Functional diversity and regulation of IL-9-producing T cells in cancer immunotherapy

Abstract

IL-9-producing T cells (T9) regulate immunological responses that affect various cellular biological processes, though their precise function remains fully understood. Previous studies have linked T9 cells to conditions such as allergic disorders, parasitic infection clearance, and various types of cancers. While the functional heterogeneity of IL-9 and T9 cells in cancer development has been documented, these cells present promising therapeutic opportunities for treating solid tumors. This review highlights the roles of IL-9 and T9 cells in cancer progression and treatment responses, focusing on potential discrepancies in IL-9/IL-9R signaling between murine tumors and cancer patients. Additionally, we discuss the regulation of tumor-specific Th9/Tc9 cell differentiation, the therapeutic potential of these cells, and current strategies to enhance their anti-tumor activities.

1. Introduction

As the signature cytokine produced from T9 cells, IL-9 was initially identified as a factor that promotes T cell proliferation. It consists of P40 and TCGF-III and can be produced by a variety of cell types, such as T helper 9 (Th9) cells, type 2 innate lymphoid cells (ILC2s) [1], cytotoxic T cell 9 (Tc9), Vδ2 T cells [2,3], Th17 cells [4], mast cells [5], osteoblasts [6], natural killer T (NKT) cells [7], regulatory T cells (Tregs) cells, and memory B cells [8]. This diverse spectrum of cellular sources suggests that IL-9’s complex expression system is involved in a variety of immune regulations and diseases. IL-9 plays an essential role in regulating various types of cells by activating specific signaling pathways such as signal transducer and activator of transcription STAT1, STAT3, or STAT5. IL-9 binds to its receptors, specifically IL-9R (IL-9 receptor) α and common γ chain, which are also used by IL-2, IL-4, IL-7, IL-15, and IL-21, to initiate a cascade of events that involve the activation of Janus Kinases (JAK)1 and JAK3 [9] the mitogen-activated protein kinase (MAPK) pathway [9], and insulin-related substrate (IRS) pathway [10] as shown in Fig. 1. The signaling pathways triggered by IL-9 are essential in the development and immune response to various conditions such as cancer, allergies, inflammatory diseases, and autoimmune disorders. The intricate network of signals and interactions between these pathways and cells contributes to the overall functioning of the immune system and the body’s ability to respond to different stimuli. In cancer, the role of IL-9 and its associated pathways is particularly significant as they can influence the growth and proliferation of cancerous cells. By regulating the activity of T cells and other immune cells, IL-9 plays a dual role in both promoting and suppressing tumor growth, depending on the specific context and condition within the tumor microenvironment (TME) [11,12].

Fig. 1. IL-9 signaling and activation pathway.

Interleukin-9 (IL-9) is important for regulating various cell types such as T cells, B cells, mast cells, and airway epithelial cells by activating specific signaling pathways like signal transducer and activator of transcription (STAT)1, STAT3, and STAT5. IL-9 binds to receptors IL-9Rα and γc to activate janus kinase (JAK)1, JAK3, mitogen-activated protein kinase (MAPK) pathway, and insulin receptor substrate (IRS) pathway to regulate target gene expression. IL-9R, interleukin 9 receptor; GRB2, growth factor receptor bound protein; SOS, son of sevenless; Raf, rapidly accelerated fibrosarcoma; MEK, mitogen-activated extracellular signal-regulated kinase; ERK, extracellular regulated kinase; PDK1, phosphoinositide-dependent kinase-1; PKB, protein kinase B; Bad, Bcl-2 antagonist of cell death; GSK-3, glycogen synthase kinase-3.

