A model of Th2 differentiation based on polarizing cytokine repression

Abstract

Conventional dendritic cells (cDCs) can integrate multiple stimuli from the environment and provide three separate outputs in terms of antigen presentation, costimulation, and cytokine production; this guides the activation, expansion, and differentiation of distinct functional T helper subsets. Accordingly, the current dogma posits that T helper cell specification requires these three signals in sequence. Data show that T helper 2 (Th2) cell differentiation requires antigen presentation and costimulation from cDCs but does not require polarizing cytokines. In this opinion article, we propose that the “third signal” driving Th2 cell responses is, in fact, the absence of polarizing cytokines; indeed, the secretion of the latter is actively suppressed in cDCs, concomitant with acquired pro-Th2 functions.

The dendritic cell-T cell three-signal activation dogma.

Mammalian primary CD4⁺ T cell activation and T cell fate specification is tightly regulated, and it is accepted that it requires three sequential signals provided by conventional dendritic cells (cDCs) (see Glossary) [1]. Signal 1 is delivered by the T-cell receptor (TCR) after recognition of cognate antigen in the context of major histocompatibility complex (MHC). Signal 2 involves the binding of costimulatory receptors, primarily CD28. Signal 3 is delivered in the form of polarizing cytokines, which signal via cytokine receptors on T cells. Signal 1 ensures the activation of antigen-specific T cells. Signal 2 amplifies the intracellular signaling process triggered by signal 1 and, together, stimulate T cell to secrete interleukin (IL)-2 and simultaneously to upregulate expression of the high-affinity IL-2 receptor (IL-2R). The binding of IL-2 to IL-2R activates intracellular signaling pathways that help T cells to proliferate. Although TCR signal strength and costimulation can contribute to modulating the subsequent fate of T cell differentiation, signal 3 (i.e., the cytokine milieu) is thought to be the most important factor for this process [1,2]. As such, the successive binding of polarizing cytokines to cytokine receptors and the triggering of signaling via different classes of signal transducer and activation of transcription (STAT) proteins lead to the expression of the lineage-specifying transcription factors and subsequent differentiation of distinct T helper (Th) cell subsets (Box 1). Specifically, IL-12 induces Th1 cell differentiation by promoting interferon-γ (IFNγ production and upregulation of T-bet [3,4]. Transforming growth factor-β (TGFβ) IL-10, and IL-2 induce and maintain Foxp3 expression and thus, support the differentiation of regulatory T (Treg) cells [5,6]. However, the presence of TGFβ along with IL-6 and IL-23 promotes the expression and maintenance of RORγt and the induction of Th17 cells [7]. The importance of polarizing cytokines in determining Th1, Th17, and Treg cell differentiation has been thoroughly demonstrated in vivo, using gene-deficient mice, where defective expression of a particular cytokine signal transduction pathway is associated with defects in the related Th cell subset [3–5,7]. Since recognition of pathogen-derived or danger signals triggers the production of IL-12, TGFβ, IL-10, IL-6, and IL-23 by cDCs [8,9], cDCs are generally believed to be sufficient for priming Th1, Th17, and induced Treg cells, thus providing the three necessary signals (Figure 1).

Box 1. CD4⁺ T cell differentiation.

CD4⁺ Th cells can differentiate into several subtypes, including T-bet⁺ Th1, RORγt⁺ Th17, Foxp3⁺ Treg, and GATA3⁺ Th2 cells [56]. Th1 cells are characterized by IFNγ production, and are important for resistance to intracellular pathogens [56]. Th17 cells produce cytokines IL-17A, IL-17F, and IL-22 and are required for host defense against specific fungi and extracellular bacteria [56]. Treg cells are involved in maintaining immune homeostasis and self-tolerance by inhibiting the activation of the immune system and preventing pathological self-reactivity [57]. Th2 cells secrete IL-4, IL-13, IL-5, and IL-9 and promote the eradication of parasitic helminths. In addition, unregulated Th2 cell responses are associated with the development of allergic diseases such as atopic dermatitis, allergic rhinitis, asthma, eosinophilic esophagitis, and food allergy [58]. The induced expression of the lineage-specifying transcription factors T-bet, GATA3, RORγt, and Foxp3⁺ allows the expression of signature cytokines, thus dictating Th cell fates and functions [59]. Beyond this, the upregulation of Bcl6 expression promotes the differentiation of T follicular helper (Tfh) [60–63] and T follicular regulatory (Tfr) [64] cells in B cell follicles, where they regulate humoral responses. Conversely, upregulation of Blimp-1 controls effector Th cell differentiation [61,63], which provides help to CD8⁺ cytotoxic T cells and activates cells of the innate immune system.

