similar effect to cell lysates of Borrelia burgdorferi in inducing IL-17A production by these CD4⁺ Th cells. In 2003, Aggarwal et al. [2] showed that IL-23 preferentially promotes IL-17A and IL-17F production by activated CD4⁺ T cells in vitro suggesting that during a secondary immune response IL-23 can promote a distinct differentiation status from well-characterized Th1 and Th2 cells. In an experimental autoimmune encephalomyelitis (EAE) model, Cua and colleagues demonstrated that it is IL-23 rather than IL-12 mediating autoimmune inflammation of the brain [3]. Furthermore, they provided in vivo evidence that IL-23 drives a pathogenic T cell population mediating inflammation in EAE and arthritis models [2, 4]. These pathogenic CD4⁺ T cells produce IL-17A, IL-17F, IL-6, and TNF. The term “Th17 cells” was proposed in 2005 by two independent groups led by Dong and Weaver, respectively [5, 6], to describe a Th cell lineage, which is distinctive from Th1 and Th2 cells. A Th17 cell is governed by its master transcription factor, retinoic acid receptor-related orphan nuclear receptor (ROR)-γt [7]. The discovery of Th17 cells heralded a major shift in T cell biology and our understanding of the mechanism of autoimmune inflammatory diseases. Cytokines produced by Th17 cells include IL-17A, IL-17F, IL-22, GM-CSF, IL-21, and TNF depending on inflammatory settings [8]. Those autoimmune diseases thought to be mediated by Th1 cells are now recognized to be mediated mainly by Th17 cells [9].

It has been clearly demonstrated by in vitro experiments that several cytokines are involved in promoting differentiation and development of Th17 cells. These include IL-6, IL-1, TNF, GM-CSF, transforming growth factor (TGF)-β, and IL-23. IL-6 is one of the cytokines involved in the initiation of CD4⁺ T cell differentiation toward Th17 cells (see Fig. 3.1) [10–12] in the presence of TGF-β.

As described above, IL-6 can replace lysates of Borrelia burgdorferi to promote OVA_322-339-specific CD4⁺ Th cells to produce IL-17A [1]. This suggests that IL-6 drives activation of a unique pathway leading to expression of IL-17A by CD4⁺ Th cells. Subsequent experiments indicate that IL-6 is essential in this process by activating signal transducers and activators of transcription (STAT)-3, which directly activates RORγt [13]. STAT-3 also suppresses TGF-β-induced forkhead box P3 (FOXP3) expression and thereby inhibits the CD4⁺ Th differentiation toward T regulatory (Treg) cell lineage [13, 14]. IL-6 also promotes IL-23 receptor expression by these CD4⁺ T cells (Fig. 3.1) and thereby potentiates these Th17 cells to receive signals from IL-23 [14, 16]. The essential role of IL-6 in generating Th17 cells is confirmed in vivo. Activation of STAT3 by inflammatory stimulus is impaired in IL-6 gene knockout mice, fails to develop Th17 cells, and is protected from development of EAE [16] and collagen-induced arthritis (CIA) [17, 18].

IL-1β also has a crucial role in the initiation of Th17 cell differentiation. IL-1R1 knockout mice fail to develop antigen-specific Th17 cells and do not develop EAE [19]. IL-1R1 expression on Th17 cells is induced by IL-6. IL-1 signaling promotes the transcription factor interferon-regulatory factor 4 (IRF4), which further reinforces the expression of RORγt [20]. Mammalian target of rapamycin (mTOR) is essential for IL-1β-induced Th17 proliferation. Thus, mTOR-deficient Th17 cells fail to proliferate in response to IL-1β. IL-1β induces phosphorylation of mTOR [21]. These results suggest that IL-6 directs the initial differentiation of Th17 cells, while IL-1β promotes the expansion of these cells when they are in competition with other T cell subsets in the context of a resource-limited tissue environment [22].

Involvement of GM-CSF in driving Th17 cells was first demonstrated in an experimental autoimmune myocarditis (EAM) model [23]. Development of EAM is a Th17 cell-mediated disease [24] and is IL-6- and IL-23-dependent [25, 26]. Mice deficient in GM-CSF fail to generate Th17 cells and do not develop EAM. This is the result of diminished production of IL-6 and IL-23 by dendritic cells [23]. Interestingly, GM-CSF deficiency did not affect the development of Th1 or Th2 cells. These results indicate that GM-CSF promotes Th17 cells by indirectly inducing IL-6 and IL-23 production by dendritic cells. GM-CSF is also an important Th17 cell effector cytokine, which is required to mediate inflammation in the EAE model. Recently, it was found that synovial CD4⁺ Th17 cells in patients with rheumatoid arthritis (RA) produce GM-CSF, which is able to induce an inflammatory dendritic cell phenotype from monocytes [27]. These results suggest a GM-CSF-driven positive feedback loop in operation in RA joints to perpetuate pathogenic Th17 cells.