IL-9 was initially identified as a T-cell growth factor linked to the Th2 response. However, it was recently indicated that IL-9-producing T cells (Th9/Tc9) can trigger tissue inflammation and impede tumor growth by activating mast cells or indirectly attracting immature dendritic cells and activated CD8⁺ T cells to tumors [13,14]. The activation of the endogenous immune system by Th9 cells was found to be more successful in regulating tumor progression than standard Th1 cells. Like Th9 cells, CD8⁺ T cells that produce IL-9 have been identified as highly effective effector T cells [15]. Adopting Tc9 cells has been proven to enhance the immune response against tumors, improving long-term survival rates and transforming transplanted cells into more active effector cells. According to studies, the success of removing tumor cells using Tc9 cells relies on IL-9’s ability to stimulate the migration of these cells toward the site of the tumor and facilitate their cytolytic effector function [15]. It is suggested that neutralizing IL-9 antibodies could impact the effectiveness of this approach. The findings have implications for the potential use of Tc9 cells in cancer therapy, as their ability to navigate toward tumor sites and exert their effector functions could be crucial for the success of such treatments. Further research is needed to fully understand the mechanisms behind these processes and to optimize the use of Tc9 cells in cancer immunotherapy.

Overall, IL-9 and IL-9-producing cells play intricate roles that differ depending on the types of cancer and stages of progression. To better understand IL-9-producing T cell-mediated effects, we will review IL-9 functional heterogeneity, the role of T9 cells in tumor therapy, and the potential divergent roles among murine and human cells, as well as primary patient tumors. Additionally, we will discuss the current strategies to improve Th9 and Tc9 cells’ immune responses in cancer immunotherapy.

2. Functional heterogeneity of IL-9/T9 cells in cancer development

IL-9/T9 cells demonstrates a variety of functions in numerous biological processes. Its diverse roles in immune responses and inflammation demonstrate its flexibility as a signaling molecule. The complex and intricate nature of IL-9’s functions underscores the importance of further research in understanding its diverse effects [16,17]. With its ability to modulate immune responses and regulate inflammatory pathways, IL-9 significantly impacts overall physiological homeostasis. Its multifunctional properties suggest a wide range of possibilities for targeting IL-9 in treating various diseases. The primary premise is that IL-9, a cytokine that stimulates lymphocyte proliferation, can potentially accelerate the course of blood cancer. In solid tissue malignancies, IL-9 may inhibit tumor growth by triggering innate or acquired immune responses [18–20]. Recombinant IL-9 or the adoptive transfer of Th9 cells both boosted survival and prevented melanoma progression. Adoptive transfer of IL-9-producing T cells (Th9 or Tc9) into tumor-bearing mice results in cancer elimination in vivo through both direct anticancer effects and the development of anticancer immune responses [21]. Nonetheless, these roles are not absolute, and some studies reported that in some other tumors, IL-9 could contribute to tumor growth as shown in Fig. 2. A study reported that IL-9 is specifically expressed by natural killer (NK)/T cells lymphoma cell lines from patients with nasal NK/T cell lymphoma, which acts as an autocrine growth factor for NK/T cell lymphoma tumors [22]. Therefore, by elucidating the complex interactions and signaling pathways involving IL-9, researchers can uncover new avenues for therapeutic development. Further exploring the specific mechanisms by which IL-9 exerts its effects can provide valuable insights for developing novel treatments and interventions.

Fig. 2. Dual function of Th9 in tumor immunology:

IL-9 from Th9 cells promotes tumor cell migration and proliferation with high IL-9 receptor expression. It also helps recruit Treg cells and mast cells that hinder antitumor immunity. Th9 cells promote antitumor immunity in melanoma by producing IL-9, IL-21, and granzyme. Th9 cells enhance antitumor immunity by activating mast cells, promoting CCR6⁺ dendritic cell recruitment, boosting CTL responses and NK cell function, and directly killing tumor cells. IL, Interleukin; CD, Cluster of differentiation; T_reg, Regulatory T cells; DCs, Dendritic cells; NK, Natural killer cells; CTLs, Cytotoxic T lymphocytes; CCR6, C-C chemokine receptor type 6.

2.1. Antitumorigenic role of IL-9

There is substantial evidence supporting IL-9’s potent anti-tumor effects. Due to its pleiotropic functions, IL-9 can exert direct and indirect anti-tumor actions (Fig. 2). Previous reports have shown that IL-9 can directly inhibit the progression of stomach cancer in nude mice [23]. Researchers have found that IL-4, IL-10, VEGF, and TGF-β levels are decreased in IL-9-treated tumor-bearing nude mice. Apart from the direct anti-tumor activity, IL-9 can stimulate innate and adaptive immune systems, as shown in Fig. 3.