Figure 1. cDC-mediated T cell activation and T helper cell differentiation.

cDCs provide three separate signals to naïve CD4⁺ T cells to induce their full activation and subsequent differentiation into distinct T helper (Th) cell subsets defined by differential expression of signature cytokines and lineage-specifying transcription factors. Signal-1 is equivalent to the binding of TCR to peptide MHC complex. Signal-2 is generated by the engagement of costimulatory receptors such as CD28. The combination of signal-1 and signal-2 is required to fully activate CD4⁺ T cells. Signal-3 is produced by the secretion of polarizing cytokines by cDCs that respond to homeostatic, danger, or pathogen-derived signals. Particular sets of polarizing cytokines induce the expression of the master transcription factors Foxp3, RORγT, or T-bet, guiding the differentiation of inducible Treg, Th17, and Th1 cells [59]. Although cDCs are necessary and sufficient to induce Th2 cells in mouse models [2,10–15], a cDC-derived cytokine that upregulates the transcription factor GATA-3 and promotes Th2 cell lineage commitment has yet to be found. Th cells that acquire a cytokine profile can then upregulate the transcription factor Bcl6 to control B cell responses [60–64] or upregulate Blimp1 to fulfill effector Th functions in peripheral tissues [61,63]. cDC, conventional dendritic cell; IFNγ interferon-γ; IL, interleukin; MHC II, major histocompatibility complex class II; TCR, T-cell receptor; TGFβ, transforming growth factor; Tfh, follicular helper T cells; Tfr, T follicular regulatory cells; Treg, regulatory T cells. Figure created with Biorender.com.

Moreover, while accumulating evidence supports the notion that cDCs are necessary and sufficient to prime Th2 cells [2,10–15], no equivalent cDC-derived cytokine or “signal 3” has been found to induce Th2 cell differentiation. Instead, Th2 cell differentiation is favored in the absence of polarizing cytokines (or signal 3) that drive differentiation into other Th cell lineages [4,16–23]. Furthermore, the acquisition of pro-Th2 functions in cDCs is associated with the suppressed production of polarizing cytokines [14,15]. Based on these findings (examined in detail below), in this opinion article, I propose that the Th2 cell differentiation pathway is an exception to the three-signal dogma for Th cell differentiation, where signals 1 and 2 are required, along with the absence or active repression of signal 3. A better understanding of this area might help us comprehend the increasing prevalence of Th2 cell-driven diseases that are associated with recent changes in lifestyle, and which might represent a trend of reduced exposure to stimuli that drive polarizing cytokine secretion.

Th2 cell differentiation requires TCR-mediated activation, CD28 costimulation, and IL-2 signaling.

