The Dichotomy of Interleukin-9 Function in the Tumor Microenvironment

Abstract

Interleukin 9 (IL-9) is a cytokine with potent proinflammatory properties that plays a central role in pathologies such as allergic asthma, immunity to parasitic infection, and autoimmunity. More recently, IL-9 has garnered considerable attention in tumor immunity. Historically, IL-9 has been associated with a protumor function in hematological malignancies and an antitumor function in solid malignancies. However, recent discoveries of the dynamic role of IL-9 in cancer progression suggest that IL-9 can act as both a pro- or antitumor factor in various hematological and solid malignancies. This review summarizes IL-9-dependent control of tumor growth, regulation, and therapeutic applicability of IL-9 blockade and IL-9-producing cells in cancer.

Introduction

Tumors develop in complex and dynamic microenvironments, which influence tumor growth, immune evasion, and metastasis. Therapeutics targeting the tumor microenvironment (TME) have garnered considerable attention as a promising approach for cancer treatment (Binnewies et al., 2018). The complexity of interactions and the diversity of the immune cells in the TME can greatly impact tumor development through the secretion of cytokines. IL-9, a pleiotropic cytokine, was originally identified as a growth factor for T cells. Lymphocytes are the primary source of IL-9; with T helper 9 (Th9) cells ostensibly acting as the central source of IL-9 in inflammatory diseases. However, IL-9 can also be produced by B cells (Takatsuka et al., 2018), Tc9, Th2 (Chang et al., 2010), Th17 (Nowak et al., 2009), Treg cells (Heim et al., 2022), mast cells (Hültner et al., 2000; Wiener et al., 2004), NKT cells (Lauwerys et al., 2000), innate lymphoid cells (Mjösberg et al., 2012; Wilhelm et al., 2011), eosinophils (Gounni et al., 2000), osteoblasts (Xiao et al., 2017), and neutrophils (Sun et al., 2018).

IL-9 signals through the IL-9 receptor (IL-9R) (Uyttenhove et al., 1988). The expression of the IL-9R can be found in many cell types, including mast cells, NKT, T cells, B cells, epithelial cells, ILCs, dendritic cells, granulocytes, and macrophages (Donninelli et al., 2020; Elyaman et al., 2009; Fawaz et al., 2007; Guggino et al., 2016; Mohapatra et al., 2016; Moretti et al., 2017; Nagato et al., 2005; Singh et al., 2013; Takatsuka et al., 2018; Turner et al., 2013; Visekruna et al., 2013; Wan et al., 2020; Zhou et al., 2011). Upon ligand binding to the IL-9R, which comprises the cytokine-specific IL-9Rα subunit and the common γ-chain subunit shared by IL-2, IL-4, IL-7, IL-15, and IL-21 receptors, activation of signal transducer and activator of transcription (STAT) complexes (Bauer et al., 1998; Demoulin et al., 1999), PI3K/AKT (Patrussi et al., 2021), and MAPK (Demoulin et al., 2003) signaling pathways facilitate IL-9 dependent gene expression.

IL-9 and IL-9-producing cells have been extensively reviewed in the context of a variety of pathologies (Angkasekwinai and Dong, 2021; Noelle and Nowak, 2010). In this review, we focus on the role of IL-9 and IL-9-producing cells in the development of cancer. First, we discuss the protumor effects of IL-9 focusing on the functional differences of IL-9 in hematological and solid tumors, highlighting the role of IL-9 and how it directly impacts tumor cell proliferation or modulates the TME to promote cancer progression. In the following sections, we address the antitumoral properties of IL-9 and IL-9-producing cells and the factors that regulate their generation and function in the TME. Finally, we discuss the prognostic value and therapeutic approaches to target IL-9 and IL-9-producing cells, and the gaps needing to be addressed in the field.

Properties of IL-9 Supporting Tumor Growth

Direct protumor effects in hematological cancers

In the past, IL-9 has been commonly associated with a protumor phenotype in most hematological malignancies. As a lymphocyte growth factor, IL-9 can promote protumor responses by directly regulating malignant murine and human lymphocytes to promote proliferation and inhibit apoptosis (Table 1). IL-9 signaling in Ba/F3 lymphoma cells can promote lymphocyte survival in vitro by promoting STAT5-dependent proliferation while concomitantly, reducing apoptosis of these cells through an IL-9-dependent STAT3/5 pathway (Demoulin et al., 2000; Demoulin et al., 1999).

Table 1.

Effects of Interleukin-9 and Interleukin-9-Producing Cells on Various Cancer Types

Cancer types	Source of IL-9	IL-9 responsive cell	Effect	References
Indirect
Leukemia (CLL, ATL)	B cell	Stromal cells	↑Metastasis ↑Cell proliferation	Chen et al. (2008), Chen et al. (2014b), Patrussi et al. (2021)
Hepatocellular carcinoma	Th9	Unknown	↑EMT	Tan et al. (2017)
Lung cancer	Th9, Tregs	Treg, Macrophages	↑Treg-mediated immunosuppression ↑ ARG1+ macrophages	Heim et al. (2022), Pasvenskaite et al. (2021)
Colorectal cancer	Th9	T cells	↑IL-6 dependent cancer cell proliferation ↓ CD8+ T cell activity	Gerlach et al. (2022), Hoelzinger et al. (2014), Niccolai et al. (2021)
Bladder cancer	Th9	CD4+ T cells and macrophages	↓CD8+ T and NK cells, ↑Immunosuppressive macrophages	Zhou et al. (2020)
Breast cancer	iTregs	Tregs	↑Treg-mediated immunosuppression ↓CD8+ T cell activity ↑Metastasis	Hoelzinger et al. (2014), Smith et al. (2011)
NSCLC	Ectopic	NSCLC cells	↑Angiogenesis	He et al. (2019)
Direct
Lung Cancer	Th9	Lung cancer cells	↑Lung cancer cell proliferation, ↑Angiogenesis and EMT	Salazar et al. (2020), Ye et al. (2012)
B cell lymphoma (DLBCL, HL, NHL)		B Cells	↑Cell proliferation	Feng et al. (2011), Fischer et al. (2003), Lv et al. (2016), Lv et al. (2013)
T cell lymphoma (ALCL, CTCL, NK-T/T cell)	T Cells	T Cells	↑Cell proliferation, ↓Oxidative Stress, ↓Apoptosis, ↑ Immunosuppression	Demoulin et al. (2001), Demoulin et al. (2000), Kumar et al. (2020), Merz et al. (1991), Nagato et al. (2005), Qiu et al. (2006), Renauld et al. (1994)
Pancreatic cancer	Unknown	Pancreatic Cancer Cells	↑Proliferation and Metastasis	Hu et al. (2017)
Hepatocellular carcinoma	Th9	HCC cells	↑Proliferation and Metastasis	Lei et al. (2017)
Leukemia (CLL)	CLL cells	CLL cells	↓Apoptosis ↑ STAT6 activation	Chen et al. (2014a)
Colorectal cancer (CAC)	Epithelial Cells	Epithelial Cells	↑Colonic epithelial cell proliferation	Tian et al. (2018)

CLL, chronic lymphocytic leukemia; ATL, adult T cell lymphoma/leukemia; NSCLC, Non-small cell lung cancer; DLBCL, diffuse large B cell lymphoma; HL, Hodgkin's lymphoma; NHL, Non-Hodgkin's lymphoma; ALCL, anaplastic large cell lymphoma; CTCL, cutaneous T cell lymphoma; CAC, colitis-associated colorectal cancer; EMT, epithelial–mesenchymal transition; ↑ indicates increased function; ↓ indicates decreased function.

Moreover, transgenic mice overexpressing IL-9 exhibited increased rates of spontaneous and chemically induced lymphoma tumorigenesis in vivo and enhanced lymphoma cell proliferation in vitro (Renauld et al., 1994). Vink et al. (1993) reported that IL-9 synergistically induced thymic lymphoma in mice exposed to X-ray radiation or N-methyl-N-nitrosourea. In that study, IL-9 was also demonstrated to synergize with IL-2, IL-4, and IL-7 to increase the rate of tumorigenesis (Vink et al., 1993). These initial studies implicate IL-9 in directly modulating tumor development in lymphoma.