Fig. 3. Antitumor role of IL-9.

Naive CD4⁺ T cells convert into Th9 cells that generate IL-9 and IL-21 when activated by cytokines (TGF-β, IL4) and transcription factors (STAT6, PU.1). IL-9 serves a variety of activities in anticancer immunology, including CCL20-induced recruitment of dendritic cells and CD8⁺ T lymphocytes to the tumor. Th9 cells release IL-21, which promotes T and B cell interactions, resulting in a greater immune response and the establishment of memory T and B cells. IL-21 induces the development of B cells into plasma cells. Th1 cells stimulate inflammatory responses by activating macrophages, NK cells, B cells, and cytotoxic T lymphocytes. STAT, Signal transducer and activation of transcription; DCs, Dendritic cells; MDSC, Myeloid-derived suppressor cell; CD, Cluster of differentiation; NK, Natural killer cells; MC, Macrophages; IFN-γ, Interferon-gamma; TFG-β, Transforming growth factor-beta; IFN-β, Interferon-beta; TNF-α, Tumor necrosis factor-alpha.

The first and most frequently reported immune-stimulatory function of IL-9 was observed in T cells. IL-9-secreting CD4⁺ T cells were found among tumor-infiltrating cells, and Th9 adoptive transfer inhibited melanoma and lung adenocarcinoma growth in an IL-9-dependent manner [24,25]. In addition to CD4⁺ T cells, the effects of IL-9 on CD8⁺ T cells have been best characterized in anti-tumor responses, including breast cancer, colorectal cancer, and melanoma. IL-9 is an immunostimulatory cytokine that modulates CD8⁺ T cell numbers and enhances cytolytic functions. A study reported that IL-9 may stimulate local CCL20 synthesis, subsequently recruiting CD8⁺ T cells to the tumor [25]. The researchers examined the role of IL-9-producing Th9 cells in patients with hepatocellular carcinoma (HCC) and discovered that the levels of circulating IL-9-producing Th9 cells were significantly increased in HCC patients compared to controls. Both peritumoral and tumor tissues showed significant elevation of IL-9-producing Th9 cells. It was also found that CCL20 promotes epithelial-mesenchymal transition-like changes in HCC cells, an effect that could be partially attenuated by STAT3 inhibition. Kalbasi et al. discovered that a synthetic IL-9/IL-9R signal allows cancer-fighting T cells to function without requiring chemotherapy or radiation [26]. The authors developed CAR-T containing an IL-2 receptor extracellular domain and IL-9R intracellular domain. The orthogonal IL-2 cytokine activates STAT1, STAT3, and STAT5 through the IL-9Rα intracellular domain (ICD), leading to stem cell-like memory and effector T cell characteristics [26]. This study indicates that by repurposing IL-9/IL-9R signaling through a chimeric orthogonal cytokine receptor, T cells can acquire additional capabilities, leading to improved effectiveness in fighting challenging solid tumors. Therefore, IL-9 is a key factor in the anti-tumor activity for T cells.

On the other hand, researchers have shown that IL-9 may activate the antitumorigenic effect of innate immune cells. IL-9 exerts its antitumor effect by targeting the activation and function of mast cells [27], thereby achieving direct cytotoxic activity and inducing the recruitment and activation of immune cells against tumor cells. Therefore, mast cell activation is a potential mechanism for the antitumor activity of IL-9 in vivo. In a B16 melanoma mice model, we found that IL-9 increases CCL20 expression in the lung tumor tissues, facilitating the recruitment of CCR6⁺ dendritic cells to enhance antitumor immunity [24]. The author’s findings indicate a distinct role for tumor-targeting Th9 cells in initiating CD8⁺ cytotoxic T cell-mediated antitumor immunity by recruiting CCR6⁺ dendritic cells in tumors, indicating that Th9 cell-based cancer immunotherapy may represent a promising treatment strategy.

