T Helper 17 Cells Overview

CD4⁺ T helper lymphocytes are mediators of cellular immunity that play a critical role in the activation of other immune cells such as B cells and cytotoxic T cells, as well as in the regulation of immune responses. CD4⁺ T helper cells are further divided into subsets characterized by cytokine secretion and effector function, which differentiate in the periphery in response to cues from innate immune cells [1]. The first two of these subsets to be described were Th1 and Th2 [2], and this dichotomy existed for several decades prior to the discovery of a new CD4⁺T helper subset, termed Th17 due to their secretion of the IL-17A cytokine [3,4]. Th17 cells play a role in host defense against extracellular pathogens, particularly at the mucosal and epithelial barriers, but aberrant activation has been linked to the pathogenesis of various autoimmune diseases [5].

Like other subsets of T helper cells, Th17 cells differentiate from naive CD4⁺ cells in the periphery in response to T cell receptor (TCR) antigen stimulation and activating cytokines secreted by antigen-presenting cells [6]. While differentiation was originally believed to be induced by IL-23, it was later demonstrated that Th17 development occurred independently of this cytokine. However, IL-23 is still thought to be important for Th17 maintenance and proliferation, and its receptor (IL-23R) is upregulated in activated Th17 cells [6]. The critical cytokine mediators of Th17 differentiation have instead been identified to be TGFβ in combination with IL-6 or IL-21 [6,7]. IL-6 and IL-21 drive expression of Th17 transcriptional regulators via STAT3 signaling, committing CD4⁺ T cells to the Th17 lineage. Defects in this signaling pathway have been associated with decreased expression of IL-23R, key Th17-associated transcription factors, and effector cytokines such as IL-17A and IL-17F [7].

A candidate master regulator of Th17 differentiation was first identified as RORγt, a member of the retinoic acid–related orphan nuclear hormone receptor family [6]. This transcription factor was found to induce expression of IL-17A and IL-17F, and its deficiency was associated with reduced, but not completely absent, Th17 development and function [6,7]. Later studies identified RORα, a related transcription factor, could also drive Th17 differentiation and cytokine expression in response to STAT3 in a similar manner as RORγt [8]. RORα and RORγt act synergistically to promote Th17 commitment, and combined deficiencies in both factors results in complete inhibition of Th17 development [8].

Other transcription factors that play a role in Th17 development are IRF4, BATF, and AHR. IRF4 is thought to be upstream of RORγt, as the ability of naive CD4⁺ T cells to upregulate RORγt expression is reduced in its absence, but its exact role in Th17 biology is not fully understood [9]. AHR is a nuclear factor shared with T regulatory cells but expressed at higher levels in Th17 cells, and while its deficiency does not impact Th17 differentiation, the production of effector cytokines, particularly IL-22, is significantly diminished [10]. Lastly, BATF has been demonstrated to be necessary for generation of Th17 cells and expression of their associated cytokines, despite the observation that BATF is not unique to the Th17 lineage and that BATF-deficient cells are still capable of inducing RORα and RORγt [11].

The Th17 lineage exhibits a high degree of plasticity and has been observed to trans-differentiate into other CD4⁺ T helper subtypes in response to changing environmental cues. T regulatory cells are another T helper subset that depends on TGFβ for its differentiation; increasing concentrations of this cytokine tend to skew naive cells towards Foxp3 expression, which is strongly inhibitory to Th17 development and instead drives commitment towards a regulatory phenotype [12]. Despite this, high levels of IL-6 and resultant STAT3 signaling can downregulate Foxp3 expression in favor of Th17-related genes in TGFβ-induced T regulatory cells, particularly in the presence of IL-1 [13]. While trans-differentiation of Th17 cells has been mainly observed with the Th1 and T regulatory subsets, evidence also exists of shared functions with Th2, T follicular helper, and TR1 cells [14]. The observation that multiple transcriptional master regulators of different CD4⁺ T helper cell subsets can be co-expressed further confirms the potential for functional flexibility between these lineages [15].

