Following signals from both antigen-presenting cells (APCs) and the micro-environment, activated CD4⁺ T cells are triggered to initiate secretion of specific effector cytokines. Since the original observation in 1986 upon antigenic stimulation naive CD4⁺ T cells can differentiate into T helper 1 (Th1) or Th2 effector T cells depending on polarizing cytokine signals (1), various additional Th subsets have been recognized. These include Th9, Th17, Th22, follicular T helper cells (Tfh), and regulatory T cells (Tregs), each characterized by a unique cytokine production profile and a key transcription factor [see for recent review Ref. (2)]. These Th subsets play a crucial role in appropriate immune responses during host defense, but are also involved in the pathogenesis of inflammatory diseases (3, 4).

Th1 cells mainly produce IFN-γ and TNF-α and are associated with the elimination of intracellular pathogens. Th1 development is facilitated either by IL-12 and STAT4 or by IFN-γ, STAT1, and the key Th1 transcriptional regulator T-box-containing protein (T-bet), encoded by Tbx21 (5). Th2 cells control helminth infections and are implicated in allergic immune responses such as allergic asthma. They are potent producers of Th2 cytokines that induce IgE synthesis (IL-4), recruit eosinophils (IL-5), and cause smooth muscle hyperreactivity and goblet cell hyperplasia (IL-13). Therefore, Th2 cells are central in the orchestration and amplification of inflammatory events in allergic asthma. The master transcription factor Gata3 is necessary and sufficient for Th2 cytokine gene expression in Th2 cells (6). Because Th2 differentiation is driven by IL-4, this raises the paradox that IL-4 is required to generate the cell type that is its major producer. But the origin of the first IL-4 required for Th2 cell induction remains unclear. While a range of cell types are able to produce IL-4, Th2 cell responses can still be generated when only T cells can make IL-4, arguing against an essential role for an external source of IL-4 (7, 8).

An accumulating number of studies suggest that the Notch signaling pathway, which also plays a crucial role in early hematopoietic development and at multiple steps of T lineage development, is essential for Th cell differentiation [for recent review see Ref. (9)]. Currently, two opposing models have been proposed that explain how Notch ligands can influence Th subset differentiation. According to the “instructive” model, Jagged and delta-like ligands (DLL) on APCs induce Th2 and Th1 differentiation, respectively (10). Alternatively, the “unbiased amplifier” model proposes that Notch ligands are not instructive but rather function to generally amplify Th cell responses (11). In this review, we will discuss these two contrasting hypotheses on the role of Notch signaling. We will focus on both Notch receptor expressing T cells and Notch ligand-expressing cells.

There are five Notch ligands: two Jagged (Jagged1 and Jagged2) and three DLL (DLL1, DLL3, and DLL4), which are bound by four receptors, Notch1–4. For these ligands to be functional, their ubiquitination by Mindbomb1 or Neuralized within the cell is required (12). Details of the Notch signaling pathway are discussed in various excellent reviews (13, 14). Briefly, following ligand–receptor binding, the Notch intracellular domain (NICD) is cleaved by a γ-secretase complex and translocates to the nucleus and binds to the transcription factor recombination signal binding protein for immunoglobulin Jκ region (RBPJκ; Figure 1). Finally, additional co-activating proteins are recruited, such as mastermind-like proteins (MAML1-3) and p300 to induce transcription of target genes. Notch signaling does not only induce Th lineage-defining transcription factors and cytokines (described below) but also general pathways critical for T cell activation, including IL-2 production, upregulation of the IL-2 receptor, and glucose uptake (15–18). Notch signaling potentiates phosphatidylinositol 3-kinase-dependent signaling downstream of the T cell receptor (TCR) and CD28 by inducing activation of Akt kinase and mammalian target of rapamycin, which enhances T cell effector functions and survival and allows them to respond to lower antigen doses (16, 19, 20). Notch signaling can be enhanced by the protein kinase PKCθ, which is crucial for TCR and CD28 signaling and regulation of the actin cytoskeleton (21). Moreover, upon TCR stimulation NICD interacts with other proteins in the cell in a non-canonical, RBPJκ-independent pathway that leads to NFκB activation (22, 23).