Clinical studies reveal that IL-9 can contribute to T cell lymphomas in human patients (Gruss et al., 1992; Merz et al., 1991; Renauld et al., 1994; Vink et al., 1993). Notably, IL-9 and IL-9R expression can be detected in a variety of T cell lymphomas, including anaplastic large-cell lymphoma (ALCL) (Merz et al., 1991), SNK-6 and SNT-8 NK/T lymphoma cell lines (Nagato et al., 2005), human anaplastic lymphoma kinase (ALK) ALCL tumors (Qiu et al., 2006), and cutaneous T cell lymphoma (CTCL) (Kumar et al., 2020). In support of the initial studies (Demoulin et al., 1999), in vitro stimulation with human IL-9 on a murine T cell lymphoma cell line expressing human IL-9R increased SOCS3 activity and inhibited apoptosis, but surprisingly failed to promote proliferation (Demoulin et al., 2001). This discrepancy suggests varying roles of mouse and human IL-9 on malignant T cell activity and is still an area needing to be investigated more thoroughly.

IL-9 acts as an autocrine growth factor in NK/T lymphomas and ALCL (Nagato et al., 2005). In NK/T lymphoma cells, blockade of IL-9 signaling using a neutralizing antibody inhibited proliferation (Nagato et al., 2005).

Similar findings were observed in ALK⁺ ALCL, in which blockade of IL-9 displayed a concentration-dependent increase in p21 expression and increased cell-cycle arrest (Qiu et al., 2006). Mechanistically, IL-9 may modulate tumor growth by regulating its metabolism. In T cell cultures of CTCL patients, treatment with IL-9 reduced oxidative stress and reduced apoptosis, which is likely dependent on a metabolic switch from oxidative phosphorylation to aerobic glycolysis (Kumar et al., 2020), implicating IL-9 as a regulator of metabolism.

IL-9 is also implicated in B cell lymphomas. In Hodgkin and Reed–Sternberg cells, IL-9 can induce B cell proliferation, and inhibition of IL-9 signaling could inhibit cell growth in vitro (Gruss et al., 1992). In patients diagnosed with Hodgkin's lymphoma, serum IL-9 expression is also increased when compared with healthy individuals (Merz et al., 1991). Interestingly, IL-9 expression was undetectable in 18 patients diagnosed with B cell non-Hodgkin's lymphoma (Merz et al., 1991) but in a study investigating the role of IL-9 in diffuse large B cell lymphoma (DLBCL), Lv et al. (2013) demonstrated that IL-9 directly affected proliferation and apoptosis by enhancing the expression of P21CIP1 genes suggesting a role for IL-9 in B cell non-Hodgkin's Lymphoma. Later studies found serum IL-9 levels were significantly elevated in patients diagnosed with DLBCL (Lv et al., 2016; Lv et al., 2013), and correlated with low albumin levels and high international prognostic index scores (Lv et al., 2016).

Furthermore, IL-9R was highly expressed in 2 different murine DLBCL cell lines (LY1 and LY8) (Lv et al., 2016). Functionally, interference with IL-9 signaling in either cell line resulted in reduced proliferation and enhanced apoptosis, and interestingly, IL-9-responsive LY1 and LY8 DLBCL cells were less responsive to chemotherapeutic treatment with rituximab, prednisolone, and vincristine (Lv et al., 2016). Altogether, these studies demonstrate a central role for IL-9 in regulating tumor growth in B cell lymphomas.

Within the last decade, little research on the role of IL-9 in leukemia has occurred and therefore, the role of IL-9 in this disease is poorly understood. In patients diagnosed with chronic lymphocyte leukemia (CLL), elevated IL-9 levels were detected in the sera of 20/47 patient samples (Chen et al., 2014b). Supporting these findings, Chen et al. demonstrated that IL-9 overexpression is mediated by IL-4-induced activation of STAT6. In this system, IL-9 directly inhibited CCL apoptosis and promoted CLL pathogenesis (Chen et al., 2014a). Altogether, these studies suggest that IL-9 likely plays a distinct protumor role in leukemia, but further investigations into the roles of IL-9 across different types of leukemia are needed. Furthermore, in hematological malignancies, the source and regulation of IL-9 secretion are not fully understood. A comprehensive understanding of the various sources of IL-9 in each malignancy could uncover novel targets for therapeutic intervention.

Direct protumor effects in solid cancers

Traditionally, IL-9 was primarily thought to directly affect hematological cancers; however, research has demonstrated that IL-9 can also directly affect tumor cells in solid cancers. Tian et al. (2018) demonstrated that IL-9 directly stimulates malignant colonic epithelial cell proliferation by upregulating c-MYC and CyclinD1 in RKO and Caco-2 colon carcinoma cell lines in vitro. Although the IL-9R composition on epithelial cells has yet to be completely defined, the direct effects of IL-9 on airway epithelial cells allude to its existence (Goswami and Kaplan, 2011; Temann et al., 1998). Furthermore, these findings are supported by increased IL-9 levels in colorectal cancer (CRC) tissue samples when compared with healthy adjacent tissues from the same individual (Gerlach et al., 2022; Tian et al., 2018). According to another study examining the effects of IL-9 in pancreatic cancer cells, IL-9 was demonstrated to directly promote proliferation by regulating the miR-200a/b-catenin axis (Hu et al., 2017).

Altogether, these data indicate a role for IL-9 in directly promoting tumor cell proliferation in solid malignancies. Alternatively, IL-9 can directly promote other tumorigenic cellular functions such as supporting chemotaxis and metastasis and preventing apoptosis. In an article from Xiao et al., they determined Th9 cells display an effector memory phenotype characterized by low expression of CD45RA and CD62L and are recruited through a pleural CCL20/CCR6 axis (Bu et al., 2013). Further supporting a role of CD4-derived IL-9 in malignant pleural effusion (MPE), Th9 cells were reported to directly increase MPE through the activation of STAT3 in A549 and SK-MES-1 lung cancer cells (Ye et al., 2012). Additionally, Ye et al. (2012) reported antiapoptotic effects as a result of IL-9 signaling in a similar STAT3/5-dependent mechanism as previously described in Demoulin et al., (1999). Furthermore, they reported IL-9 could promote the migration of A549 lung cancer cells through an ICAM/LFA1 and/or VCAM-1/Integrin-B-dependent mechanism (Ye et al., 2012).

In vitro studies showed that IL-9 had negligible effects on proliferation but enhanced expression of angiogenesis-related gene vascular endothelial growth factor (VEGF) and microvessel density in lung cancer cells which promote tumor growth by increasing vascularization (He et al., 2019). Supporting the role of IL-9 in promoting metastasis and angiogenesis, IL-9-producing Th9 and Th17 cells were reported to induce metastasis and epithelial–mesenchymal transition (EMT) of human and murine lung cancer cells by upregulating the expression of MMP-3, MMP-13, as well as several genes involved in angiogenesis and cell adhesion. Although their work demonstrates a cooperative role of Th9 and Th17 cells to promote metastasis in lung cancer, they point out that Th9 cells function distinctly from Th17 cells. Particularly, they demonstrated that Th9 cells promote chemokine-mediated inflammation and angiogenesis, whereas the IL-9 and IL-17-producing Th17 cells influenced oxidative stress response and Ras-signaling pathways (Salazar et al., 2020) to support tumor cell homeostasis in the TME.

Interestingly, in a study examining the effect of IL-9 on liver cancer cells in vitro, IL-9 also upregulated the expression of MMP-2, MMP-9, and VEGF by activating JAK2/STAT3 signaling in the hepatocellular carcinoma line SMMC-7721 further supporting the role of IL-9 in metastasis (Lei et al., 2017). Altogether, these studies highlight the dynamic range of IL-9-responsive cancer cells and further support the effects of IL-9 directly on malignant cells beyond those found in hematological cancers.

Indirect protumor effects in the TME

An increasing number of studies have shown that IL-9 can support the growth of many cancers by altering the TME and regulating the functions of IL-9-responsive cells (Table 1). As alluded to in earlier discussions, IL-9 may act on IL-9-responsive cells, such as myeloid cells, mast cells, T cells, and nonhematopoietic cells, to indirectly enhance tumorgenicity. Interestingly and unlike what is generally observed in most other hematological cancers, IL-9 can enhance leukemia progression by indirect means. Chen et al. (2008) demonstrated that the human T cell lymphotropic virus type I (HTLV-I) Tax protein induces IL-9 expression in primary adult T cell leukemia (ATL) cells. In one of the first reports of IL-9 acting indirectly to promote the growth of leukemic T cells, they reported that IL-9R-expressing CD14⁺ monocytes mediate the proliferation of ATL cells through a mechanism not yet fully understood (Chen et al., 2008).