The effector function of CD4⁺ T helper cells is mediated primarily by secreted cytokines, and the specific cytokine profile produced by each subset has been one of the main defining characteristics uses to phenotype each population [1] (Figure 2). Th17 cells were the first subset identified as distinct from the original Th1/Th2 paradigm that had persisted for decades and were initially characterized based on their expression of IL-17A homodimers in response to IL-23 [1,3]. Th17 cells were later observed to produce another homodimeric IL-17 family member, IL-17F, as well as the heterodimeric protein IL-17AF, which consists of one subunit of IL-17A and one of IL-17F [16]. IL-17-mediated responses are best characterized in epithelial cells, endothelial cells, and fibroblasts, which respond to IL-17R signaling by secretion of TNFα, IL-1β, IL-6, IL-8, CXCL1, CXCL8, and G-CSF, driving neutrophil recruitment and inflammatory responses [17].

The cytokine IL-22, a member of the IL-10 family, was also found to be secreted by Th17 cells, although its induction is different to that of IL-17A [18]. IL-22 expression is highly dependent on IL-23 signaling more so than signaling of the Th17 differentiation cytokines TGFβ and IL-6, suggesting that it is secreted by more fully differentiated Th17 cells [18]. Its receptor, which is a heterodimeric complex of IL-22R1 and IL-10R2, is expressed predominantly on nonhematopoietic cells, and interaction of IL-22 with its target cells contributes to host defense against extracellular pathogens, secretion of antimicrobial peptides such as β-defensins, and promotion of wound healing [17].

The pleiotropic cytokine IL-21 is expressed by various CD4⁺ T cells, including Th17 cells, and has also been demonstrated to play a critical role in the development and function of these cells. In Th17 cells, it can function in an autocrine manner to further augment Th17 commitment by inducing RORγt expression via STAT3 signaling in a similar manner as IL-6 [19]. In addition to driving Th17 responses, IL-21 promotes cellular immunity by enhancing the function of Th1 cells, CD8⁺ cytotoxic T cells, and NK cells [20]. The maturation, terminal differentiation, and function of B cells are also dependent on IL-21 signaling [21].

While Th17 cells are critical for maintenance of mucosal immunity, their dysregulation has been implicated in the pathogenesis of autoimmune inflammation. It was their role in driving this inflammation that first suggested the presence of a third subset of CD4⁺ T helper cells distinct from the classical Th1/Th2 model. In the original model, Th1 cells were thought to be the main mediators of autoimmunity, but it was demonstrated that the absence of their main Th1 effector cytokine, IFNγ, or their activating cytokine, IL-12, exacerbated models of autoimmune inflammation, such as experimental autoimmune encephalomyelitis (EAE) [22]. The IL-23/IL-17 axis, rather than IL-12/IFNγ, was identified as the main pathway mediating this inflammation [23], and later studies characterized Th17 cells as a new CD4⁺ T helper subset associated with this axis [3,4]. Th17 cells have since been demonstrated to play a role in the progression of other autoimmune diseases, such as rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel disease [5]. The Th17 cytokines IL-17A and IL-17F trigger the production of pro-inflammatory cytokines in target tissues, which not only mediate inflammation through the recruitment of innate immune cells such as neutrophils, but also promote further Th17 activation in a positive feedback manner [5]. The precise environmental cues that lead to Th17 differentiation determine whether they develop towards a tissue-protective or pathogenic phenotype, as research suggests that differentiation triggered by high levels of IL-6 and IL-1β results in a more pro-inflammatory role [13]. Current studies continue to explore Th17 cells and their cytokine products as therapeutic targets for autoimmunity.

