The Multifaceted Role of Th17 Lymphocytes and Their Associated Cytokines in Cancer

2.1. Th17 Differentiation

Th17 cells are defined as CD4⁺ T lymphocytes secreting substantial amounts of interleukin 17A (IL-17A) and expressing the transcription factor retinoic acid receptor-related orphan receptor gamma t (RORγt), which seems to act as a molecular determinant for their polarization [35, 36]. In addition, Th17 produce IL-21 and IL-22 [37] and, depending on the differentiation/environmental conditions secrete variable amounts of TNFα, IFNγ, and/or GM-CSF [32, 38–41]. Th17 foster B lymphocyte-mediated immunity [3], contribute to the migration and activation of macrophages [42], neutrophils [43], and regulate the activation and expansion of CD8⁺ T cells [41, 44].

Th17 can be generated in vitro from naïve CD4⁺ T lymphocytes using specific cytokines. In mice, the combination of TGFβ, IL-6, and IL-23, in presence of TCR and CD28 signals (antigen presenting cells, plate-bound anti-CD3 plus anti-CD28 or anti-CD3/anti-CD28 coated microbeads), is required and sufficient to drive the differentiation of naïve CD4⁺CD25⁻ T cells into Th17 (Figure 1) [45]. Studies have indicated that IL-6, by inhibiting TGFβ-driven induction of FoxP3, impairs Treg differentiation, leading to IL-17-producing RORγt⁺ lymphocyte generation. However, other reports have also shown that, in IL-6^−/− mice, Treg depletion increases the susceptibility of the animals to experimental autoimmune encephalomyelitis (EAE) as a result of enhanced pathogenic Th17 responses [39, 46]. These last observations suggest that Th17 lymphocytes can be generated in absence of IL-6. IL-21 was further identified as an alternative proinflammatory cytokine capable of suppressing TGFβ-mediated induction of FoxP3 expression in the absence of IL-6 [39, 47]. IL-21, produced in large amounts by Th17, promotes an autocrine amplification feedback loop enhancing Th17 generation especially in the absence of IL-6 [48]. The IL-23 receptor (IL-23R) is composed of IL-23R and IL-12Rβ1 [49]. Naïve CD4⁺ T lymphocytes express very low levels of IL-23R. Conversely, Th17 are characterized by the expression of the IL-23R. It is therefore not surprising that, although IL-23 is not required for the initial Th17 lineage commitment, this cytokine fosters Th17 expansion and survival and contributes to their stabilization and proinflammatory properties [50]. Indeed, in IL-23p19-deficient mice, the number of Th17 is substantially decreased compared to their wild-type counterparts [51, 52]. In addition, IL-23 appears essential for the pathogenic properties of Th17 as demonstrated in collagen-induced arthritis (CIA) and EAE models [51, 53]. IL-23 is also essential for the generation of Th17 in prolonged in vitro cultures [50]. The proinflammatory cytokine IL-1β has also been reported as another important factor in the polarization of Th17 cells in proinflammatory environments. IL-1β induces interferon regulatory factor 4 (IRF4), which is a critical regulator of the IL-21 autocrine signaling loop [54, 55].

In humans, the conditions that would drive optimal Th17 differentiation remain unclear. Several reports have indicated that TGFβ may not be necessary for the generation of these cells [56–58] while other studies have argued for a critical role of this cytokine in Th17 differentiation [47, 59, 60]. A study by Yang et al. indicated that the combination of TGFβ with IL-21 but not IL-6 was effective in inducing Th17 differentiation [47]. Other reports have suggested that IL-1β alone or in combination with TGFβ is also required for human Th17 production [61]. Similar to the observations made in mice, the addition of IL-23 supports Th17 proliferation and stabilization [62].

2.2. Th17 Plasticity

Th1 and Th2 cells are relatively stable and terminally differentiated subsets: they essentially do not transdifferentiate into other specialized CD4⁺ T helper cell lineages. On the other hand, one of the most striking characteristics of Th17 is their high degree of plasticity and their remarkable ability to give rise to other populations of either proinflammatory effector cells such as Th1 [63] or immunosuppressive FoxP3⁺ Treg [64]. Interestingly, Th17 may themselves originate from FoxP3⁺ Treg cells that have undergone “reprogramming” in specific environmental conditions [65]. Intermediary cell subpopulations expressing both FoxP3 and RORγt and demonstrating immunosuppressive activity have been identified [66].