To provide more mechanistic insight into the regulation and function of IL-9 in leukemia, Hamilton et al. (2021) demonstrated that upregulation of p66Shc increased IL-9 production in CLL cells which, in turn, promoted the expression of IL-9-responsive stromal cell-derived chemokines, CCL2, CXCL9, CXCL10, CXCL11, CXCL13, and CCL19, which directly enhanced recruitment and survival of malignant CLL cells in an Eu-TCL1 mouse model of human CLL (Patrussi et al., 2021). It is likely that IL-9, in addition to directly acting on IL-9R⁺ leukemic cells, indirectly promotes the expression of chemokines from immune and nonimmune cells to enhance leukemia progression.

In the context of solid malignancies, the protumor function of IL-9 is primarily associated with the modulation of the TME. Recent studies revealed that macrophages are a central IL-9-responsive cell type associated with lung cancer (Fu et al., 2022). In a metastatic B16 and orthotopic LLC model of lung cancer, genes associated with vasculature development in Il9r^−/− interstitial macrophages from tumor-bearing mice were significantly downregulated when compared with tumor-bearing wild-type interstitial macrophages, thereby supporting a role for an IL-9/macrophage axis in modulating the TME. Furthermore, they demonstrated that IL-9 enhances the expression of arginase 1, a hallmark marker of immunosuppressive macrophage polarization, and the expression of IL-6 (Fu et al., 2022), a cytokine that prevents JAK3/STAT3-dependent tumor cell apoptosis and promotes tumor cell growth (Chonov et al., 2019). In this context, it is likely that IL-9 transcriptionally alters macrophages to promote immunosuppression by regulating arginine metabolism and producing tumor-promoting factors.

IL-9 can also regulate adaptive immune cells to enhance tumor growth. It was reported that patients diagnosed with muscle-invasive bladder cancer (MIBC) and increased infiltration of IL-9-producing cells had overall worse survival and correlated with an overall increase in tumor-promoting Treg cell, immunosuppressive macrophage, and mast cell infiltration resulting in a decrease in antitumor CD8⁺ T cell response (Zhou et al., 2020). Interestingly this study reported a tumor-promoting function that is inconsistent with findings from (Purwar et al., 2012) where they found mast cells to have antitumor activity in a B16 model of melanoma, highlighting the situational diversity of IL-9 in tumor immunity.

In addition to diminishing CD8⁺ T cell responses, IL-9 can support lung cancer cell immune evasion by enhancing IL-9-responsive Treg immunosuppressive IL-10 production in the TME (Heim et al., 2022). Further supporting the role of IL-9 in modulating Treg cell function in the TME, Smith et al. (2011) demonstrated that activation of 4-1BB alters the immunosuppressive capacity of iTregs by preventing an IL-9-dependent autocrine feedback loop to maintain the number and suppressive function of iTregs.

Additionally, the finding from Zhou et al. examining the role of IL-9 in CRC and breast demonstrated IL-9 inhibited early activation of antitumor adaptive immunity in CD8⁺ and CD4⁺ T cells (Hoelzinger et al., 2014). Moreover, in another study, it was demonstrated that IL-9 indirectly promotes epithelial cell proliferation in colorectal neoplasia by regulating the expression of claudin2/3 and IL-6 expression in T cells (Gerlach et al., 2022). Despite substantial evidence of the protumor effects of IL-9-responsive innate and adaptive immune cells on tumor development, many studies still lack the fundamental details of underlying mechanisms such as the regulation of the IL-9R and the conditions in which IL-9 promotes a protumor phenotype.

More recently it has become increasingly apparent that the microbiome is implicated in many pathologies, including cancer (Fan and Pedersen, 2021; Flemer et al., 2018). In a recent study, Niccolai et al. (2021) demonstrated a bidirectional crosstalk between the microbiota and the expression of several cytokines, including IL-9 in CRC. They reported that expression of IL-9 was positively correlated with Prevotella spp. and negatively correlated with Bacteroides spp with each correlation hinting toward IL-9/microbiota-mediated antitumoral dysregulation. Their findings suggest a microbiota/tumor immunity axis in CRC that can be influenced by IL-9 (Niccolai et al., 2021), a finding warranting further mechanistic investigation. In summary, the situational dependence of IL-9 in promoting tumor growth depends on the expression of the IL-9R on the tumor cell or nearby cells found in the tumor environment.

Antitumor Activities of IL-9 and IL-9-Producing Cells

Direct cytotoxicity against cancer cells

IL‑9 and IL-9-producing cells exert antitumor effects through multiple mechanisms, such as direct cytotoxicity against tumor cells and promoting antitumor immune responses of other immune cells. In human melanoma, IL-9 inhibits HTB-72 and SK-Mel-5 melanoma cell growth and induces the apoptosis of HTB-72 cells in vitro. The IL-9-mediated growth inhibition and apoptosis of HTB-72 cells are correlated with the upregulation of antiproliferation molecule p21 and proapoptosis molecule TNF-related apoptosis-inducing ligand (TRAIL) (Fang et al., 2015). However, IL-9 had a marginal effect on the growth and survival of HTB-65 or CRL-11147 melanoma cells. This study suggests that the direct antitumor effects of IL-9 on human melanoma cells are dependent on specific cell types (Fang et al., 2015) and likely the expression of its receptor. Furthermore, IL-9 also inhibited the migration and invasion of the human gastric cancer cell line sgc-7901 in vitro (Cai et al., 2019). In human cervical cancer tissue, the infiltration of PU.1⁺ Th9 cells and IL-9R⁺ cells is significantly increased in human cervical cancer tissue.

Furthermore, IL-9 suppressed proliferation, enhanced apoptosis, and stimulated the expression of MHC-I and E-cadherin on HeLa cell lines (Chauhan et al., 2019). The IL-9-induced MHC-I expression was also reported in mouse CMT165 and human A549 lung tumor cells [47], suggesting that IL-9 is not only inhibiting tumor cell proliferation and inducing apoptosis but also preventing immune evasion of lung tumor cells. More recently, Sreya Das et al. (2021) demonstrated IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological 3-dimensional invasion assays. Mechanistically, these cells express moderate IL-9R, and IL-9 inhibits the metastatic potential of these tumor cells by controlling extracellular matrix remodeling and cellular contractility. In mouse squamous cell carcinoma, IL-9 induces the apoptosis of squamous cancer (SqC) cells in vitro, possibly due to the high IL-9R expression on SqC cells (Miao et al., 2017). The proapoptotic effects of IL-9 were also reported in mouse GL261 glioma cells (Zheng et al., 2017).

A recent mouse lung cancer study demonstrated that IL-9 suppressed tumor growth in CMT167 but not the Lewis lung carcinoma (LLC) mouse lung tumor model. IL-9R expression is detected on CMT167 cells but not LLC cells, and IL-9R knockdown in CMT167 abolished IL-9-mediated tumor rejection, suggesting that IL-9 has a direct antitumor effect on the tumor cells. Further mechanistic analysis indicated that IL-9 upregulates surface MHC-I expression on CMT167 cells through the activation of the ERK1/2 pathway, which mounts an enhanced CD8⁺ T cell-mediated antitumor response. In addition, IL-9 also enhanced PD-L1 on CMT167 suggesting that IL-9 treatment can potentially be combined with anti-PD1/PDL1 therapy (Feng et al., 2011). Altogether, these studies suggest that IL-9 can directly target IL-9R⁺ tumor cells and regulate tumor apoptosis, proliferation, migration, and marker expression that enhances antitumor immunity.

As the major IL-9-producing cell population, Th9 cells have been reported for their direct cytotoxicity against some tumors. Purwar et al. (2012) found Ag-specific OT-II Th9 could mediate direct cytotoxicity against ovalbumin-expressing (B16-OVA) melanoma cells. The killing capacity of Th9 cells seems to be granzyme B dependent and Ag specific, as inhibition of granzyme-B expression diminished the cytotoxic effects. Supporting this, EL4 tumor cells without ovalbumin expression are resistant to OT-II Th9-dependent cytotoxicity (Purwar et al., 2012). In agreement with Purwar's results, studies from Lu et al. (2018) further demonstrated that Th9 cells with high EOMES and granzyme expression exert cytotoxic effects directly on melanoma cells and thereby eradicated advanced late-stage tumors.