TCR/CD28-driven upregulation of the autocrine growth factor IL-2 and the high-affinity IL2R is crucial for the optimal expansion of recently activated T cells. In addition, IL-2 signaling has been implicated in regulating T cell polarization by rendering activated T cells more receptive to subsequent cytokine stimulation [24,25]. In particular, IL-2-induced activation of STAT5 has long been known to contribute to initial Th2 cell lineage commitment. Specifically, in vitro mechanistic studies using murine primary T cells with defective expression of members of the IL-2 or IL-4-mediated signaling pathways have shown that IL-2-STAT5 signaling is essentially required to promote transcription of the Il4ra gene, which encodes the IL-4 receptor alpha chain (IL-4Rα). This led to increased surface expression of the IL-4 receptor (IL-4R), thereby enhancing IL-4 responsiveness [26]. Furthermore, STAT5 DNA binding studies in activated human and murine T cells have shown that IL-2 stabilizes the accessibility of the Il4 locus, licensing early IL-4 production [26,27]. Thus, IL-2 signaling contributes to initiating and stabilizing the Th2 cell phenotype by promoting a positive feedback loop that first increases IL-4 secretion and responsiveness, and subsequently through IL-4-STAT6 activation, upregulates GATA3 to promote sustained IL-4 production [26,27] (Figure 2A). Recent studies using a mouse model of house-dust-mite (HDM)-induced allergic airway inflammation have corroborated the importance of IL-2 signaling for the induction of Th2 cell responses to natural allergens [21]. Overall, in vitro and in vivo data suggest that Th2 cell differentiation can proceed with adequate TCR- and CD28-derived signals, driving strong IL-2 production, and that it does not require any other external stimulus or cytokine. However, IL-2 production and signaling are required for early T cell activation preceding any T cell fate decision. Therefore, the question is: how can Th2 cell commitment be specifically regulated? One possibility is that the duration of IL-2 signaling determines the initial Th2 cell lineage commitment. In vivo analysis of allergen-specific T cells in mice has shown that IL-2 signaling during the first 24 to 48 hours post-stimulation is required for full T-cell expansion; however, the prolongation of IL-2 signaling beyond this time period is particularly necessary for Th2 cell differentiation but dispensable for polarization toward other lineages, such as Th17 and Th1 cell subsets [21]. In summary, these novel studies open the prospect that Th2-biased cell responses might be more reliant on prolonged IL-2 signaling than other Th cell responses. Going one step further, it is possible that CD4⁺ T cells default into the Th2 cell pathway in the presence of prolonged IL-2 signaling but in the absence of positive signals (or cytokines) that drive differentiation into other Th cell lineages. This hypothesis was initially suggested based on experimental models in mice [4,28], but recent evidence from human studies reinforces this idea, as discussed below.

Figure 2. cDC-mediated Th2 cell priming (key figure).

(A) Ligation of TCR and CD28 receptors on naive CD4⁺ T cells induces IL-2 secretion and upregulates the expression of the alpha chain of IL-2 receptor (IL-2Rα or CD25), leading to surface expression of high-affinity IL2R [24]. IL-2-IL-2R interactions promote activation and proliferation and prepare CD4⁺ T cells to receive subsequent differentiation signals [24,25]. Prolonged IL-2 stimulation in the absence of polarizing cytokines favors Th2 cell lineage commitment by promoting the expression of the alpha chain of the IL-4 receptor (IL-4R) and IL-4, allowing for an IL-4-positive feedback loop that initiates and preserves the Th2 cell phenotype [21,26,27]. (B) Polarizing cytokines secreted by cDCs upon pathogen detection or under inflammatory or homeostatic conditions repress the Th2 cell differentiation program in mice. IL-12 suppresses Th2 cell priming by inducing T-bet and inhibiting GATA3 in CD4⁺ T cells [4,16,29,30]. IL-6 upregulates suppressor of cytokine signaling 3 (SOCS3) in CD4⁺ T cells, which inhibits IL-2 signaling and early Th2 cell commitment [21]. TGFβ signaling prevents the Th2 cell differentiation program by unknown mechanisms [20]. cDC, conventional dendritic cell; IL, interleukin; MHC II, major histocompatibility complex class II; STAT5, signal transducer and activation of transcription 5; TCR, T-cell receptor; TGFβ, transforming growth factor. Figure created with Biorender.com.

Th2 cell differentiation proceeds in the absence of DC-derived signal 3.

IFNγ, IL-12, IL-6, and TGFβ are key cytokines that control Th1, Th17, and inducible Treg differentiation. In addition, various in vitro and in vivo studies in experimental model systems and in humans with loss and gain-of-function mutations have shown that the expression of these cytokines or the associated lineage-specifying transcription factors is essential to repressing Th2 cell differentiation, as detailed below.