2.2. Roles of IL-9/T9 cells in tumorigenesis and progression

The role of IL-9/T9 in tumorigenesis and progression has primarily been investigated in mouse tumor models (Fig. 2); however, its effects on human patient tumors remain unclear. Our analysis of patient data from the Human Protein Atlas database did not provide any evidence that IL-9 plays a significant role in tumor tumorigenesis, immunosuppression, or progression in patients.

2.2.1. Hematological malignancies

IL-9 plays a tumorigenic impact on most hematological cancers due to its ability to act as a lymphocyte growth factor that promotes activation and proliferation [28]. Numerous studies have shown that IL-9 and IL-9R play a significant role in driving oncogenesis in various blood cancers, such as diffuse large B-cell lymphoma (DLBCL), Hodgkin’s lymphoma, chronic lymphocytic leukemia, and large cell anaplastic lymphoma [29–31]. High IL-9Rα production in cancer cells facilitates cell survival and growth. Renauld et al. presented findings showing that mice overexpressing IL-9R were susceptible to developing thymic lymphomas [32]. Although most mice exhibited no significant histological or morphological changes in their lymphoid system, around 7 % developed thymic lymphomas between 3 and 9 months of age. The authors found that IL-9 expression is crucial for optimal tumor growth. Furthermore, the in vitro growth stimulation of IL-9 on cell lines from these tumors lacking the transgene suggested an autocrine mechanism driving their proliferation in vivo. Elevated levels of IL-9R were identified in various human hematological malignancies, such as NKT cell lymphoma, Hodgkin’s lymphoma, and large cell anaplastic lymphoma, and were associated with an unfavorable prognosis [33]. IL-9 overexpression in cutaneous T cell lymphoma (CTCL) is dependent on STAT3/5 and promotes the survival of tumor cells. Furthermore, IL-9 secretion is controlled by STAT3/5, silencing STAT5 or blocking IL-9 with neutralizing antibodies enhanced cell death following UVB or psoralen + UVA (PUVA) treatment in vitro studies. Mice lacking IL-9 showed decreased tumor growth, increased numbers of Tregs, and enhanced stimulation of CD4⁺ and CD8⁺ T lymphocytes [34].

In addition to these published results, we analyzed the RNA-seq/scRNA-seq data from the Human Protein Atlas database (www.proteinatlas.org) to assess the RNA expression level of IL-9R on human immune cells, cancer tissue, and cancer cell lines (Fig. 4). Our findings indicate that only leukemia and lymphoma cancer cell lines exhibit some levels of IL-9R expression, while IL-9R is not expressed in lymphoma tissue from patients (Fig. 4). Unexpectedly, only human eosinophil and B cells produce low levels of IL-9R, whereas IL-9R expression appears to be harmful in other types of human normal immune cells. This low expression pattern likely influences the binding interaction of the expressed cytokines, necessitating further assessment of the effect of IL-9 on blood cancers. Furthermore, our investigations revealed no evidence of IL-9 stimulating the proliferation of numerous B-cell lymphoma cell lines in vitro [35]. Nonetheless, our lab’s findings revealed that tumor antigen-specific IL-9-producing T cells exhibited potent cytotoxicity against these B-cell lymphoma cell lines [35]. To be more specific, NALM-6 tumor cells were intravenously injected into NSG mice, and seven days later, when the tumor burden developed, mice received 4 × 10⁶ CD3⁺ T9 or T1 CAR-T cells via the tail vein. T9 CAR-T cells were more effective in significantly reducing tumor burden and prolonging tumor-bearing mice survival than T1 CAR-T cells. These results showed that T9 CAR-T cells display a strong activity against B cell lymphoma [35]. Mechanistically, T9 CAR-T cells displayed distinct cytokine expression profiles and reduced markers of exhaustion and terminal differentiation. GSEA analysis revealed that human Th9 CAR-T cells are enriched in the central memory stage, while Th1 CAR-T cells are enriched in the effector memory stage. Consistent with these findings, flow cytometry analysis showed a higher frequency of central memory subsets (CCR7⁺CD45RO⁺) in T9 CAR-T cells. Tc9 cells are central memory T cells, which may have contributed to their prolonged persistence, enhanced self-renew and survival, and better antitumor ability in vivo after adoptive transfer. Another contributor to the anti-B-cell lymphoma activity of T9 CAR-T cells is their hyperproliferative capacity. In vitro expansion showed that the Th9 polarizing condition helps T cells acquire increased proliferative capacity. This is evident by the enrichment of molecular signatures related to G2M transition, DNA replication, and cell cycle checkpoint and by decreased frequency of apoptosis in T9 CAR-T cells. After a second exposure to tumor cells both in vitro and in vivo, T9 CAR-T cells also exhibited increased proliferative capacity compared to T1 CAR-T cells, thus exerting a robust anti-B-cell lymphoma activity [35].