The cytotoxic properties of Ag-specific Th9 cells were also shown in mouse SqC squamous tumor cells. Results from the coculture of Th9 cells and SqC cells suggested Th9 cells markedly induced SqC cell apoptosis, and attenuation of IL-9 signaling by a neutralizing anti-IL-9 antibody abolished the proapoptotic effects mediated by Th9 cells (Miao et al., 2017).

The direct killing effect of Tc9 cells has also been examined in B16 melanoma (Liu et al., 2019; Lu et al., 2014). Although Tc9 exhibited lower specific cytolytic activity compared with Tc1 cells in vitro, Tc9 cells mediated enhanced antitumor response and displayed greater persistence in vivo (Lu et al., 2014). In hematopoietic malignancies, T9 cells (T cells differentiated under Th9-skewing conditions) could lyse allogeneic GFP–MLL-AF9 leukemic cells and MOLM14 leukemic cells. Moreover, IL-33 further enhances cytolytic molecule (Granzyme B and Perforin) expression and cytolytic activity of T9 cells (Ramadan et al., 2017). In another GVHD study, in vitro-generated Th9 cells selectively mediate the antitumor effect toward allogeneic B cell malignancies (Reisser et al., 2020). In summary, Th9/Tc9 cells secrete cytolytic molecules to mediate direct cytotoxicity against tumors in an antigen-specific and IL-9-dependent manner.

Activation of innate antitumor immunity

IL‑9 and IL-9-producing cells have been shown to have various effects on nontumoral cells in TMEs that foster antitumor immunity. The first in vivo study on the antitumor activities of IL‑9 and IL-9-producing cells was carried out by Purwar et al. (2012) who investigated the antitumor effects of Th9 cells in a mouse model of B16 melanoma. The results indicated that adoptively transferred Th9 cells were highly efficient in inhibiting tumor growth. The antitumor efficacy of Th9 cells was superior to all other helper T cell subsets. Importantly, blocking IL-9 using neutralizing antibodies abolished the beneficial effect of adoptively transferred Th9 cells, underscoring the importance of IL-9 in antitumor immunity in this melanoma model (Purwar et al., 2012). To further study the role of IL-9 in melanoma progression, subcutaneous B16 tumor growth was tested in Il9r KO mice. The absence of IL-9R resulted in faster tumor growth compared with WT mice. Conversely, the administration of recombinant IL-9 reduced B16 and LLC tumor burden in WT mice, demonstrating that the antitumor effects of IL-9 were not restricted to melanoma (Purwar et al., 2012).

Furthermore, the antitumor activities of IL-9/Th9 were preserved in B16 tumor-bearing Rag1-deficient mice but not in mast cell-deficient (kit W-sh) mice indicating that mast cells are essential for IL-9-mediated antitumor immunity in melanoma (Purwar et al., 2012). The role of mast cells in Th9 cell-driven antitumor immunity was further explored in a study by Abdul-Wahid et al. (2016) who established a vaccination strategy in a mouse metastatic colon carcinoma model using MC38 CEA tumor cells. In this study, IL-9 and mast cells were required for Th9-dependent antitumor immunity, as IL-9 blockade or depletion/inactivation of mast cells prevents the ability of the vaccine to protect mice against tumor metastasis (Abdul-Wahid et al., 2016). These results indicate that Th9 cells could enhance mast cell activity through IL-9, resulting in tumor inhibition.

In addition to mast cells, NK cells were also involved in Th9-driven innate antitumor immunity. IL-21 secreted by IL-1β-induced Th9 cells enhanced IFN-γ secretion from NK cells and CD8⁺ T cells, which were responsible for B16 melanoma elimination (Végran et al., 2014). In summary, Th9 cells can trigger innate antitumor immunity through IL-9 and IL-21 secretion to limit tumor growth.

Activation of adaptive antitumor immunity

While IL-9 promotes innate antitumor immunity, other studies clearly demonstrated that IL-9-mediated antitumor immunity includes the activation of adaptive immune cells. Several studies suggested IL-9/Th9 could promote the recruitment and activation of DCs and CD8⁺ T cells in the TME. In a B16 tumor metastasis model, adoptive transfer of tumor-specific Th9 cells provoked a strong antitumor response and prevented the development of lung tumor foci (Lu et al., 2012). Tumor-infiltrating Th9 cells produce IL-9, which induces lung tissue to produce CCL20 that recruits CD8⁺ DCs into the tumor setting, where they uptake tumor antigens and become activated (Lu et al., 2012). These activated DCs migrate to tumor-draining lymph nodes and activate cytotoxic CD8⁺ T lymphocytes (CTLs) that can migrate into tumor lesions guided by CCL20 to eradicate tumor cells (Lu et al., 2012). The same group later found that Th9-derived IL-3 could prolong the survival of myeloid DCs through the upregulation of antiapoptotic protein Bcl-xL and activation of p38, ERK, and STAT5 signaling pathways in DCs (Liu et al., 2019; p. 3).

In agreement with the above studies, Kim et al. (2015) also demonstrated that DTA-1, an agonistic antibody against tumor necrosis factor receptor (TNFR)-related protein (GITR), induces IL-9 expression, which strengthens tumor-specific CD8⁺ CTL responses by promoting the cross-presentation of tumor antigens and the upregulation of costimulatory and MHC class II molecules on tumor-infiltrating DCs in a B16 melanoma model. Furthermore, human IL-9-expressing Th9 cells in breast cancer patients potently elicited antitumor responses by enhancing CD8⁺ T cell-mediated cytotoxicity through IL-9 and IL-21-dependent pathways (You et al., 2017).

Beyond Th9 cells. IL-9-producing ILC2s can also inhibit the growth of CT26 CRC cells by activating CD8⁺ T cells (Wan et al., 2021). Furthermore, blocking ILC2s promoted tumor growth in CT26 tumor-bearing mice, and recombinant IL-9 reversed the tumor-promoting effects of blocking ILC2s suggesting that ILC2s could exert antitumor effects by secreting IL-9 (Wan et al., 2021). In addition to CD8⁺ T cells, tumor-reactive CD4⁺CD8⁺ double-positive (DP) T cells from melanoma patients are also responsive to IL-9 stimulation (Parrot et al., 2016).

A study by Parrot et al. (2016) revealed that IL-9R expression in DP T cells was significantly higher than its expression in CD8 single-positive T cells and that IL-9 protected DP T cells against apoptosis and promoted proliferation. Furthermore, IL-9 enhanced their inflammatory cytokine production and increased the levels of granzyme B/perforin as well as degranulation capacity in vitro, which lead to a strengthened cytotoxic response of DP T cells against human melanoma cells (Parrot et al., 2016).

Engineered T cells

Chimeric antigen receptor (CAR)-T cells and TCR-Engineered T Cells have emerged as a revolutionary new pillar in cancer therapeutics (Greenbaum et al., 2021; June et al., 2018). Liu et al. (2020) demonstrate that human CAR-T cells polarized and expanded under Th9-culture conditions (T9 CAR-T) have stronger antitumor activity against established hematologic and solid tumors in vivo when compared with T1 CAR-T cells. T9 CAR-T cells display a central memory phenotype, express lower levels of exhaustion markers, and produce IL-9 but little IFN-γ in vitro. After adoptive transfer into the tumor-bearing mice, T9 CAR-T cells differentiate to IFN-γ and granzyme-B secreting effector memory T cells while maintaining long-lived hyperproliferative features (Liu et al., 2020).

In line with Liu's observation, a recent study from Xue et al. (2021) and colleagues demonstrate that murine and human Th9 cells, but not Th1, Tc1, or Th17 cells, express tumor-specific TCRs or CARs that facilitate the eradication of advanced tumors that contain antigen loss variants . This superior antitumor capacity of engineered Th9 cells is due to the enhanced direct killing of tumor cells and bystander antitumor responses mediated by the intratumor release of IFN-α/β.