T-bet can be redundantly upregulated by IL-12 and IFNγ [4]. Furthermore, T-bet is essential for Th1 cell differentiation, but T-bet also plays a crucial role in suppressing the Th2 cell-associated program [4,16,29,30]. Indeed, loss of function approaches carried out in mice following Toxoplasma gondii infection or during homeostatic proliferation have shown that deletion of T-bet in T cells results in the activation of the Th2 cell differentiation program [4]. In contrast, overexpression of T-bet in in vitro Th2-polarized murine CD4⁺ T cells results in the loss of the Th2 cell phenotype [29]. Recent human studies likewise support a role for T-bet in actively preventing Th2 cell development. Human T-bet deficiency causes increased production of the Th2 cytokines IL-4, IL-5, and IL-13 due to the Th2 skewing of T-bet-deficient CD4⁺ T cells, a phenotype that is reversed ex vivo upon expression of wildtype T-bet (via retroviral transduction) [16,31,32]. Furthermore, single-cell transcriptome analyses have revealed that the acquisition of a T-bet/IFN signature in allergen-responsive T cells in healthy subjects is associated with the absence of a Th2-biased phenotype and protection from allergy [33]. Genome-wide ChIP-sequencing (seq) and RNA-seq analyses of in vitro activated wildtype and Tbx21^−/− primary murine CD4⁺ T cells have shown that T-bet directly suppresses Th2 cell polarization by inhibiting the expression and function of GATA3 through epigenetic repression at the Gata3 locus [4] as well as via protein-protein interactions [30].

IL-6 induces the development of Th17 cells in concert with TGFβ. However, human studies also suggest an important role for IL-6 in controlling Th2 skewing. Specifically, loss-of-function mutations in human genes affecting IL-6 signaling, including the IL6 receptor (IL6R) [17], Glycoprotein 130 (GP130) [18,34], and STAT3 [19,35,36], have led to increased Th2 cell development, IL-4-dependent Immunoglobulin (Ig)E class switching, and manifestations of allergy, as evidenced by eosinophilia and atopic dermatitis [17,37]. In addition, genome-wide association studies (GWAS) in asthmatic and allergic humans found that the IL6R locus is associated with atopic status among asthmatics, suggesting that IL-6 signaling dysregulation increases the risk of Th2 cell-biased immune responses and allergy [38,39]. Studies in mice have shown that IL-6 signaling in recently activated T cells following in vivo allergen exposure, prevents Th2 cell lineage commitment. Mechanistically, IL-6 suppresses prolonged IL-2 signaling during early T-cell activation, thereby inhibiting a shift toward a Th2 cell profile – a phenotype that is impaired in mice with defective IL-6 or excessive IL-2 signaling in allergen-specific T cells [21]. Moreover, IL-6-driven inhibition of IL-2 responsiveness in responding T cells is mediated by the upregulation of the suppressor of cytokine signaling 3 (SOCS3) and subsequent inhibition of Janus kinase (Jak)1-STAT5 signaling induced by IL-2 [21]. Thus, these data indicate that IL-6-driven SOCS3 acts to inhibit a prolonged IL-2 response in newly activated T cells and suggest that this pathway is an essential mechanism for suppressing Th2 cell development.

From another angle, TGFβ triggers Foxp3 expression in naïve T cells and therefore, induces Treg cell differentiation [40,41]. However, patients with mutations in the receptor subunits of TGFβ are strongly predisposed to developing aberrant Th2 cell responses and allergic diseases, including allergic rhinitis, asthma, food allergy, eosinophilic gastrointestinal disease, and atopic dermatitis [20,42,43]. TGFβ signaling in these patients is dysregulated, as evidenced by the slightly delayed, but overall enhanced, TGFβ-driven intracellular signaling in in vitro stimulated CD4⁺ T cells [20]. Although this altered TGFβ signaling does not affect Foxp3 expression and Treg development and function, it promotes naïve CD4⁺ T cells skewing towards a Th2 phenotype in a cell-autonomous manner [20]. These data suggest that under homeostatic conditions, TGFβ signaling not only promotes Treg differentiation but may also suppress the Th2 cell differentiation program. Furthermore, the findings indicate that distinct signaling pathways downstream of TGFβ can control the induction of Treg cells and the repression of the Th2 pathway. TGF-β can signal by activating both canonical SMAD-dependent and non-canonical SMAD-independent signaling pathways [44]. The TGF-β-SMAD pathway controls Foxp3 transcription and Treg development [40], while non-canonical signaling often cooperates with and modulates the activity of the SMAD-dependent pathway [44]. T cells from patients with mutations in the TGFβ receptor subunits leading to dysregulated TGFβ signaling and enhanced Th2 cell differentiation, show increased SMAD-dependent and dysregulated SMAD-independent signaling in response to in vitro stimulation with TGFβ [20]. However, the exact mechanism by which dysregulated TGFβ signaling promotes Th2 cell differentiation is still unclear. Thus, although studies on the mechanism are still lacking, data suggest that intrinsic TGFβ signaling in T cells is required to suppress the Th2 cell differentiation pathway.