TGFβ appears as a master regulator of the balance between Th17 and suppressive Treg differentiation. The role of TGFβ in Th17 polarization has, however, been questioned. Although some studies have indicated that TGFβ is required for the production of IL-17 by Th17 cells [60], others have reported that TGFβ may not be essential for the induction of Th17 [58]. Additional reports have demonstrated that the generation of Th17 and the development of Th17-mediated EAE are impaired in transgenic animals with T lymphocytes deficient in functional TGFβ receptor [67] or when TGFβ expression is ablated in T cells [68]. TGFβ alone induces the expression of FoxP3 and RORγt [69]. However, in the presence of IL-6, IL-21, or IL-23, FoxP3 expression is inhibited while RORγt expression is induced, resulting in Th17 generation instead of Treg. While it has been established that TGFβ is required for the initial production of IL-17 and for the induction of IL-23R expression [47, 57, 60], high concentration of TGFβ conversely impairs the expression of IL-23R [69]. Therefore, the outcome of the balance between Treg versus Th17 generation is likely dictated by the strength of the signals provided by TGFβ and thus depends, at least partially, on the concentration of this cytokine in the environment of the differentiating cells. Large amounts of TGFβ primarily promote the development of cells endowed with immunosuppressive activity (possibly even in the presence of low doses of IL-6 or IL-21), while intermediary or low concentration of TGFβ in combination with the proinflammatory cytokines IL-6 or IL-21 drives primarily the differentiation of naïve CD4⁺ T cells into Th17 (Figure 1). This scenario may explain the observed Th17-Treg plasticity. Recent reports have also indicated that, in specific conditions, fully differentiated FoxP3⁺ Treg may undergo “reprogramming” into effector helper T cells. These reprogrammed Treg are not immunosuppressive, produce proinflammatory cytokines (among which IL-2, TNFα, or IL-17), and may play an important role in anti-tumoral CD8⁺ T cell activation [70]. Importantly, it has been shown that reprogrammed Treg may lose or maintain FoxP3 expression [70, 71]. Additional studies have reported that, in presence of IL-2 and IL-1β, human Th17 may be preferentially differentiated from naïve FoxP3⁺CD25⁻CD4⁺ Treg rather than from naïve FoxP3⁻CD25⁻CD4⁺ T cells [72, 73]. In these studies, Th17 differentiation was enhanced by IL-23 and TGFβ [73].

Th17 may also redifferentiate into Th1 lymphocytes. Indeed, IL-17-producing CD4⁺ T lymphocytes expressing the Th1 lineage-specific transcription factor Tbet and producing IFNγ have been described (Figure 1) [74]. Tbet⁺ Th17 cells have been identified in patients with multiple sclerosis [38] and IFNγ-producing human Th17 cells have been described [57]. These intermediary Th17/Th1 lymphocytes have been reported as pathogenic [32, 75] and as outlined in Section 3 can exhibit anti-tumoral activity [41, 74]. Initially, the recognition that IL-23 and IL-12 shared the common IL-12p40 (IL-12β) subunit and the observation that IL-23 induced not only IL-17 but low amounts of IFNγ led to the speculation that Th17 cells developed as a distal branch of the Th1 lineage. However, a recent study reported that Th17 precursors may produce IFNγ, independently of IL-23 and IL-12 signaling. These Th17 precursors are also capable of responding to IL-23 and IL-12 and, in the absence or in presence of low concentrations of TGFβ, can differentiate into cells characterized by enhanced production of IFNγ and minimal IL-17A and IL-17F secretion [76]. These studies confirmed that Th17 lymphocytes belong to a distinct cell lineage susceptible, however, to reprogramming into Th1 cells. This observed stability or plasticity of Th subsets seems controlled by epigenetic modifications regulating the expression of key transcription factors and cytokines specific for a dedicated Th lymphocyte lineage.

Thus, Th17 lymphocytes represent a highly heterogeneous cell population with a remarkable flexibility in their ability to differentiate into immunosuppressive Treg or effector proinflammatory Th1 depending on the environmental conditions. Since Treg are known to suppress anti-tumor immune responses and promote cancer development while Th1 enhance anti-tumoral immunity, it is therefore not surprising that Th17 have been reported to exhibit both pro- and anti-tumor activities.