Further mechanistic investigation illustrated that tumor-specific Th9 cells lack the expression of ATP-degrading ectoenzyme CD39, resulting in increased intratumor accumulation of extracellular ATP, which promotes monocyte infiltration and stimulates their IFN-α/β production by inducing eATP-endogenous retrovirus-Toll-like receptor 3/mitochondrial antiviral signaling pathway activation (Xue et al., 2021). These results suggest that engineered T cells polarized under Th9-culture conditions represent a unique T cell subset endowed with an unprecedented capacity to eliminate tumor cells (Xue et al., 2021). In summary, IL-9 and IL-9-producing cells not only mediate direct cytotoxicity against cancer cells but also interact with other immune cells and enhance their antitumor immunity (Table 2).

Table 2.

Antitumoral Effects of Interleukin-9 and Interleukin-9-Producing Cells on Various Cancer Types

Cancer types	Models/tumor lines	Effectors	Responders	Functions/effects	References
Melanoma (Hu)	In vitro/HTB-72 & SK-Mel-5 In vitro/M314 In vivo/PDX	IL-9 TCR-Th9	Tumor cells DP T cells	Apoptosis, cell cycle arrest, upregulation of P21 and TRAIL in tumor cells; Antiapoptosis, enhanced proliferation, cytotoxicity of DP T cells; TCR Th9 cell-mediated cytotoxicity.	Fang et al. (2015), Parrot et al. (2016), Xue et al. (2021)
Melanoma (Ms)	In vitro/B16F10 In vivo/B6F10	Th9 Th9/Tc9 (ACT)	Tumor cells mast cells NK cells CD8 T cells DCs	Granzyme B-dependent and Ag-specific cytotoxicity; IL-9-induced mast cell activation; IL-9 and IL-21-dependent NK cell and CD8 T cell activation; Recruitment of CCR6+ DCs and CD8 T cells; DC activation and cross-presentation.	Lu et al. (2014), Purwar et al. (2012), Végran et al. (2014), Kim et al. (2015), Lu et al. (2012)
Colon cancer (Ms)	In vivo/CT26 In vivo/MC38.CEA	IL-9/Th9/ILC2	DCs, CD8 T cells Mast cells	DCs activation; IL-9 and IL-21-induced CD8 T cell activation; IL-9-induced mast cells.	Kim et al. (2015), Abdul-Wahid et al. (2016)
Lung Cancer (Ms, Hu)	In vivo/CMT165 In vitro/A548	IL-9	Tumor cells	Upregulation/activation of MHC-I on tumor cells, enhanced CD8 CTL cytotoxicity.	Feng et al. (2011)
Breast cancer (Hu)	In vitro/MDA-MB-231 & MCF-7 In vitro/autologous tumor cells	IL-9/Th9	Tumor cells CD8 T cells	Inhibited ECM remodeling capacity, actomyosin contractility and metastasis of tumor cells; IL-9 and IL-21-dependent CD8 T cell cytotoxicity.	Das et al. (2021), You et al. (2017)
Squamous cancer (Ms)	In vitro and in vivo/SCC VII	IL-9/Th9 (ACT)	Tumor cells	IL-9-induced apoptosis.	Miao et al. (2017)
Liver cancer (Hu)	In vivo/HepG2	CAR T9 (ACT)	Tumor cells	CAR T9 cell-mediated cytotoxicity.a	Liu et al. (2020)
Ovarian cancer (Ms)	In vivo/ID8	CAR-Th9 (ACT)	Tumor cells	CAR Th9 cell-mediated cytotoxicity.	Xue et al. (2021)
Cervical cancer (Hu)	In vitro/HeLa	IL-9	Tumor cells	Apoptosis, Inhibited proliferation and metastasis; Upregulation of MHC-I and e-cadherin.	Chauhan et al. (2019), Das et al. (2021)
Gastric Cancer (Ms)	In vitro/SGC-7901	IL-9	Tumor cells	Inhibited migration and invasion, P53 and P21 upregulation.	Cai et al. (2019)
Glioma (Ms)	In vitro and in vitro/GL261	IL-9/Th9 (ACT)	Tumor cells	IL-9-induced apoptosis.	Zheng et al. (2017)
Leukemia & lymphoma (Ms, Hu)	In vivo/MLL-AF9 leukemic cells In vitro/MOLM14 In vivo/A20 In vivo/K562 & NALM6	Th9/T9 (ACT) CAR T9 (ACT)	Tumor cells	GVL effects. CAR T9 cell-mediated cytotoxicity.	Ramadan et al. (2017), Reisser et al. (2020), Liu et al. (2020)

Hu, human; Ms, mouse; DP T cells, CD4⁺CD8⁺ double-positive T cells; PDX, patient-derived xenograft; ACT, adoptive cellular therapy; ECM, extracellular matrix; GVL, graft-versus-leukemia.

The regulators of IL-9 and IL-9-producing cells in antitumor immunity

Various cytokines, cosignaling receptors, transcription factors, metabolism modifiers, and exogenous regulators promote or impede the transcription of IL-9 and thereby, modulate Th9/Tc9 differentiation and their antitumor activities (Fig. 1). TGF-β and IL-4 are 2 key cytokines that support IL-9 production and Th9 cell differentiation (Dardalhon et al., 2008; Veldhoen et al., 2008), but other cytokines have also been reported to promote Th9 cell differentiation and enhance their antitumor activities. Végran et al. (2014) found that IL-1β induced IRF1 expression in Th9 cells, which enhanced the secretion of the cytokines IL-9 and IL-21 from Th9 cells. A study from Xue et al. (2019) also underscored the importance of IL-1β signaling in driving Th9 cell development. Authors found IL-1β- and IL-4-polarized Th9 cells were less exhausted, exhibited an increased cytotoxic T effector gene signature and tumor-killing function, and mediated a stronger antitumor response in a B16 melanoma model than classic Th9 cells (Xue et al., 2019).

FIG. 1.

Regulation of IL-9 and Th9/Tc9 cell-mediated antitumor immunity. The diagram summarizes the positive regulators (in red font) and negative regulators (in blue font) that influence the antitumor effects of IL-9 and Th9/Tc9 cells. These regulators include transcription factors, such as PU.1 and STAT5; cytokines, such as IL-4 and TGF-β; cosignaling receptors, such as GITR and Fas; tumor vaccines; other immune cells; and many others. The figure illustrates the complex and multifaceted nature of IL-9 and Th9/Tc9 cell-mediated antitumor immunity regulation. BATF, basic leucine zipper ATF-like transcription factor; DBP, the D site of albumin promoter (albumin D-box)-binding protein; Foxo1, forkhead box protein O1; HIF1α, hypoxia-inducible factor 1-alpha; ID3, inhibitor of DNA binding 3; Foxp1, forkhead box P1; IRF1/8, interferon regulatory factor1/8; EGFR, epidermal growth factor receptor; PP2A, serine/threonine protein phosphatase 2A; Cbl-b, Cbl proto-oncogene B; Fas, fas cell surface death receptor; FasL, Fas ligand; PD1, programmed cell death 1; PDL1, PD1 ligand. Figure was made in BioRender.com

Bi et al. (2017) found that pretreatment of mouse naive CD4⁺ T cells with IL-7 further enhanced their differentiation into Th9 cells and augmented their antitumor activity in a B16 melanoma lung metastasis model. IL-7 activated the STAT5 and PI3K-AKT-mTOR signaling pathways, and consequently, the transcriptional regulator Foxo1 was translocated to the nucleus where it bound to the Il9 promoter and induced the production of IL-9 (Bi et al., 2017). Interestingly, they also found another transcriptional regulator, Foxp1, in adjacent conserved Forkhead box transcription factor-binding sites competed with Foxo1 for binding to the Il9 promoter and inhibited IL-9 transcription (Bi et al., 2017).

Similar to IL-7, TNF-α and IL-33 were also found to enhance Th9 cell differentiation in vitro (Jiang et al., 2019; Ramadan et al., 2017). Adoptively transferred Th9 cells differentiated with these cytokines exert enhanced antitumor activities compared with classic Th9 cells. TNF-α not only enhanced IL-9 secretion from Th9 cells through STAT5 and NF-κB signaling in a TNFR2-dependent manner but also promoted Th9 cell survival and proliferation (Jiang et al., 2019).

