As a crucial component of the transforming growth factor-β superfamily, the TGF-β controls Th9 cell development via dual mechanisms that are SMAD-dependent and SMAD-independent (6, 60, 61). In the SMAD-dependent pathway, TGF-β binds to its receptors to promote a SMAD2/3/4 heterotrimeric complex, which translocates to the nucleus, where it binds to transcription factors, thereby controlling the expression of target genes and subsequently promoting Th9 development (62). SMAD7 inhibits this process by preventing the SMAD complex from binding to DNA (63). In the non-SMAD pathway, TGF-β and IL-4 synergistically activate TAK1, downregulate ID3 expression, promote the binding of E2A/GATA binding protein 3 (GATA3) to the IL-9 promoter, and increase the transcriptional activity of IL-9 (5). Furthermore, suppression of sirtuin 1 (SIRT1) deacetylase expression by TAK1 leads to increased IL-9 secretion and glycolytic activity, whereas overexpression of exogenous SIRT1 has the opposite effect (64).

Signal transducer and activator of transcription 6 (STAT6) and GATA3 are key regulators of Th9 cell differentiation (6, 7, 65). IL-4 induces GATA3 expression by activating STAT6 phosphorylation and driving Th2 cell differentiation (6). However, when IL-4 cooperates with TGF-β, it suppresses TGF-β-induced generation of Foxp3⁺, Tregs through the IL-4-STAT6-GATA3 axis while simultaneously downregulating the production of Th2-type cytokines (e.g., IL-4, IL-5, IL-13). This process consequently induces the generation of IL-9-secreting Foxp3^- effector T cells, ultimately promoting Th9 cell differentiation (6). Paradoxically, IL-4 negatively regulates Th9 differentiation under specific conditions.IL-4-induced cytokine-inducible SH2-containing protein (CIS) suppresses activation of STAT3, STAT5, and STAT6, thereby inhibiting Th9 cell differentiation. Consequently, deficiency of CIS in T cells significantly enhances both Th2 and Th9 cell differentiation. This indicates that IL-4 inhibits Th9 differentiation by inducing CIS (66).

Cytokines play pivotal roles in Th9 cell differentiation through complex and diverse regulatory mechanisms, exhibiting both promoting and inhibitory effects. Among cytokines that enhance Th9 differentiation, IL-2 serves as a critical regulator. IL-2 controls Th9 differentiation and IL-9 secretion through two distinct mechanisms, which involve either the activation of STAT5 or the inhibition of B-cell lymphoma 6 (BCL6). IL-2-mediated phosphorylation of STAT5 results in the direct binding of STAT5 to the IL-9 promoter and subsequent IL-9 transcription, whereas a shortage or blockage of IL-2 inhibits IL-9 synthesis (11, 12). IL-2 can also promote Th9 differentiation by suppressing expression of the transcriptional repressor BCL6, which competes with STAT5 to bind to the IL-9 promoter (11). Jiang et al. elucidated that TNF-α comprehensively enhances Th9 cell differentiation, survival, and proliferation through the TNFR2-STAT5 and NF-κB signaling pathways (67). Furthermore, cytokines including IL-36γ from the IL-1 family (68), IL-1 (69), IL-2 (11, 70), IL-6 (71), IL-10 (7), IL-23 (72), IL-25 (73), IL-33 (74), IFNα/β (75), and TSLP (76, 77) contribute to Th9 cell induction. However, their precise molecular mechanisms require further elucidation. Conversely, certain cytokines suppress Th9 cell differentiation. Murugaiyan et al. have demonstrated that IFN-γ directly inhibits Th9 cell differentiation through STAT1-mediated signaling; moreover, it has induced dendritic cells to produce IL-27, which has partially suppressed Th9 cell development through STAT1 and T-bet (78).

As receptors that recognize innate immunological patterns, Toll-like receptors (TLRs) control T cell growth and have a direct impact on Th9 differentiation (79). Studies have shown that in the presence of TGF-β and IL-4, the activation of TLR2 promotes Th9 cell differentiation by upregulating the transcription factors BATF and PU.1 (14). Moreover, a negative correlation between the activity of TLR2 and serum vitamin D levels has been observed, suggesting that vitamin D may negatively regulate Th9 differentiation by inhibiting the TLR2 pathway (34).

