Identity crisis of Th17 cells: Many forms, many functions, many questions

Highlights

• Th17 cells are potent regulators linked to induction and persistence of inflammation.

• The human Th17 compartment is heterogeneous with pro- and anti-inflammatory constituents.

• Th17 cells acquire additional effector functions in inflamed tissue.

• Development and plasticity of Th17 cells are regulated by cytokines and metabolic checkpoints.

Abstract

Th17 cells are a subset of CD4⁺ effector T cells characterized by expression of the IL-17-family cytokines, IL-17A and IL-17F. Since their discovery nearly a decade ago, Th17 cells have been implicated in the regulation of dozens of immune-mediated inflammatory diseases and cancer. However, attempts to clarify the development and function of Th17 cells in human health and disease have generated as many questions as answers. On one hand, cytokine expression in Th17 cells appears to be remarkably dynamic and is subject to extensive regulation (both positive and negative) in tissue microenvironments. On the other hand, accumulating evidence suggests that the human Th17 subset is a heterogeneous population composed of several distinct pro- and anti-inflammatory subsets. Clearly, Th17 cells as originally conceived no longer neatly fit the long-standing paradigm of stable and irrepressible effector T cell function. Here we review current concepts surrounding human Th17 cells, with an emphasis on their plasticity, heterogeneity, and their many, tissue-specific functions. In spite of the challenges ahead, a comprehensive understanding of Th17 cells and their relationship to human disease is key to ongoing efforts to develop safer and more selective anti-inflammatory medicines.

Introduction

Inflammation is the fundamental process of immune activation. Despite its “killer” reputation, inflammation is required for immune memory and vaccine responses, protection from infectious microorganisms, and even tissue maintenance and repair in the absence of pathogenic infection [1], [2]. Of course, inflammation is also a common underlying pathophysiology observed in autoimmunity, cardiovascular disease, cancer, metabolic syndromes such as diabetes, and fibrosis [3], [4], [5], [6], [7]. A key determinant of whether or not inflammation turns pathologic is its ability to be resolved; in autoimmunity, for example, pathologic tissue damage is mediated by chronic, unresolved inflammation resulting from loss of immune tolerance to host tissues and T and B cell autoreactivity. On the other hand, it is also clear that not all inflammatory responses are the same. Qualitatively distinct inflammatory responses are coordinated at the level of antigen presenting cells (APCs) and carried out by CD4⁺ T helper (Th) cells, which differentiate from multipotent naïve precursor cells into a variety of phenotypically and functionally distinct effector subsets [8], [9]. Cytokine production (i.e., effector function) by Th cell subsets, in turn, orchestrates various aspects of innate and adaptive immune function.

Th cell differentiation is an instructive process that starts with the presentation of peptide antigens by APCs to T cells via MHC class II molecules. Cognate antigen recognition by naïve Th cell clones induces an elaborate network of activation signals through the T cell antigen receptor (TCR) complex, which combine with positive or negative co-stimulatory signals provided by major T cell co-stimulatory receptors (CD28, CTLA-4) and their ligands expressed on APCs (B7.1, B7.2) [10]. Whereas these signals initiate T cell activation and can influence effector T cell differentiation – depending on the strength and duration of TCR and co-stimulatory signals [11], [12], [13] – additional environmental cues, in the way of local cytokines, are required to specify T cell fate decisions. Cytokines bind to multimeric receptors expressed on Th cells, and regulate T cell differentiation via activation of JAK/STAT signaling pathways. STAT proteins reside in the cytoplasm of resting cells, and undergo rapid dimerization and nuclear translocation following their recruitment to activated (i.e., ligand-bound) cytokine receptors and phosphorylation by receptor-associated JAKs. Once in the nucleus, STAT proteins induce the expression of lineage-specifying transcription factors and coordinate downstream transcriptional programs that underlie Th cell differentiation. There are 7 mammalian STAT proteins (STAT1-4, STAT5a, STAT5b, STAT6), and all but STAT2 play essential roles in Th cell differentiation. A number of outstanding reviews are available elsewhere that detail JAK/STAT signaling pathways and their many functions during Th cell differentiation [14], [15].

Th cell differentiation was long considered a binary process, leading to either IFNγ-producing Th1 cells or Th2 cells that secrete IL-4, IL-5 and IL-13. Whereas Th1 cells develop in an IL-12/STAT4- and IFNγ/STAT1-dependent manner that requires activation of the “master” Th1 transcription factor T-bet (TBX21), Th2 differentiation requires IL-4/STAT6 and the Th2-specifying transcription factor, GATA-3 [16], [17], [18], [19]. Th1 cells, by virtue of IFNγ production, activate CD8⁺ cytotoxic T lymphocytes, NK cells, and phagocytes to regulate immunity against intracellular bacterial pathogens and viruses, whereas Th2 cells and their effector cytokines provide help to B cells, regulate class-switch antibody recombination, and recruit eosinophils for immune responses against many extracellular bacteria and large parasitic worms (i.e., helminthes) (reviewed in [20]). For nearly 20 years, the Th1/Th2 paradigm was thought to explain all aspects of immunity and inflammation. However, starting with the discovery of Th17 cells in the mid-2000s, the repertoire of effector T cell lineages has expanded dramatically, and now includes Th17 cells, induced T regulatory (iTreg) cells, T follicular helper (Tfh) cells, as well as Th22 and Th9 cells that express IL-22 and IL-9, respectively [9], [21], [22]. With the exception of Th17 cells, the regulation and in vivo functions of these new T cell subsets are only beginning to be unraveled.