Th17 cell infiltration has been observed in a variety of malignant tumors in comparison to healthy tissues, although the specific mechanism of their recruitment is unknown and their impact on overall prognosis is variable [14]. The IL-17 cytokines have been associated with higher angiogenesis, and therefore increased tumor growth and metastasis in some models, and the potential for Th17 cells to trans-differentiate into a more immunosuppressive phenotype has also been observed to play a role in tumor immune evasion [14]. However, the ability of Th17 cells to recruit CD8⁺ cytotoxic T cells and dendritic cells to tumor sites is demonstrated to promote tumor clearance, as is their observed ability to convert towards an IFNγ-secreting Th1 phenotype in the presence of some environmental factors [14]. Overall, the role that Th17 cells play in the progression of cancer appears to be highly dependent on the specific tumor microenvironment. Exploiting this plasticity to manipulate them towards anti-tumor responses may prove to be a beneficial strategy in the development of cancer immunotherapies.

The following describes some commonly used methods and tools available from Thermo Fisher Scientific for the isolation and study of human and mouse Th17 cells.

Flow cytometry analysis using cells isolated from peripheral blood, tissues, and tumors has been widely used to study Th17 biology and function. Characterization of Th17 cells by flow cytometry has typically relied on intracellular staining for either key effector cytokines such as IL-17A or master transcriptional regulators such as RORγt, in combination with pan-CD4 surface phenotyping markers. The inherent plasticity and instability of these cells has made attempts to identify a single surface phenotyping marker for Th17 cells difficult, although some candidates have been suggested. CCR6, the chemokine receptor that drives cellular migration in response to CCL20, is expressed by Th17 cells and implicated in their recruitment to inflamed tissues [24]. However, there exists heterogeneity in the CD4⁺ CCR6⁺ population, which can include not only Th17 cells but also the related Th22 and Th17.1 subsets [24]. Plasticity between the Th17 and T regulatory lineages further complicates the use of CCR6 in combination with CD4 as the sole surface phenotyping markers for Th17 cells. In humans, the co-expression of CD39 with CD161, the human homologue of the mouse NK cell marker NK1.1, has also been presented as a potential marker of the Th17 lineage [25]. A nonexhaustive list of Th17 cellular markers for flow cytometry can be found in Table 1.

Optimized multicolor immunophenotyping panels (OMIPs) can be useful references when building flow cytometry panels, not only providing published combinations of markers and fluorochromes, but also suggested gating strategies for analysis. Table 2 lists the OMIPs relevant to Th17 biology.

Table 1. Markers for characterizing Th17 cells by flow cytometry. Markers are relevant to both human and mouse biology unless otherwise stated in the “Additional information” column.

Table 2. Optimized multicolor immunophenotyping panels (OMIPs) relevant to the characterization of Th17 cells.

Immunoassays are valuable tools for quantifying levels of soluble proteins in cell culture media and biological fluids. They are commonly used to study cytokine expression, and this can include the effector cytokines secreted by activated Th17 cells. Immunoassays for Th17 research can include both enzyme-linked immunosorbent assays (ELISA), which detect levels of a single analyte in the solution, or complex, multiparameter panels that quantify multiple proteins in the same sample using the Luminex platform. Thermo Fisher Scientific offers a variety of Invitrogen ProcartaPlex multiplex panels for detection of Th17 and other T helper cell cytokines via Luminex technology. A list of those relevant to Th17 biology can be found below in Table 3.

Table 3. Invitrogen ProcartaPlex Luminex Panels for Th17 study.

Th17 cells can be differentiated in vitro from CD4⁺ T cells using recombinant cytokines to induce polarization and functional antibodies to prevent signaling from cytokines that promote Th1/Th2 lineage development. We recommend starting the culture with lymphocytes selected to be CD4⁺ by cell sorting or magnetic bead enrichment kits, then consulting a detailed differentiation protocol to determine the appropriate time points and reagent concentrations, as expression of certain Th17 markers may differ depending on the precise culture conditions. Table 4 lists reagents commonly used to culture Th17 cells in vitro.

Table 4. Reagents for in vitro culture of Th17 cells. Consult protocols for recommended concentrations and timing.