Although some interleukins, such as IL-35 and IL-1β, can functionally replace TGF-β under certain circumstances, they still need to work in concert with IL-4 to induce Th9 differentiation. For example, IL-35 has been shown to promote Th9 differentiation through activation of IRF4 via the IL-12Rβ2/GP130 receptor. However, the lack of synergistic effects between TGF-β1 and IL-35 at optimal doses indicates that they probably share partial downstream signaling pathways. In the presence of IL-4, IL-35 can replace TGF-β1 to induce the differentiation of naïve CD4⁺ T cells into Th9 cells, although the underlying mechanisms remain unclear and require further elucidation (15). Other studies have shown that when combined with IL-4, IL-1β can act through the IL-1R signaling pathway to replace TGF-β and promote Th9 differentiation (13). Initiating synergistic effects requires the presence of TGF-β or its alternative components, whereas IL-4 plays a crucial role in Th9 differentiation. Surprisingly, TGF-β and IL-4 are not required for IL-33 to promote Th9 differentiation. For example, dectin-1 signaling in dendritic cells has been shown to promote the production of IL-33, which then acts through its suppression of tumorigenicity 2 (ST2) receptor to induce differentiation of CD4⁺ T cells into Th9 cells, which produce IL-9 (80).

BAFT, a key transcription factor for Th9 development, binds to the IL-9 promoter and controls its production by forming a complex with IRF4 downstream of the TGF-β/IL-4 signaling pathway. While co-expression of BAFT with IRF4 synergistically increases IL-9 production, BATF deficiency dramatically decreases IL-9 production (16). TL1A stimulation has been shown to upregulate Basic leucine zipper ATF-like transcription factor 3 (BATF3), a homolog of BATF, which combines with BATF and IRF4 to form a complex that enhances IL-9 promoter binding and dramatically increases IL-9 secretion (17).

IRF4, a transcription factor that is essential for Th9 differentiation, drives transcription by binding directly to the IL-9 promoter. IRF4-depleted CD4⁺ T cells are unable to differentiate into Th9 cells, highlighting the importance of IRF4 in this process (18). Meanwhile, TGF-β and IL-4 have been shown to promote the conversion of Th2 to Th9 cells through the SMAD3/SMAD4 signaling pathway and induction of IRF4 expression (19). Interferon Regulatory Factor 8 (IRF8) and IRF4 exhibit structural similarities, and have been shown to work together with BATF and PU.1 to form a transcriptional complex that binds to the IL-9 promoter to increase IL-9 expression (81).

PU.1 is a fundamental member of the ETS transcription factor family and the first transcription factor to be found to play an essential role in the specific production of IL-9 in Th9 cells. Studies indicate that PU.1-deficient mice exhibit reduced IL-9 expression. Conversely, PU.1 confers upon Th9 cells the capacity for robust IL-9 production (20). PU.1 suppresses Th2-specific gene expression downstream of TGF-β and IL-4 signaling through inhibition of GATA3 (20). Furthermore, PU.1 has been shown to form a complex with the histone acetyltransferase General Control Non-derepressible 5 (GCN5), which increases histone H3/H4 acetylation levels in the IL-9 promoter region, thereby markedly increasing transcriptional activity and chromatin accessibility (21, 82). Another member of the ETS family, ETS variant transcription factor 5 (ETV5), promotes histone H3K27 and H4K16 acetylation by recruiting the histone acetyltransferase P300, thereby enhancing IL-9 transcription (22). ERG has also been shown to combine with PU.1 and ETV5 to produce a complex that increases the transcriptional activity and binding effectiveness of the IL-9 promoter (23).

Members of the FOX family are involved in regulating the differentiation of Th9 cells. One member, Forkhead box O1 (FOXO1), is a key positive regulatory factor that binds directly to the IL-9 promoter to boost its transcription. FOXO1 has also been shown to promote IRF4 expression, which greatly increases the efficiency of Th9 differentiation (24, 25). In contrast, Forkhead box P1 (FOXP1) has been identified as a negative regulator of Th9 differentiation since it inhibits IL-9 production and prevents Th9 differentiation. In a recent study, IL-7 signaling was found to induce FOXO1 to displace FOXP1 in the nucleus and promote IL-9 production (83). Forkhead box O4 (FOXO4), another member of the FOX family, has also been shown to promote Th9 differentiation (83). Conversely, Th9 differentiation is suppressed by Forkhead box P2 (FOXP2) and Forkhead box P3 (FOXP3). FOXP2 has been shown to adversely affect Th9 differentiation by downregulating the expression of BATF and IRF4 and subsequently destabilizing the core transcriptional complex necessary for Th9 development (26). Th9 differentiation is further negatively regulated by the binding of FOXP3 to GATA3, which inhibits the transcription of Th9-associated genes (7).