Overall, although cross-regulation between Th cell subsets is not unique to the Th2 cell pathway [45], this mechanism appears to be of vital importance in regulating the Th2 cell balance. As such, primary cytokines and transcription factors that promote the Th1, Th17, and inducible Treg cell pathways can independently suppress Th2 cell differentiation. Importantly, a defect in any of these three pathways fosters the development of unwanted Th2 cell responses, showing the non-overlapping roles of these pathways in preventing inappropriate Th2 responses and the need for their synchronized actions (Figure 2B).

cDCs repress signal 3 to induce Th2 cell differentiation.

The observation that initial Th2 cell commitment can proceed successfully in the presence of TCR/CD28 signaling leading to IL-2-driven activation, but is conversely strongly inhibited by the presence of key polarizing cytokines, has led to the hypothesis that CD4⁺ T cells default to the Th2 cell pathway in the absence of signals driving differentiation into other Th cell lineages [2,4,28]. However, evidence suggests that more than being a default pathway, Th2 cell development in response to Th2-polarizing stimulation might be the result of active suppression of the production of polarizing cytokines, thus enabling Th2 differentiation [14,15]. For instance, many studies have shown that cDCs are required for the induction of Th2 cell responses [2,10,11]. cDCs can be divided into two ontogenically distinct subsets, type-1 cDCs (cDC1s) and type-2 cDCs (cDC2s). Although cDC1s particularly excel in eliciting effector CD8⁺ T cell and Th1 cell responses, cDC2s harbor remarkable functional plasticity and can influence almost all types of CD4⁺ T cell responses. Thus, cDC2s are known to induce Th2 cell responses in specific immune settings, as shown in experimental mouse models of helminth infection and allergic airway inflammation [2]. However, cDC2s can also suppress Th2 cell development in other immunological contexts; for example, after respiratory exposure of mice to allergens containing microbial-derived products [22,23]. The functional specialization of cDC2 and their subsequent ability to promote or prevent Th2 cell responses are crucially influenced by the signals received in the microenvironment [2]. The combined use of cDC-specific conditional knockout mice and in vivo murine models has established that the functional adeptness of cDC2s in the induction of Th2 cell responses requires IL-13 signaling, activation of STAT6 [14], and the induced functional activation of the transcription factors interferon regulatory factor 4 (IRF4) [15] and krüppel-like factor 4 (KLF4) [13]. Furthermore, Group 2 innate lymphoid cells (ILC2) are an early source of IL-13 in mice after Th2-directing stimulation, such as helminth infection or allergen exposure [46]. However, ILC2s can also produce homeostatic IL-13 in specific tissues, such as the murine skin [14]; this may suggest that skin environment is intrinsically predisposed to Th2 induction. By utilizing mouse strains with targeted depletion of ILC2, IL-13 expression by ILC2 has been shown to essentially contribute to the licensing of cDC2 for the induction of Th2 cell responses [14,46]. Ultimately, although the data suggest that IL-13/STAT6, IRF4, and KLF4 control the ability of cDC2 to induce Th2 cell responses, it is unclear how these transcription factors cooperate to license the DC signals that drive the development of Th2 responses. However, one shared function is that these transcription factors suppress the capacity of cDC2s to produce polarizing cytokines by different mechanisms. Specifically, detailed characterization of gene expression in the Th2-promoting cDC2 subset from mouse skin and lymph nodes indicates that IL-13/STAT6 signaling and IRF4 expression in cDC2 suppress the ability to sense bacterial and fungal components by repressing the expression of pattern recognition receptors (PRR), particularly Toll-like receptors (TLRs) [14,15]. TLR activation triggers the induction of pro-inflammatory and polarizing cytokines such as IL-12, IL-23, and IL-6, wherein the TLR-interacting adaptor molecule myeloid differentiation primary response 88 (MyD88) recruits various signaling molecules and transcription factors. In primary macrophages and in macrophage cell lines, IRF4 interacts with MyD88 and acts as a negative regulator of TLR signaling [47], while KLF4 cooperates with STAT6 to sequester coactivators required for nuclear factor-κB (NF-κB) activation downstream of TLR activation [48]. Overall, I argue that these data suggest that the molecular basis by which cDC2s may promote Th2 responses is the activation of a transcriptional program that actively suppresses the capacity to produce polarizing cytokine, such as IL-6, IL-23, and IL-12 (Figure 3), while preserving their capacity to provide costimulatory signals. On the one hand, this is not incompatible with the possibility that cDC2s additionally provide positive signals to induce Th2 cell differentiation, apart from the costimulation that leads to IL-2 production. However, presently, these possible positive signals have not been yet identified.