T helper 2-promoting stimuli including helminth eggs, prostaglandin E2, cholera toxin, and allergens, such as house dust mite (HDM), birch pollen, and cockroach allergens, were shown to induce Jagged expression on APCs, as summarized in Table 1. Conversely, microbial Th1-inducing stimuli, e.g., dengue virus, respiratory syncytial virus (RSV), bacterial LPS, and the TRL9 ligand CpG, up regulate the Notch ligands DLL1/DLL4 on APCs (Table 1). Other studies, however, do not show exclusive upregulation of either DLL or Jagged molecules, but rather upregulation of Notch ligands of both families upon stimulation (10, 24–33). Interestingly, whereas surface induction of DLL requires MyD88, this is not the case for Jagged induction (10, 34–38). LPS can promote both Th1 and Th2 responses, which are MyD88 dependent and Myd88 independent, respectively, but the molecular mechanisms responsible for Jagged induction by LPS are unknown (39–41). Together, although there is also evidence that particular stimuli can induce both Th1 and Th2 differentiations, many studies support an instructive role of DLL and Jagged expression on APCs.

As summarized in Table 1, considerable evidence supports an “instructive model” whereby pathogens direct Th1 and Th2 differentiation via upregulation of DLL or Jagged ligands on DCs (Figure 1). This implies that different Notch ligands induce distinct cellular responses in T cells, largely by the same signaling components. Although it has been speculated that different ligands might induce qualitatively different signals, e.g., RBPJκ-dependent or independent, or signals that differ in strength or kinetics (96), the molecular mechanisms involved are currently unknown.

It has been shown that DLL4 induces a stronger Notch signal than DLL1 or Jagged1 (86). Also, the ability of ligands to induce Notch signaling is dependent on the glycosylation status of the extracellular domain of Notch: Notch receptors carrying N-acetylglucosamine preferentially signal via delta ligands, while Jagged binding is inhibited (97). Absence or overexpression of Fringe glycosyltransferase proteins alters Th1 and Th2 differentiation (60, 98). Another possibility would be that different ligands preferentially activate different Notch receptors, which may each have unique downstream nuclear targets to induce distinct cellular programs. Indeed, it has been reported that whereas Notch1 and Notch2 activate Th2 differentiation, Notch3 promotes Th1 differentiation and IFN-γ production (46, 53). The expression of all these Notch receptors is induced on T cells upon TCR stimulation (62, 99, 100). Because different NICDs have different target gene preferences (101), distinct ligand–receptor combinations may produce quantitatively or qualitatively distinct signals (102). However, this is not supported by the findings that both Th1 and Th2 differentiation is affected in T cells that are Notch1/Notch2 double-deficient (53, 62) and that retroviral expression of Notch1 as well as Notch3 was associated with increased Th1 responses (46, 61). This issue is further complicated by the observation that individual Notch receptors are up regulated with different kinetics (103). It is, therefore, conceivable that they have distinct functions depending on the phase of the response.

Several studies are in apparent conflict with the “instructive model.” For example, DLL were reported to promote Th2 responses or Jagged ligands were implicated in Th1 induction (60, 104). Neither Jagged1 nor DLL1 could instruct Th2 or Th1 cytokine differentiation in vitro in the absence of polarizing cytokines (105). Importantly, Bailis et al. showed that Notch signaling simultaneously induced Th1, Th2, and Th17 gene transcription, also under polarizing conditions that were described to favor only one of the differentiation outcomes (11). In addition, Notch signaling via DLL4 was shown to boost antigen sensitivity of CD4⁺ T cells via promoting co-stimulatory signals in T cells (16). Together, this would suggest that Notch acts as a co-stimulating factor that orchestrates multiple Th cell programs by sensitizing cells to exogenous cytokines, thereby ensuring that activated CD4⁺ T cells overcome a Th cell commitment threshold. In support of a role for Notch as an unbiased amplifier (Figure 1), Notch signaling was shown to be required for optimal T cell expansion, CD25 and IL-2 induction in vitro of both Th1 and Th2 cells (15, 16, 18, 105). Finally, Notch signals promote survival by enhancing anti-apoptotic signals and glucose uptake (17, 106).