3.1. IL-17-Producing Cells, IL-17, and Major Th17-Associated Cytokines

3.2. Th17 Lymphocytes in Cancer: Foes or Allies?

As previously outlined, the role of Th17 lymphocytes in cancer is still highly controversial (Figure 2). An important distinction should be made between “endogenous” Th17 cells present in cancer patients or mouse tumor models, which develop under the pressure of the complex tumor environment, and the adoptively transferred Th17 cells generated in vitro under well-defined cytokine conditions.

Th17 lymphocytes have been detected in patients with different types of malignancies, such as ovarian, pancreatic, or gastric cancers, but the role of these cells in disease progression and their prognosis value has been controversial [37, 117, 118]. Whether Th17 lymphocytes are induced de novo from naïve CD4⁺ T cells or recruited at the tumor site or originate from “reprogrammed Treg” (see previous section and below) remains to be elucidated. In a report evaluating the nature of tumor-associated Th17 lymphocytes in ovarian cancer patients, it was demonstrated that the percentage of these cells correlated with the number of IFNγ ⁺ CD4⁺ T cells, IL-17⁺ IFNγ ⁺, IFNγ ⁺ CD8⁺ T cells as well as NK cells and inversely correlated with the frequency of immunosuppressive Treg cells [37]. In another report focusing on prostate cancer patients, highly differentiated Th17 cells correlated with slower disease progression [119], which was contradicted by results from others obtained in hormone resistant prostate cancer patients [120]. In additional studies, an association between increased numbers of tumor-associated Th17 lymphocytes and survival was observed in ovarian and lung cancer patients [37, 121]. Similarly, a significant increase in Th17 cell numbers in the tumor environment has been reported in the mouse ID8 ovarian, Pan02 pancreatic, and B16 melanoma cancer models [42, 122, 123]. The physiological significance of this increase has been disputed. In an ovarian cancer model, TNFα-mediated induction of IL-17-producing CD4⁺ cells led to the recruitment of myeloid cells into the tumor microenvironment and resulted in enhanced tumor growth [42]. In contrast, induced production of IL-6 in the tumor microenvironment, as a result of either indoleamine 2,3-dioxygenase (IDO) inhibition or the transduction of tumor cells with the IL-6 gene, led to the conversion of Treg to Th17 cells and regression of mouse B16 melanoma [123] or Pan02 pancreatic tumors [122], respectively.

Multiple studies have investigated the impact of in vitro generated Th17 cells on tumor growth following adoptive transfer, with variable outcomes. Initial studies by Muranski et al. have evaluated the effects of Th17 generated from CD4⁺ T cells isolated from TCR transgenic mice specific for the TRP-1 melanoma epitope. Administration of these Th17 lymphocytes led to the eradication of established B16 melanoma. The therapeutic effects of these cells were, however, substantially mediated by IFNγ [124]. In line with these results, subsequent studies indicated that adoptive transfer of in vitro generated Th17 lymphocytes impaired tumor development by eliciting robust tumor-specific CD8⁺ T cell responses. Th17 cell therapy promoted the homing of dendritic cells to the tumor site and the draining lymph nodes [41]. Supporting the anti-tumoral role of in vitro polarized Th17, in a more recent study Muranski et al. demonstrated that adoptive Th17 cell therapy has the potential to eliminate established tumors. The anti-tumoral efficacy of these Th17 lymphocytes was dependent on their ability to produce both IFNγ and IL-17. Interestingly, the administered Th17 differentiated into cells, which exhibited a stem cell-like phenotype and Th1 properties (Tbet, IFNγ expression) but retained their ability to produce IL-17. Importantly, the therapeutic efficacy of Th17 lymphocytes generated from Tbet^−/− or IFNγ ^−/− or IL17^−/− mice was severely impaired [74]. In agreement with these reports, our own results have suggested that the adoptive transfer of Th17 efficiently combined with chemotherapy to treat established murine mammary carcinoma [125]. In one study, the possibility that in vitro generated Th17 cells may exhibit immunosuppressive function and promote tumor progression through the expression of ectonucleotidases has been proposed [126].

It should, however, be emphasized that in the majority of these studies, the generated populations of CD4⁺ T cells were heterogeneous in nature and were not a pure subset of Th17 lymphocytes, advocating for a cautious interpretation of the above results. In addition, the concentration of cytokines (TGFβ, IL-6, and IL-23) represents a major source of variability between protocols used to generate Th17 cells in vitro. Therefore, standardized procedures are still needed to generate and purify homogeneous populations of CD4⁺ T cells producing high levels of IL-17.