In the context of allogeneic hematopoietic cell transplantation, Ramadan et al. (2017) found that T cells polarized under Th9 culture conditions with IL-33 (T9₃₃) enhanced antileukemic activity in vivo compared with conventionally differentiated T9 cells without the induction of graft-versus-host disease. Moreover, IL-33-treated human Th9 cells, which featured increased granzyme B and perforin production, also exhibited stronger antileukemic activity compared with conventional Th9 cells (Ramadan et al., 2017).

In addition to cytokines, cosignaling receptors such as tumor necrosis factor receptor superfamily (TNFRS) members have been reported to promote Th9 cell-mediated antitumor immunity. Two studies have demonstrated that GITR engagement promotes Th9 cell differentiation (Kim et al., 2015; Xiao et al., 2015). Il-Kyu and colleagues found GITR signaling enhanced Th9 cell differentiation in a TNFR-associated factor 6 (TRAF6)- and NF-κB-dependent manner and suppresses the generation of inducible Tregs in vitro (Kim et al., 2015). GITR-triggered IL-9 production in Th9 cells facilitates antitumor CTL responses in vivo. Furthermore, IL-9 is required for agonistic anti-GITR antibody-mediated melanoma and colon tumor regression (Kim et al., 2015). Importantly GITR costimulation also enhances human Th9 differentiation in vitro (Kim et al., 2015). A study by Xiang et al. (2015) suggested GITR ligation subverts Tregs to boost Th9 antitumor immunity through the regulation of histone acetylation and STAT6 activation.

Besides GITR, another TNFRSF member, Fas, has also been demonstrated to enhance the antitumor potential of Th9 cells (Shen et al., 2019). Its ligation during Th9 cell differentiation enhances IL-9 secretion and antitumor efficacy (Shen et al., 2019). The PD-1/PD-L1 pathway controls the induction and maintenance of immune tolerance within the TME (Patsoukis et al., 2020). A study from Nonomura et al. (2016) found that upon the polarization of human PBMCs with Th9-skewing conditions, IL-9 secretion from CD4⁺ T cells was significantly increased in the presence of anti-PD1 or anti-PD-L1 antibodies suggesting that PD-1/PD-L1 engagement suppressed human Th9 cell differentiation. As the underlying mechanism is unclear, further studies are warranted to explore the role of PD-1/PD-L1 in IL-9 secretion. Altogether, these results suggest that certain cytokine and cosignaling receptors could enhance the antitumor function of Th9Tc9 cells, and targeting these signaling pathways in Th9/Tc9 cells could be a potential therapeutic strategy for cancer treatment.

Transcription factors play central roles in the regulation of IL-9 and Th9 cell function (Kaplan, 2017). In an early study from Purwar's group, they revealed high IL-9 expression in RORγt-deficient T cells and further reported that melanoma growth inhibition in the Rorc^−/− mice was partially dependent on IL-9, suggesting transcription factor, RORγt, is a negative regulator for Il-9 production (Purwar et al., 2012).

Our laboratory has been working on the transcription factors required for the acquisition of an IL-9-secreting phenotype in T helper cells; one of our studies illustrated that STAT5 was the earliest transcription factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cells (Fu et al., 2020). The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows the acquisition of the IL-9-secreting phenotype. Under Th17-polarizing conditions, overexpression of a constitutively active form of STAT5 (caSTAT5) and BATF skewed Th17 cells to IL-9-secreting cells. Additional studies have identified PU.1 as an essential transcription factor of IL-9 secretion in Th9 cells (Chang et al., 2010), and had a crucial role in their antitumor properties.

A study from Rivera Vargas et al. identified that polarizing Th9 cells underwent selective autophagy leading to the degradation of PU.1, reducing IL-9 production and antitumor effects (Rivera Vargas et al., 2017). Importantly, blocking autophagy during Th9 cell differentiation enhanced PU.1 expression and favored their antitumor activity in a B16 melanoma model and MC38 colon cancer model (Rivera Vargas et al., 2017). Végran et al. showed that IL-1β-induced IRF1 expression enhanced Th9 cell effector functions by increasing IL-9 expression and enhancing antitumor potential. Their subsequent study also found IRF8 was required for optimal IL-9 production during Th9 cell differentiation and controlled antitumor effects (Humblin et al., 2017). IRF8 deficiency impaired the antitumor activity of Th9 cells upon adoptive transfer in B16 melanoma models (Humblin et al., 2017). When dissecting the downstream signaling of TGF-β and IL-4 signaling in Th9 cells, 2 groups found novel transcription factors that negatively regulated Th9 cell differentiation (Nakatsukasa et al., 2015; Park et al., 2022).

Nakatsukasa et al. (2015) reported that DNA-binding inhibitor Id3 inhibited Th9 cell differentiation, as deletion of Id3 increased IL-9 production from Th9 cells. Mechanistically, TGF-β1 and IL-4 downregulated Id3 expression through the activation of the kinase TAK1, thereby promoting Il9 gene transcription by enhancing the binding of the transcription factors E2A and GATA-3 in the Il9 promoter region. Importantly, Id3-deficient, TGF-β-treated T cells inhibited B16 melanoma growth in vivo in an IL-9-dependent manner, suggesting that Id3 negatively regulates Th9 cell-mediated antitumor immunity (Nakatsukasa et al., 2015).

In a recent study, Sang et al. found 2 new transcription factors downstream of TGF-β and IL-4 signaling, D-binding protein (DBP) and E2F8, which controlled Th9 differentiation (Park et al., 2022). They identify DBP and E2F8 as an activator and a repressor, respectively, for Il9 transcription in both mouse and human Th9 cells. siRNA knockdown of Dbp or E2f8 in Th9 cells promotes or suppresses the antitumor activity of Th9 cells that were adoptively transferred into the B16 melanoma-bearing mice and MCA205 fibrosarcoma-bearing mice (Park et al., 2022). Altogether, various studies clearly illustrated a direct link between transcription factors that regulate IL-9 production from Th9 cells and their antitumor activities.

Regulation of metabolic pathways such as glycolysis plays a crucial role in helper T cell differentiation (Palmer et al., 2015); however, an understanding of the metabolic events during Th9 cell differentiation is still limited. A study by Wang and colleagues found histone deacetylase SIRT1 could negatively regulate Th9 cell glycolytic activity through the activation of mTOR/HIF-1α signaling (Wang et al., 2016). SIRT1 overexpression in CD4⁺ T cells inhibited IL-9 production and glycolysis while SIRT1 deficiency in Th9 cells enhanced their IL-9 secretion and their antitumor activities in a B16 melanoma model, indicating that Th9 cells count on an mTOR-HIF1a-dependent glycolytic pathway for the full acquisition of their effector functions (Wang et al., 2016). The importance of HIF1a signaling was underscored by another study focused on epidermal growth factor receptor (EGFR)-HIF-1α signaling in Th9 development (Roy et al., 2021). Roy et al. (2021) uncovered that EGFR is required for IL-9 induction. EGFR signaling induced HIF-1α, which binds and transactivates IL-9 and NOS2 promoters in Th9 cells.

Loss of EGFR or HIF-1α abolished Th9 cell differentiation and inhibited their antitumor activities upon adoptive transfer into B16 melanoma tumor-bearing mice (Roy et al., 2021). By proteomic analysis of murine Th0 and Th9 cells, Amit Awasthi's group identified the catalytic subunit of protein phosphatase (PP2A) as an essential enzyme for Th9 cell differentiation (Roy et al., 2020). Pharmacological inhibition of PP2A activity reduced IL-9 secretion from Th9 cells and impaired their antitumor function of adoptively transferred Th9 cells in a melanoma model (Roy et al., 2020).

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ubiquitin-protein ligase that negatively regulates receptor tyrosine kinase signaling, thereby limiting the antitumor reactivity of T and NK cells. Schanz et al. (2021) found that Cblb-deficient Th9 cells exert superior antitumor activity in B16 melanoma. Accordingly, blocking IL-9 in melanoma cell-exposed Cblb^−/− mice reversed their tumor rejection phenotype, suggesting that Cblb is a negative regulator for Th9 cell-mediated antitumor immunity (Schanz et al., 2021). Compared with Th9 cells, the metabolic activities in Tc9 cells are even less investigated.