It is conceivable that minor differences in experimental design or conditions form the basis of the discrepant results that support one of the two opposing models for Notch function in Th differentiation. Many studies on Notch ligands on APCs have employed GM-CSF cultured bmDCs (Table 1), which were recently shown to contain not only DCs, but also monocyte-derived macrophages (107). In our own studies, we found that Jagged expression was required for the induction of a Th2 response in the lung when in vitro HDM-pulsed bmDCs were used for allergen sensitization, but not when mice were in vivo sensitized by endogenous airway DCs (32). Moreover, studies are complicated by the finding that Notch ligands are not only induced on DCs, but also on macrophages, B and T cells, or lymph node stromal cells (24, 95, 99, 108). Stimulation via CD46 and CD3 was shown to up regulate Jagged1 on human T cells (109), suggesting that T cells can provide Notch signals to each other. However, it is of note that normally several mechanisms, including lateral inhibition, are used to regulate Notch activity when similar cell types express both ligand and receptor. By lateral inhibition signal-sending cells actively repress their Notch signaling pathway (110), which would hamper concerted Notch-mediated differentiation and polarization of adjacent T helper cells. Finally, Notch receptors can become activated independent of ligand binding (111). Indeed, spontaneous Notch cleavage has been observed upon TCR triggering (15, 18, 22). Ligand-independent Notch signaling would also be supported by the recent identification of a PKCθ-dependent mechanism that enhances Notch activation (21). More experiments targeting Notch ligands in various cells types are required to determine how the Notch signaling pathway is activated in T cell subsets in vivo.

Another concern is that some gain-of-function approaches, involving overexpression of Notch receptors or ligands, may be associated with strong or prolonged, less physiological Notch signals. In this context, it is interesting that variable Notch signal strength allows induction of distinct responses by the same signaling pathway (112, 113), paralleling previous experiments demonstrating Th1 or Th2 cells are induced by strong or weak TCR signals, respectively (114, 115). Therefore, in studies on the effects of Notch ligands on Th differentiation, it may be critical to use a range of antigen doses. Finally, since it has recently been shown that Th2 inflammation also crucially involves IL-4-producing Tfh cells (116, 117), findings of impaired in vivo Th2 cell differentiation may point at Tfh rather than Th2 defects and should, therefore, be interpreted with care.

Given the increasing number of characterized Th subsets, it is unlikely that Notch signaling simply acts as a bimodal molecular switch for the induction of either Th1 and Th2 differentiation, based on DLL and Jagged expression on DCs, respectively. Nevertheless, many studies described above support the notion that individual Notch ligands have differential effects on Th cell differentiation, which cannot be explained by the unbiased amplifier model. The two models, however, may not necessarily be mutually exclusive. Effects of Notch signaling could be quite different during induction and during maintenance of Th subset differentiation. Moreover, the finding that there is quite some plasticity between Th subsets (2) and that Th2 differentiation may involve a Tfh phase has further complicated the role of Notch signaling in Th differentiation. We also conclude that the elucidation of the role of Notch ligands on particular cell types requires comprehensive in vivo studies, using cell-specific knockout of individual Notch ligands or combinations.

Since Notch signaling is involved in the differentiation of basically all Th subsets, it could serve as a potential therapeutic target, for example, by inhibiting Th2 responses in allergies or Th1/Th17 responses in autoimmune diseases. However, because effects of GSI are not limited to Notch signaling, it will be valuable to develop more specific compounds targeting Notch signaling components. Indeed synthetic, cell-permeable stabilized peptides that target a critical protein–protein interface in the Notch transactivation complex (118–120) as well as specific antibodies that target Notch receptors (121–123) or Notch ligands (24, 124) have been designed. Promising results were obtained with Notch pathway blocking antibodies in cancer patients (125) and future studies should explore whether these antibodies are beneficial for allergic or autoimmune patients.

Interestingly, GSI administration during only the challenge in asthma models was sufficient to decrease Th2 cytokine production (58, 59). These findings imply that Notch signaling is not likely critical to initiate IL-4 production in activated T cells and thus the initial source of IL-4, for example in AAI, remains unclear. While several cells including basophils, Tfh cells, NKT cells, and ILC2 are capable of producing IL-4 (55, 116, 126–131), mice deficient for NKT cells, ILC2, or basophils are still capable of inducing Th2 responses (132–134), suggesting that IL-4 production by Tfh cells could be crucial for Th2 cell induction. Nevertheless, the finding that in animal models allergic disease symptoms are reduced by GSI administration during challenge only indicates that Notch signaling is important in maintaining rather than inducing Th2 cell responses. This makes Notch signaling an interesting target for development of therapeutic strategies in allergic asthma.

IT and MP performed the literature research, wrote the paper, and designed the figure. RH critically revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.