IL-21 is produced by natural killer (NK) cells and T cells. IL-21 signals through a unique IL-21R chain and the shared common γ-chain (shared by IL-2, IL-4, IL-7, IL-9, and IL-15). IL-21 is an autocrine growth cytokine for the expansion of Th17 cells. IL-21 together with TGF-α is able to induce the differentiation of Th17 cells, and IL-21 can replace IL-6 in this process [28]. In the absence of IL-6, IL-21 together with TGF-α was able to inhibit the development of inducible Treg and to promote the differentiation of Th17 cells [28]. During the initial Th17 differentiation, IL-6-induced IL-21 acts as a positive amplification loop to enforce Th17 differentiation [29, 30]. In vivo, however, the role of IL-21 in the induction of Th17 cells remains controversial. It was reported that the absence of IL-21 or IL-21R had no significant difference on the development of Th17 cells [31, 32]. Thus, IL-21 might be an alternative pathway in inducing and expanding Th17 cells [33].

TGF-β is generally considered an anti-inflammatory cytokine. The role of TGF-β in Th17 cell differentiation is complex. The effect of TGF-β on promoting IL-17A production by CD4⁺ T cells was first described by Veldhoen et al. [12]. TGF-β in combination with IL-6 initiates naive CD4⁺ T cells to differentiate into IL-17A-producing cells. This effect is amplified by IL-1β and TNF [12]. T cell-specific deletion of the TGF-β gene and expression of the nonfunctional TGF-β receptor confirmed that endogenous TGF-β induces Th17 cell development in vivo [34–36]. One caveat associated with deficient TGF-β signaling is excessive production of IL-4 and interferon (IFN)-γ, which are potent suppressors of Th17 cell development and population expansion [34, 35]. So the alternative explanation is that TGF-β indirectly induces Th17 development by suppressing Th1 or Th2 development. Indeed, in some experiments, in the absence TGF-β, when IL-4 and IFN-γ activities are neutralized, Th17 cell differentiation can be initiated and fully developed when IL-23 is added [5, 6]. These results suggest that TGF-β can be spared in the initiation of Th17 differentiation. That is, IL-6 alone can induce Th17 cell development in T-bet- and STAT6-deficient mice [37]. In humans, conflicting results were generated in studies of in vitro development of Th17 cells from naive CD4⁺ T cells by different groups. Thus, TGF-β is dispensable to development of Th17 cells in some but not in the other experiments [38–42]. Interestingly, Th17 cells generated by TGF-β and IL-6 are less pathogenic. In contrast, Th17 cells generated by IL-6 and IL-23 are highly pathogenic and able to induce severe EAE [15].

The role of IL-23 in Th17 cell survival is indispensable [2, 4]. Mice with the IL-23 p19 gene knockout generate no or few Th17 cells and do not develop EAE or CIA [2, 4]. However, IL-23 is not required for the initial differentiation of Th17 cells, but is essential for clonal expansion and stabilization of Th17 cells [12]. Naive CD4⁺ T cells do not express IL-23R. IL-23 alone cannot initiate the differentiation of Th17 cells from naive CD4⁺ T cell precursors [2, 10]. Earlier studies indicate that IL-23 acts on activated and memory CD4⁺ T cells to induce IL-17A production [1]. Upon IL-6-mediated initial differentiation, Th17 cells express IL-23R [15]. IL-23 signaling further increases IL-23R expression (Fig. 3.1) [15] to reinforce the action of IL-23 on Th17 cells. This fact is particularly relevant to therapeutic strategy design for targeting IL-17/Th17 pathway. In Th17 cell-mediated disease, pathogenic Th17 cells are already formed and need constant IL-23 signaling for survival. Therefore, interrupted IL-23 signaling would be effective to eliminate pathogenic Th17 cells. It has been suggested that Th17 cells activated by TGF-β and IL-6 play a more physiological role in mucosal defense and barrier tissue integrity and may also curtail immunopathogenic responses (Fig. 3.1) [43–45], while IL-23-activated Th17 cells play a role in chronic tissue inflammation during infection, granuloma formation, and autoimmune conditions (Fig. 3.1). These have been demonstrated in experiments in neutralizing IL-23 activity in disease models [46, 47] and provide evidence for the rationale of IL-23-targeted therapy in human disease (see below).