Covalent histone modifications and DNA methylation are two of the many ways that epigenetic regulation affects Th9 cell development. At the level of histone modifications, Chromobox protein homolog 4 (Cbx4) increases the transcriptional activity of the Hypoxia-inducible factor-1 alpha (HIF-1α) protein, stabilizing it through SUMOylation, and thus promoting IL-9 expression (84). IL-7 induces the histone acetyltransferase p300 through the STAT5-PI3K-AKT-mTOR pathway, thereby promoting histone acetylation at the IL-9 promoter region and activating transcription (83). By sponging miR-155-5p, the long non-coding RNA LINC00240 has been shown to control DNMT1-mediated PU.1 promoter methylation at the DNA methylation level. While high expression of LINC00240 inhibits this process, low expression alleviates PU.1 inhibition and promotes Th9 differentiation by lowering methylation levels (27).

The lncRNA HOTAIRM1 has been shown to promote Th9 differentiation by competitively binding to miR-148a-3p and reducing its transcriptional repression on IRF4 (85). In contrast, miR-143/145 expression levels are significantly inversely correlated with Th9 differentiation, with miR-143/145 reducing the IL-9 transcriptional pathway by targeting and suppressing Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression (28). Finally, the tumor suppressor gene miR493-5p inhibits Th9 cell development by inhibiting the expression of FOXO1 (29).

Distinct metabolic characteristics are closely associated with Th9 differentiation. Extracellular ATP provides energy support for Th9 development by activating the purinergic receptor mTOR-HIF-1α signaling axis, which in turn induces the generation of NO (30). Furthermore, interactions between amphiregulin and the EGFR strengthen the differentiation process by increasing the ability of HIF-1α to bind to the IL-9 promoter (31). Notably, when compared to other Th subsets, Th9 cells show significantly more glycolytic dependence. In addition to reducing glycolytic capacity, HIF-1α deficiency also interferes with the tricarboxylic acid cycle, pentose phosphate pathway, and fatty acid metabolic network (30). Dysregulation of these metabolic pathways severely disrupts the energy homeostasis of Th9 cells, leading to significant inhibition of their differentiation.

The metabolic microenvironment further regulates Th9 differentiation through metabolitic homeostasis. While succinate stabilizes HIF-1α through succinylation modifications to promote Th9 differentiation, α-ketoglutarate (α-KG) suppresses Th9 differentiation by promoting degradation of HIF-1α (31). Butyrate from gut bacteria and polyamines from the host have opposing effects. Butyrate reduces IL-9 synthesis by activating FOXP3 (33), whereas polyamines positively regulate IL-9 production by increasing GATA3 expression (32). The regulatory network of metabolites provides novel treatment targets for metabolically-based immunomodulatory methods by highlighting the critical role of microenvironmental metabolic homeostasis on Th9 differentiation.

Lipid metabolism plays a role in regulating Th9 differentiation through two independent pathways. First, Th9 differentiation and IL-9 production are markedly increased by the expression of Acetyl-CoA carboxylase 1 (ACC1), a fatty acid synthase, and 3 - Hydroxy - 3 - methylglutaryl - CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis (86). Second, Th9 differentiation is negatively regulated by inhibiting ACC1-mediated fatty acid synthesis via the retinoic acid receptor-TGF-β-SMAD signaling axis. The crucial role of lipid metabolic homeostasis in Th9 differentiation is highlighted by the fact that exogenous oleic acid reverses the effects of ACC1 inhibitors or lipid uptake blockers, which markedly increase IL-9 release (87, 88). Furthermore, the mechanosensory receptor Piezo1 regulates HIF-1α signaling by modulating mitochondrial oxidative phosphorylation, while its functional deficiency suppresses Th9 differentiation (36). These studies reveal a novel mechanism through which the physical microenvironment governs Th9 differentiation.

Vitamin D, a crucial nutrient, inhibits Th9 differentiation by downregulating the expression of transcription factors including PU.1, IRF4, and BATF, and decreasing histone acetylation levels at the IL-9 promoter region (35). In contrast, sphingosylphosphorylcholine activates SMAD3, STAT5, and β-catenin signaling pathways by increasing mitochondrial reactive oxygen species and positively regulating Th9 differentiation (37). These studies show that the metabolic network is closely associated with transcription factor activity and epigenetic modifications. However, since recent studies have focused on single metabolic pathways, future studies need to analyze the mutual effects between different metabolic axes to provide a theoretical basis for the development of precision therapies targeting metabolic pathways.