Figure 3. Transcription factor-guided functional specialization of type-2 cDCs for Th2 cell priming.

Mouse research indicates that IL-13 activates STAT6 and stimulates the expression and functional activity of the transcription factors IRF4 and KLF4 in type-2 cDCs (cDC2s) [12–15,46]; this ultimately promotes the functional specialization of cDC2s to support Th2 cell differentiation by reducing the ability of cDC2s to produce polarizing cytokines IL-12, IL-6, and IL-23 by three independent mechanisms: 1) IL-13/STAT6 signaling and IRF4 expression repress the expression of TLRs [14,15]; 2) IRF4 interacts with MyD88 and acts as a negative regulator of TLR signaling [47]; 3) KLF4 cooperates with STAT6 to sequester coactivators required for NF-kB activation downstream of TLR activation [48]. In contrast, the priming of Th2 cells by cDC2s is prevented by transcriptional regulation that enhances the production of polarizing cytokines. Induced expression of T-bet promotes sustained IL-12 production, suppressing Th2 cell priming and promoting a Th1-like profile [22,23]. Activation of CREB and RORγt represses IRF4 and KLF4 expression and promotes IL-6 and IL-23 production, thus promoting Th17-biased responses while suppressing Th2 cell priming [49,50]. AP1, activator protein 1; cDC, conventional dendritic cell; CREB, cAMP-response element binding protein; IRF4, interferon regulatory factor 4; IL, interleukin; IL-13R, IL-13 receptor; KLF4, krüppel-like factor 4; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor-κB; STAT6, signal transducer and activation of transcription 6; TLR, toll-like receptor; TRAF6, TNF receptor associated factor 6. Figure created with Biorender.com.

On the other hand, transcriptional regulation of cDC2s leading to the production of the polarizing cytokines IL-6, IL-23, and IL-12, can repress the ability of cDC2 to induce Th2 cell responses. As such, in a mouse model of allergen-induced airway inflammation, intrinsic upregulation of the transcription factor T-bet was required for cDC2s to produce IL-12 and to suppress Th2 cell priming and lung inflammation, instead promoting a Th1-like profile [22,23]. In addition, the expression of RORγt [49] and the activation of cAMP-response element binding protein (CREB) signaling [50] in cDC2s from mice stimulated with various PPR and cAMP-depended stimuli, repressed the expression of IRF4 and KLF4, and promoted IL-6 and IL-23 production, thus favoring Th17-biased responses while suppressing Th2 cell priming (Figure 3). Thus, teasing out the exact transcriptional networks that control the production and repression of different polarizing cytokines by cDC2s represent an important challenge in the field. Tackling this challenge may propel a better understanding of the functional plasticity of cDC2s and enable the manipulation of cDC2 programming to modulate Th2 cell immunity.