In summary, data from in vitro and in vivo experiments indicate that TGF-β, IL-6, and IL-1β are essential cytokines that initiate Th17 cell differentiation; IL-21 drives Th17 cell growth in an autocrine fashion; GM-CSF is a Th17 effector cytokine, which also promotes Th17 cell differentiation; and IL-23 is the cytokine that promotes Th17 cell differentiation and is essential for the pathogenic inflammatory function of Th17 cells.

IL-27 is a heterodimeric cytokine comprised of Epstein–Barr virus-induced gene 3 (EBI3) and IL-27p28 subunits. IL-27 signals through IL-27Rα and gp130 [48]. IL-27 is a potent inhibitor of IL-17 production. IL-27 receptor-deficient mice infected with Toxoplasma gondii develop severe neuroinflammation, which is mediated by Th17 cells [49]. In vitro, IL-27 is able to completely suppress IL-6 and TGF-β−induced de novo Th17 differentiation but has a lesser inhibitory effect to counter IL-23-mediated development of Th17 cells [49–51]. IL-27 fails to suppress IL-17A from encephalitogenic Th17 cells [50]. These results suggest that IL-27 may have an impact during Th17 differentiation but have a limited effect on already committed Th17 cells.

The role of IFN-γ in Th17 cells is complex. Historically, IFN-γ was considered the effector cytokine of Th1 cells mediating many of the autoimmune inflammatory diseases, which are now confirmed to be mediated by Th17 cells. IFN-γ knockout mice develop severe EAE and CIA with increased number of Th17 cells [52–54]. Arthritis in IFN-γ-deficient mice is suppressed by neutralizing IL-17A antibodies [53]. In vitro de novo differentiation of Th17 cells is suppressed by the presence of IFN-γ [5, 6]. These results clearly indicate the inhibitory effect of IFN-γ on the development of Th17 cells. However, other studies provided evidence indicating that IFN-γ might be required for pathogenicity of Th17 cells [15, 55, 56]. Those Th17 cells inducing EAE co-express IFN-γ. IL-17A and IFN-γ co-expressing Th17 cells also exist in human diseases [57, 58]. It appears that the so-called Th17 cells are plastic and are able to shift toward Th1-like cells [24, 45, 59–61]. In humans, those IL-17A and IFN-γ co-expressing Th17 cells may represent a transient status of the Th17 cells and are dependent on DNA methylation status of Rorc2 and Il17a genes [62]. The heterogeneity of Th17 cells may originate from the Th17 precursors, which are CD4⁺ CD161⁺. These CD4⁺ CD161⁺-naive T cells can be differentiated into Th17 cells or non-classic Th1 cells, which express RORγt but are able to produce IFN-γ as well as IL-17A [62, 63]. However, whether and how much IFN-γ contributes to the pathogenicity of these Th17 cells is unclear and is difficult to delineate with certainty.

The signature Th2 cytokine, IL-4, is also a potent inhibitor of Th17 cells [5, 6]. IL-4-mediated inhibition of Th17 cells is STAT6 dependent but not GATA3 dependent. The inhibitory effect of IL-4 takes effect during the initial de novo differentiation stage but has no impact on fully differentiated Th17 cells [64]. This highlights that as with IL-27, IL-4 may only have limited application as a therapeutic agent to treat Th17-mediated conditions.

The differentiation and development Th17 cells are finely controlled by transcription factors (Fig. 3.2).

Of these transcription factors, RORγt is the master controller to determine the CD4⁺ T cell fate to become Th17 cells. ROR are members of the nuclear receptor family of intracellular transcription factors [65, 66]. The ROR subfamily has three members in mammals: RORα, RORβ, and RORγ [67]. RORγ is encoded by the Rorc gene. RORγ is expressed in many tissues such as the heart, kidney, liver, lung, brain, and muscle. RORγt was first identified by He et al. [68] as a thymus-specific isoform of RORγ that is expressed predominantly in CD4⁺ CD8⁺ double-positive thymocytes [69]. In fact, RORγt is a splice variant of RORγ, which is different only at the N-terminus [68]. Unlike the wide tissue expression of RORγ, RORγt is expressed exclusively in lymphoid cells [70]. RORγt is critical in regulating gene expression during the development of T cells and the formation of the secondary lymphoid organ [71–73]. Rorc gene-deficient CD4⁺CD8⁺ thymocytes undergo early apoptosis, and the animals fail to develop lymph nodes, Peyer’s patches, and lymphoid tissue inducer cells (LTi) [72, 73]. In vitro, as a result of the absence of Rorc in CD4⁺ T cells, IL-17 expression was greatly impaired under Th17-polarizing conditions. This has been seen in humans with RORγt deficiency [74]. Conversely, overexpression of RORγt in naive CD4⁺ T cells was sufficient to induce the expression of IL-17A, IL-17F, and IL-22 [7]. RORγt is essential for the expression of IL-17 as well as the differentiation of Th17 in mouse and human CD4⁺ T cells [7, 39]. RORγ-deficient mice produce few Th17 cells and fail to develop EAE [7]. Therefore, the role of RORγt is similar to transcription factors such as T-bet and GATA3 in Th1 and Th2 differentiation, respectively, and hence, RORγt is the master transcriptional factor for Th17 differentiation [60, 75]. Agents targeting RORγt are effective in inhibiting Th17 differentiation and suppression of disease models of inflammation (see below). RORγt promotes IL-17 expression by directly binding the promotor region of Il17 genes at multiple sites [7, 76, 77].