Studies from Yi's group underscored a key contribution of lipid metabolism in Tc9 cell differentiation and effector functions (Ma et al., 2018; Xiao et al., 2022). They first revealed that Tc9 cells have a unique pattern of cholesterol synthesis and efflux gene expression resulting in significantly lower cholesterol content than Tc1 cells (Ma et al., 2018). Manipulating cholesterol content during the Tc9 differentiation significantly downregulated IL-9 gene expression and Tc9 cell mediated antitumor response in vivo. Mechanistically, cholesterol or its derivatives downregulated IL-9 expression by activating liver X receptors (LXRs), leading to LXR sumoylation and reduced p65 binding to the Il9 promoter. Notably, IL-9 was indispensable for Tc9 cell longevity and antitumor functions (Ma et al., 2018). To further study the mechanisms by which IL-9 regulates lipid metabolism in Tc9 cells, they performed RNA-sequencing and functional validation in tumor-infiltrating Tc9 cells in several tumor models and found that Tc9 cells exhibited unique lipid metabolic programs.

Tc9 cell-derived IL-9 activated STAT3, enhanced fatty acid oxidation and mitochondrial activity, and endowed Tc9 cells with decreased lipid peroxidation and resistance to tumor or reactive oxygen species-induced ferroptosis in the TME. Accordingly, inhibiting the IL-9/STAT3/fatty acid oxidation pathway in adoptively transferred Tc9 cells impaired their survival and antitumor activities in B16 and MC38 tumor models (Xiao et al., 2022). Importantly, human Tc9 cells also had lower lipid peroxidation levels compared with other T cell subsets. In melanoma patients, tumor-infiltrating CD8⁺ T cells exhibited downregulated IL9 expression and upregulated lipid peroxidation and ferroptosis-related genes when compared with circulating CD8⁺ T cells (Xiao et al., 2022). These results suggest that the IL-9/STAT3/fatty acid oxidation pathway inhibited lipid peroxidation, which negatively regulates Tc9 cell longevity and antitumor effects. Furthermore, inhibiting T cell lipid peroxidation could be a potential therapeutic target for enhancing T cell-based cancer immunotherapy.

As adoptively transferred Th9/Tc9 exhibited superior antitumor activities, there are also studies focused on the induction of endogenous antitumoral Th9/Tc9 cells in the TME. Immunization with tumor vaccines and immunomodulation reagents has been reported for antitumoral Th9/Tc9 generation in vivo. Siqing's group has been working on Th9/Tc9 inductions by DC-based tumor vaccines (Chen et al., 2018; Liu et al., 2019; Zhao et al., 2016). They first demonstrated DCs activated by dectin-1 agonists potently promoted Th9 cell differentiation in vitro. These activated DCs mediate enhanced antitumor immunity that relies on Th9 cells and IL-9 in a B16 melanoma model (Zhao et al., 2016). The subsequential studies from their group further revealed that IL-33 is the key cytokine for dectin-1-activated DC-induced Th9 cell differentiation and antitumor activities (Chen et al., 2018). In addition, IL-33 was also found to drive the antitumor immunity of DCs through the induction of Tc9 cells in B16-tumor-bearing mice (Liu et al., 2019). In echo with Siging's studies, Kim et al. (2019) reported that GM-CSF-activated monocyte-derived DCs converted tumor-specific naive helper T cells into the Th9 subset in mouse melanoma, lung cancer, and colon cancer models.

Besides DC vaccines, vaccination with modified tumor cells and tumor antigens can also trigger an antitumor Th9 response in vivo. We have discussed that vaccination against carcinoembryonic antigen (CEA) generated a CEA-specific Th9 response that prevented the engraftment of tumor cells through the activation of mast cells (Abdul-Wahid et al., 2016). A study by Do et al. found that live tumor cells ectopically expressing a membrane-bound form of IL-9 (MB-IL9) provoked CD4⁺ and CD8⁺ T cell activation and increased their cytotoxic activity in a CT26 colon cancer model; however, the toxicity of MB-IL9-expressing tumor cells need to be addressed (Do Thi et al., 2018). Using a similar approach with the live tumor cells, Chen and colleagues developed a tumor vaccine derived from irradiated hepatocellular carcinoma (HCC) cells, which could effectively suppress HCC tumor growth by increasing Th9 cell numbers in HCC-bearing mice (Chen et al., 2021).

Staphylococcal enterotoxin B (SEB) is a superantigen that causes polyclonal T cell activation and cytotoxicity. Two independent groups reported that administration of SEB along with SqC or glioma cell extracts facilitated Th9 cell development in vivo (Miao et al., 2017; Zheng et al., 2017). Both studies showed that SEB together with tumor extracts led to elevated serum IL-9 levels, increased IL-9 secretion from CD4⁺ T cells, and eventually promoted the development of tumor-specific Th9 cells that triggered tumor eradication in an IL-9-dependent manner.

In addition to the tumor vaccine and immunoregulatory reagents, Almeida et al. revealed a key contribution of the gut microbiota in the generation of antitumoral Th9/Tc9 cells in a mouse melanoma lung metastasis model (Almeida et al., 2020). In tumor-bearing mice, the gut microbiota drives the induction of Th9/Tc9 cells, probably through enhancing TGF-β and IL-4 secretion from adjacent tissues. Th9/Tc9 cells then exert antitumor activities by directly killing tumor cells or activating other immune cells (Almeida et al., 2020). In summary, IL‑9 and IL-9-producing cells exert antitumor activities through direct cytotoxicity, provoking innate and adaptive antitumor immunity in various cancer types. Furthermore, targeting cytokines, cosignaling receptors, transcription factors, or utilizing tumor vaccines that promote antitumor functions of IL-9-producing cells may bolster naturally occurring IL-9-producing cells thereby enhancing antitumor immunity.

Prognostic Value of IL-9 and Th9 Cells in Cancer Patients

Although preclinical studies indicated that IL-9 or IL-9-producing cells have contradictory roles in various cancer types, the prognostic value of assaying IL-9 and Th9 cells in cancer patients has been underscored by numerous clinical studies. In a prospective study with 46 melanoma cancer patients treated with anti-PD-1 therapy, markedly increased cell counts of circulating Th9 cells posttreatment were reported to be associated with an improved clinical response for patients with metastatic melanoma, highlighting that peripheral Th9 cells might serve as a biomarker to predict the response to anti-PD-1 therapy (Nonomura et al., 2016). Interestingly, in another study supporting these findings, Zhou et al. (2020) reported an increased CD8⁺ antitumor response as a result of anti-PD1 treatment in MIBC patients with increased intratumoral IL-9⁺ cells. In another clinical trial with 74 metastatic melanoma patients, baseline serum levels of IL-9 predicted response to tumor-infiltrating lymphocyte (TIL) adoptive cell therapies (ACT). They reported that 78% of patients with a serum IL-9 level of 5.3 pg/mL or above responded to therapy (Forget et al., 2018).

Furthermore, IL-9 in conjunction with its role as a lymphocyte growth factor may also play a role in drug resistance in hematological malignancies and could act as a marker for patients likely to develop therapeutic resistance (Hamilton et al., 2021). Altogether, these findings indicate that IL-9 can be a potential pretherapy predictive biomarker for positive clinical outcomes using checkpoint inhibitors in metastatic cancers. Studies on CRC patients also indicated IL-9 as a potential biomarker for CRC progression. Three studies reported lower IL-9 levels in the colorectal tumor tissue compared with normal tissue, and that IL-9 expression is negatively correlated with tumor–node–metastasis (TNM) staging (Huang et al., 2015; Wang et al., 2019; Wang et al., 2018). Furthermore, lower levels of IL‑9 in plasma were detected in colon cancer patients (Huang et al., 2015). The patients with positive IL-9 expression possessed longer survival time compared with those not expressing the protein (Wang et al., 2019) suggesting that higher IL-9 expression may result in a more favorable prognosis.

In a study on a cohort of 66 patients diagnosed with CRC, increased serum IL-9 levels were observed when compared with healthy controls. IL-9, together with Eotaxin, G-CSF, and TNF-α are “good” to discriminate between CRC patients and control subjects (Yamaguchi et al., 2019). Besides melanoma and CRC, IL-9 and Th9 cells have also been reported as antitumoral prognostic biomarkers for other solid cancers, including epithelial ovarian cancer (Habel et al., 2023), endometrial carcinoma (Tong et al., 2020), breast cancer (You et al., 2017), and gastric cancer (Fang et al., 2020).