Concluding remarks

The last century has witnessed an allergy epidemic with substantial epidemiological data confirming a concerning increase in the prevalence of Th2-driven allergic diseases, including asthma, rhinitis, atopic dermatitis, and food allergy [2,51]. Importantly, Th2-driven allergic disorders often have an early onset, suggesting that during early childhood, there is a predisposition to induce Th2-biased immune responses [2,22]. In addition, our current lifestyle contributes to exacerbating this trend [2,51]. Elucidating the signals that drive or prevent the development of Th2 cell responses to allergens and the cellular networks involved in these processes may help us better understand the genetic determinants as well as the environmental factors that influence allergic disease risk. Furthermore, this information can be crucial for developing prevention approaches that safely, effectively, and specifically target factors influencing Th2 cell fate decision to reduce the risk of allergic disorders. The evidence described in this opinion suggests that the repression or suboptimal production of different polarizing cytokines, including IL-12, IL-23, IL-6, or TGFβ, by cDCs may favorTh2 cell development [4,14–22]. In contrast, the induced secretion of these cytokines seems pivotal for preventing heightened Th2 cell immunity [21–23]. The sensing of danger or microbial products triggers the polarizing cytokine response [1,2,28], and therefore, the factors influencing this process can crucially impact Th2 cell balance. Studies in mouse models have suggested that infants are poorly responsive to stimulation with pathogen-derived products and, consequently, cDC2s have a decreased ability to produce polarizing cytokines and suppress allergic Th2 cell responses [22,23]. Furthermore, epidemiological studies indicate that urbanization is linked to restricted exposure to microbial products in common circulating allergens, and to an increased risk of developing Th2-driven allergen-induced diseases, particularly during the first years of life [2,52–55]. However, further research is required to assess the effect of quantity but also exposure to a variety of microbial contaminants on allergens, as well as other environmental interactions that ensure the production of the full spectrum of polarizing cytokines. To analyze these processes, it will also be necessary to delve into the different requirements that may exist from birth to adulthood. These studies are only beginning to uncover the role and underlying mechanisms by which individual polarizing cytokines can suppress Th2 cell responses, the influence of environmental factors in controlling the functional heterogeneity of cDCs, and their impact on Th2 cell-mediated immunity (see Outstanding Questions).

Outstanding questions.

• In addition to establishing a specific Th program, does each polarizing cytokine induce an associated signaling pathway in activated T cells that represses Th2 lineage commitment? Mouse studies have shown that IL-12 and IL-6 initiate signaling pathways in activated CD4⁺ T cells to prevent the Th2 cell program during Th1 and Th17 cell responses. It is unclear whether other polarizing cytokines, such as IL-23, TGFβ, or IL-10, can activate independent signaling pathways aimed at repressing Th2 cell differentiation.

• How are cDCs, particularly cDC2s, instructed to induce or repress the production of different polarizing cytokines? Recent studies in mouse models have begun to show that specific transcriptional networks control the functional specialization of cDC2s by regulating the secretion of particular polarizing cytokines and the induction of corresponding Th-cell responses while repressing or promoting Th2 cell differentiation. Further studies are needed to fully understand the specific signals that drive cDC2 diversification and their ability to induce or suppress the production of IL-12, IL-6, IL-23, IL-10, and TGFβ.

• During infancy/childhood, is there an attenuated ability to produce polarizing cytokines? Could this substantially contribute to a greater tendency to prime Th2 cell responses and translate into a higher risk of developing allergies? Studies in humans and mice have shown that during infancy, there is decreased production of polarizing cytokines in response to microbial stimulation. Mechanistic studies in mice suggest that this hypo-responsiveness to microbial stimulation and subsequent low secretion of polarizing cytokines, particularly IL-12, may contribute to an increased risk of developing allergen-specific Th2 cell responses during childhood. Further studies are warranted to assess whether during infancy and early childhood, higher doses and/or diverse exposure to aeroallergen-contaminating environmental microbial products is required to prevent the early development of Th2 cell allergic responses.

Significance.

A precise understanding of the signals driving and licensing T helper 2 (Th2) cell responses is key to manipulating Th2 cell immunity in health and disease. The current paradigm assumes that the polarizing signals provided by dendritic cells dictates the choice of T helper cell response. However, recent evidence suggests that the induction of Th2 cell responses might follow a different path requiring the suppression of polarizing signals.

Highlights.