Another related retinoic acid nuclear receptor, RORα, is also expressed in Th17 cells both in vitro and in vivo. RORα expression is induced by TGF-β and IL-6 via STAT3. CD4⁺ T cells deficient in Rora have impaired Th17 differentiation but are not completely abolished. Mice deficient in RORα produce fewer Th17 cells in vivo and develop less severe EAE, but disease incidence was not affected [78]. Interestingly, RORα deficiency impairs IL-17A but not IL-17F expression. Co-overexpression of RORα and RORγ significantly increases Th17 differentiation [79]. These results suggest that RORα is a transcription factor that synergizes RORγt function but is not essential for Th17 cell differentiation.

The transcription factor STAT3, which is preferentially activated by IL-6, IL-21, and IL-23, is capable of inducing RORγt and regulating Th17 cell development [13, 80]. Deficiency of STAT3 in CD4⁺ T cells impaired Th17 cell differentiation in vivo, and overexpression of a constitutively active STAT3 could increase IL-17A production [13, 81]. STAT3 might affect the production of IL-17 by increasing the expression of RORγt and RORα [13, 78]. Furthermore, STAT3 also binds directly to the Il17 and Il21 promoters and leads to the expression of IL-17A and IL-21 [82, 83]. Therefore, STAT3 and RORγt seem to cooperate to induce IL-17A production.

Earlier studies have demonstrated that multiple other transcription factors also participate in regulating the development of Th17 cells. These include IRF4, BATF, and RUNX1 [75]. The importance of IRF4 and BATF was confirmed by a gene-deficient approach. BATF-deficient CD4⁺ T cells fail to maintain RORγt expression, and mice with BATF deficiency are resistant to induction of EAE [84]. Similarly, IRF4-deficient mice were shown to have impaired Th17 responses and were resistant to EAE [85]. Interestingly, deficiency in either of BATF or IRF4 results in defective Th1, Th2, Th9, and T follicular helper (Tfh) cell development [86–89]. Several studies indicate that in the early event of Th0 activation, BATF and IRF4 cooperate to allow the accessibility of chromatin by other lineage-specific transcription factors to determine the fate of Th cell differentiation. During Th17 cell differentiation, upon TCR signaling, the so-called pioneer factors, BATF and IRF4, bind Il17a gene promoter region and regulatory enhancer in multiple sites [90, 91]. IL-6 signaling recruits STAT3, BATF, IRF4, and STAT3 complex along the co-activator histone acetyltransferase p300 to promote the expression of Rorc [91]. TCR and IL-6 signaling also promotes the expression if hypoxia-inducible factor 1α (HIF1α), a key sensor of hypoxia [92]. HIF1α directly binds and drives transcription of Rorc and forms a complex with RORγt and p300 to drive Il17 gene expression. HIF1α also binds to FOXP3 to induce proteasomal degradation of FOXP3 [92]. IRF4, BATF, STAT3, RORγt, and HIF1α complex further induces expression of Il23r. This allows IL-23 to induce maturation of Th17 cells. IL-23 signaling activates and recruits B lymphocyte-induced maturation protein 1 (BLIMP1) to the STAT3-RORγt transcription factor complex to enhance the expression of Th17 cell signature genes (Fig. 3.2) [22].

It is interesting to note that RORγt only directly controls Il17a, Il17f, and Il23r genes. For example, a lack of RORγt does not affect p300 recruitment. This renders RORγt an exceptional drug target as therapeutic intervention would not be expected to perturb the genetic regulatory programs shared by other cell types [91].