Although most studies have focused on the antitumoral value of IL-9, the prognostic and therapeutic value of protumoral IL-9 cannot be overlooked in both hematological and solid cancers. To date, several articles have proposed IL-9 as a biomarker for tumor progression in hematological (Fischer et al., 2003; Glimelius et al., 2006; Kumar et al., 2020; Lv et al., 2016; Lv et al., 2013) and solid tumors (Carlsson et al., 2011; Fu et al., 2022; Pasvenskaite et al., 2021; Tan et al., 2017; Zhou et al., 2020); however, further studies are needed to refine the predictive value of IL-9 concentrations and Th9 cell numbers in larger cohorts of patients with various cancers and treatment types.

Therapeutic Implications of IL-9 and Th9/Tc9 in Cancer Patients

Preclinical studies have documented the pleiotropic role of IL-9 in tumor immunity, suggesting that targeting IL-9 has the potential for therapeutic application in the clinic. As IL-9 possesses both protumor and antitumor properties, the contribution of IL-9 to tumor progression must be assessed on a case-by-case basis before any use of IL-9-based therapies in patients. In the context of its antitumor activities, the eradication of tumors by Th9/Tc9 cells may be due to their distinctive features as T cells. No studies have yet demonstrated the effectiveness of adoptive transfer of other IL-9-producing cells in eliminating advanced tumors. However, the superior antitumor capability of Th9/Tc9 ACT has been demonstrated. The results of animal studies with CAR T9 cells and TCR-Th9 cells suggest that ACT with engineered T cells generated under Th9-skewing conditions are a promising immunotherapy for solid cancers. In addition to ACT, targeting the regulators of IL-9 and IL-9-producing cells may represent another strategy to boost IL-9 and IL-9-producing cell-mediated antitumor immunity.

Therapeutic intervention targeting pathogenic IL-9 has been tested in other pathologies such as allergic airway disease where treatment with a humanized monoclonal antibody targeting IL-9 has been shown to reduce asthma exacerbation (Parker et al., 2011; White et al., 2009). Furthermore, in many murine cancer models, it has been demonstrated that blockade of IL-9 using a monoclonal antibody can impair tumor growth, EMT, metastatic spread, and increase overall survival (Chen et al., 2008; Heim et al., 2022; Salazar et al., 2020). Interestingly, Fu et al. (2022) demonstrated the potential for targeted immunotherapy by successfully using nanoparticles to preferentially deliver Il9r siRNA to macrophages that facilitate IL-9-dependent tumor growth in the lung, effectively reducing tumor burden and prolonging survival. Overall, the utility of IL-9-neutralizing antibodies as an immunotherapy depends on the cancer type and the context in which IL-9-responsive cells are functioning due to the ability of IL-9 to function as a protumor or antitumor factor.

Due to the seemingly tissue and cancer-specific effect of IL-9 on tumor development, anti-IL-9 therapeutic strategies will likely require targeted delivery to IL-9-responsive cells to effectively curb tumor growth without causing adverse effects to antitumor immunity mediated by IL-9. In summary, the potential prognostic and therapeutic potential of IL-9 and Th9 cells is clearly documented; however, further studies are needed to investigate the contexts in which IL-9 functions as a protumor or antitumor factor.

Conclusion and Prospects

Preclinical studies have clearly suggested that IL-9/Th9 cells exhibit protumoral properties in certain types of tumors in contrast to established antitumor activities and although human Th9/Tc9 cells have been identified in cancer patients and human IL-9 and IL-9-producing T cells are promising biomarkers for cancer prognosis, the in vivo functions of IL-9 and Th9/Tc9 in cancer patients are still elusive. Several studies have shown that human Th9 cells exhibit unique phenotypic characteristics compared with mouse Th9 cells. Specifically, human Th9 cells produce very low levels of IL-10 (Putheti et al., 2010; Wong et al., 2010), while mouse Th9 cells have been observed to produce higher levels of this cytokine (Dardalhon et al., 2008; Veldhoen et al., 2008). Further exploration is needed to fully understand the functional differences between human and mouse IL-9-producing T cells. To harness the therapeutic potential of targeting IL-9-expressing or Th9 cells in cancer immunotherapy, it is critical to understand the precise mechanisms that regulate their anti- and protumor properties in various cancer types existing in specific tissues.

Conspicuously, IL-9 has been recognized for its dual functions depending on cancer type, the expression pattern of its cognate receptor on tumor and immune cells, and other experimental conditions contributing to diverse cancer outcomes. Nevertheless, further studies are still required to better understand how the TME contributes to the functions of IL-9 and IL-9-producing cells in tumor immunity. Moreover, the immune-related adverse effects on the host must be taken into consideration when enhancing the functions of IL-9-producing cells in vivo or performing Th9/Tc9 cell-based ACT.

The findings regarding Th9/Tc9-based ACT and modulation of IL-9/Th9/Tc9 in animal tumor models have shed light on novel cancer immunotherapies. However, many questions still need to be addressed before translating the preclinical findings to clinical applications in cancer treatment. The most prominent challenge is characterizing the impact of IL-9 signaling on cancer progression in patients. It is worth noting that the timing of IL-9 exposure may be a crucial factor in determining its effect on tumor burden. As demonstrated with other cytokines such as IFN-γ (Gocher et al., 2022), early-stage tumors are more easily influenced by cytokine signaling when compared with advanced clinical-grade tumors. Research into the effects and functions of IL-9 in advanced and established tumor settings is an area needing to be addressed. Moreover, clinical data to determine the impact of IL-9 on tumor growth is lacking. The single-cell genetic and proteomic profiling of IL-9 signaling-related molecules in TME may provide important clues to understanding the precise role of IL-9 in cancer patients.

The second challenge is the management of immune-related adverse events (irAE), which may range in severity from mild to life threatening in clinical settings. While in studies with inbred animals, irAE associated with IL-9-based immunotherapies have shown minimal toxicity, it is not yet clear how that will translate to patients. Given the fact that IL-9 and IL-9-producing cells play pathogenic roles in allergic and autoimmune diseases, the potential of irAE induced by IL-9-based therapies is a concern. Further preclinical studies are needed to improve our understanding of how IL-9-based therapies will affect disease severity in these settings. Furthermore, the IL-9/Th9-responsive cells in TME are not completely defined. A deeper understanding of the variety of cellular responses to IL-9 will be beneficial to establish a network between IL-9 signaling and other cancer-related signaling pathways in TME and could eventually lead to the development of novel combinational therapies for cancer treatment.

In summary, the role of IL-9 in solid tumors is highly debated and seemingly inconsistent potentially attributed to tissue-dependent roles of IL-9-responsive cells found in the TME and the type of cancers that respond to IL-9 treatment. Considerable insights from preclinical and clinical studies have helped clarify the pleiotropic roles of IL-9 and IL-9-producing cells in tumor immunity (Fig. 2); however, more research is needed to better characterize the role of IL-9 in regulating tumor immunity, and importantly, its clinical relevance to developing therapeutics. Future studies on the biology of IL-9, IL-9 producing and responding cells, and its clinical implications will improve our knowledge of their roles in tumor immunity and may eventually lead to the development of efficacious immunotherapy for cancer patients.

FIG. 2.

Effect of IL-9-producing cells in cancer. In cancer, IL-9-producing cells may have protumor or antitumor effects depending on the cancer type and the IL-9-responsive cells in the tumor microenvironment. On the left side, the diagram shows both direct and indirect mechanisms that promote tumor growth, as well as the underlying mechanisms behind them. The diagram on the right illustrates that antitumor effects can be achieved through direct cytotoxicity, activation of the innate/adoptive immune response, and the use of engineered T cells. Figure was made in BioRender.com.

Authors' Contribution

A.C. and J.Z.: Writing—Original draft preparation.: A.P. and M.H.K.: Writing—article review and editing.

Author Disclosure Statement

The authors declare no competing interests.

Funding Information

Funding for the preparation of this review included grants from the Indiana University Simon Comprehensive Cancer Center and the 100 Voices of Hope Foundation and support from the Brown Center for Immunotherapy. A.P. was supported by T32 AI060519. A.C. was supported by T32 HL007910.