• Current dogma holds that functional differentiation of helper T cells expressing lineage-specifying transcription factors and signature cytokines requires three signals provided by dendritic cells (DCs).

• TCR signaling (or signal 1) defines specificity, costimulation (or signal 2) ensures full T activation and expansion, and polarizing cytokines (or signal 3) upregulate specific master transcription factors thereby guiding T cell differentiation into distinct functional helper subsets.

• However, in vitro and in vivo studies provide evidence suggesting that Th2 cell differentiation may follow a particular DC-T cell two-signal activation model, in which signals 1 and 2 are required in the absence of signal 3.

• We propose a model that bypasses the need for signal 3, and instead, Th2 cell commitment requires the active suppression of polarizing cytokine secretion by DCs.

Glossary

CD28

receptor expressed on T cells that binds to CD80 (B7.1) and CD86 (B7.2) proteins expressed on activated dendritic cells and provides costimulatory signals necessary for T cell activation, proliferation, and survival.

ChIP-seq analysis

technology to map genome-wide locations of DNA-associated proteins such as transcription factors or modified histones.

Ig class switching

biological mechanism in B cells which allows the switch in immunoglobulin production from one isotype to another, such as from IgM to the IgG, IgE, or IgA isotypes.

Conventional dendritic cells (cDCs)

innate cells residing in peripheral tissues and secondary lymphoid organs whose primary function is to capture, process, and present protein-derived peptides to T cells and mediate their activation and functional polarization.

Effector T helper cells

activated CD4⁺ T cells that migrate to peripheral tissues where they secrete signature cytokines and activate local immune responses to eliminate the pathogens that caused their activation.

Group 2 innate lymphoid cells (ILC2)

type of innate lymphoid cells that produce type-2 cytokines, such as IL-13, and are involved in early responses against allergens and helminth infections.

High-affinity IL2 receptor (IL-2R)

heterotrimeric protein comprised of α chain (IL-2Rα or CD25), β chain (IL-2Rβ or CD122), and γ chain (IL-2R γ or common y chain); binds IL-2 with high affinity on activated T cells and Treg cells.

Major histocompatibility complex class II

group of genes encoding proteins found on the surfaces of antigen-presenting cells, such as dendritic cells, whose primary function is to bind peptide fragments derived from proteins and display them on the cell surface for recognition by the appropriate CD4⁺ T cell.

Pattern recognition receptors (PRR)

class of germline-encoded receptors that recognize molecules frequently found in pathogens (so-called pathogen-associated molecular patterns or PAMPs). Activation of PRRs results in the expression of a variety of polarizing and proinflammatory cytokines and the upregulation of costimulatory molecules; this is crucial for the initiation of innate immunity and a prerequisite for subsequent activation and shaping of adaptive immunity.

RNA-seq analysis

technology used for the detection and quantitative analysis of messenger RNA molecules in a biological sample.

Single-cell transcriptome analysis

technology used to examine RNA transcripts of individual cells, providing a high-resolution view of cell-to-cell variation.

T follicular helper (Tfh) cells

specialized subset of CD4⁺ T cells defined by the expression of the transcription factor Bcl6; they provide help to B cells and are essential for the germinal center formation, antibody isotype switching, affinity maturation, and the development of high-affinity antibodies and memory B cells.

T follicular regulatory cells (Tfr)

subset of Treg cells that suppress Tfh cells and B cells; characterized by co-expression of the transcription factors Bcl6 and Foxp3.

Toll-like receptors

type of PRR that initiate immune responses by sensing PAMPs. In mammals, a total of 13 TLRs (TLR1–13) have been found; however, humans express only 10 (TLR1–10) of them. Examples of PAMPs recognized by TLRs include bacterial cell surface lipopolysaccharides (LPS), lipoproteins, and peptidoglycans; proteins such as flagellin from bacterial flagellae; single and double-stranded RNA of viruses; unmethylated CpG islands of bacterial and viral DNA.

Type-2 conventional dendritic cells (cDC2s)

distinct from cDC1s; characterized by surface expression of CD11b and CD172α (SIRPα) and the requirement for the transcription factor IRF4 for their development from common DC progenitors.