The concept of a distinct, pro-inflammatory, IL-17-producing effector T cell subset began with work from Sedgwick and colleagues [23], who showed that IL-23 and not IL-12 is the critical driver of autoimmunity in mice. IL-23 is an IL-12-related heterodimeric pro-inflammatory cytokine comprised of IL-12 p40 and the unique IL-23 p19 subunit [24], [25]. Whereas mice lacking only IL-23 (p19^−/−) were completely protected from experimental autoimmune encephalomyelitis (EAE), animals specifically lacking IL-12 (p35^−/−) remained susceptible to disease, despite a defect in Th1 cell development [23]. IL-23 was found to induce IL-17A-expressing effector T cells, and these cells were responsible for IL-23-mediated EAE [26], [27]. However, because IL-23 did not induce IL-17A expression in naïve T cells in vitro, the notion of Th17 cells remained enigmatic. Shortly thereafter, pioneering work from the Littman, Stockinger, and Kuchroo, labs described the in vitro differentiation of IL-17-expressing Th17 cells, using combinations of IL-6, IL-21, and TGFβ (i.e., TGFβ1) [28], [29], [30], [31], [32]. Because TGFβ had previously only been considered an anti-inflammatory cytokine that regulated, among other things, development of FOXP3-expressing iTreg cells [33], Weaver et al., thus coined Th17 cells, “an effector lineage with regulatory ties” [34]. Whereas TGFβ alone induces FOXP3, the combination of TGFβ and IL-6 induces Th17 cell development via STAT3-dependent induction of the Th17-specific orphan nuclear receptor, RORγt (RORC in humans) [31], [35], [36]. In turn, STAT3 and RORγt synergize with other, general transcription factors, including the AP-1 family member BATF, IRF4, NFκB, and NFAT to regulate transcription and chromatin remodeling at the IL17A/IL17F locus [37]. In addition to IL17A/F, Th17 cells express a cast of pro-inflammatory cytokines, such as IL-22, TNFα and GM-CSF and, importantly, they selectively express the IL-23 receptor (IL-23R) [38]. In this way, IL-23 regulates the growth and inflammatory function of Th17 cells following their initial differentiation (discussed below). Indeed, many more molecules and pathways have since been defined that both enhance and inhibit the development of Th17 cells, and those are reviewed elsewhere [38], [39]. Here, we focus on the regulation of Th17 cell effector function and plasticity post-initial differentiation, particularly as it relates to inflammation in human autoimmunity and cancer.

Unlike inbred mice housed in barrier facilities, humans have a large, heterogeneous, and highly variable (person-to-person) endogenous effector/memory T cell compartment. Thus, the immune system as seen in human peripheral blood affords a unique view into the natural diversity of T cell effector lineages and provides important and complimentary information to that derived from artificial in vitro T cell culture systems and experimental animal models of autoimmunity. Among the most important insights coming from the study of endogenous human effector/memory T cells is that effector T cell lineages co-express unique lymphocyte homing receptors together with lineage-defining cytokines and lineage-specifying transcription factors [40], [41], [42], [43], [44]. For example, IFNγ-producing Th1 cells are highly enriched within human memory cells that express the chemokine receptor CXCR3 [40], [43]. Th2 cells not only express IL-4, IL-5, and IL-13; they also express the chemokine receptor CCR4 and the chemotactic receptor of prostaglandin D2, CRTH2 [40], [43], [45]. Th17 cells uniformly express the inflammatory chemokine receptor CCR6 [46], [47], though these cells are further heterogenous and can express additional chemokine receptors in combination (see below). Co-regulation of effector cytokines and lymphoid homing receptors confers tissue-specific T cell effector function. For example, tissues produce a variety of chemokines, both at steady-state and upon infection, stress, or damage. Chemokine receptors thus serve to direct T cell traffic into organs and tissues where their effector cytokines are needed for immune responses against specific microbes or for tissue maintenance and repair (reviewed in [48]). Like most aspects of the immune system, chemokines are also a double-edged sword that can contribute to chronic and autoimmune inflammation. The molecular link between effector cytokines and lymphoid-homing receptors comes by virtue of lineage-specific transcription factors (e.g., T-bet, GATA-3, RORγt), which activate chemokine receptor gene expression in a subset-specific manner; forced expression of T-bet, GATA-3, RORγt in cultured human naïve T cells is sufficient to induce CXCR3, CCR4/CRTH2, and CCR6 expression in Th1, Th2, and Th17 cells, respectively [49], [50], [51].