Fig. 4. IL-9R expression overview from Human Protein Atlas data analysis.

The encoded protein of this gene serves as a cytokine receptor that facilitates the physiological impacts of interleukin 9 (IL9). The functional IL9 receptor complex relies on this particular protein as well as the interleukin 2 receptor, gamma (IL2RG), which serves as a common gamma subunit shared among the receptors of numerous cytokines. The binding of the ligand to this receptor results in the activation of diverse JAK kinases and STAT proteins and mediates various biological responses. A depicts RNA-seq data presenting an average FPKM (number of Fragments per Kilobase of exon per Million) reads and nTPM (normalized transcripts per million) values, derived from the Human Protein Atlas database (www.proteinatlas.org) for A: immune cells, B: tumor tissue and C: cancer cell lines. The scatter dot plot and bar graph are the average nTPM or FPKM values from various test samples.

2.2.2. Solid tumors

Few reports indicate that IL-9 and IL-9-producing T cells also have positively affected the development of different solid tumors. A study stated that IL-9 stimulates pancreatic cancer cell proliferation and migration through the miR-200α/β-catenin axis. Overexpression of miR-200α in pancreatic cancer cells lowers β-catenin expression, resulting in the development of pancreatic cancer [36]. The authors found elevated levels of Th9, Th17, and Th1 cells in malignant pleural effusion (MPE) compared to blood samples. The rise in Th9 cells within MPE can be attributed to the influence of cytokines and regulatory T cells. Moreover, a separate study linked increased levels of IL-9 in the blood to the progression of breast cancer. Furthermore, an increased number of Th9 cells in the pleural cavity was associated with an elevated risk of death in patients [37]. These Th9 cells were shown to enhance the growth and migration of lung cancer cells by activating STAT3 [36]. Studies revealed that mice deficient in IL-9 had increased levels of active T cells producing IFNγ [38]. It was found that the elimination of endogenous IL-9 enabled the sensitization of host T cells to tumors, leading to their early rejection without the requirement of vaccines or immunomodulatory therapies. Specifically, IL9-deficient mice acquired immunologic memory, which actively protected them from residual disease and tumor rechallenge, an effect linked to the activation of CD8⁺ T cells. Depletion of either CD8⁺ or CD4⁺ T cells abolished the benefits of IL-9 loss to tumor control. Adoptive transfer experiments showed that T cells from tumor-rejecting IL9-deficient mice retained their effector competency in wild-type animals. The same study revealed the ability of IL-9 to enhance the proliferation and migration of pancreatic cancer cells [38]. Moreover, it was also reported that IL-9 can directly increase the survival and proliferation of lung cancer cells [39]. According to a different study, the expression of IL-9 in tissue linked with colitis-associated cancer (CAC) was notably higher than in para-tissues. Upregulating the expression of c-myc and cyclin D1, lentiviral vector-mediated IL-9 overexpression in the colon cancer cell lines RKO and Caco2 may encourage the proliferation of colon cancer cells [40]. Patients with breast cancer have elevated levels of IL-9 in their serum, causing tumor growth [37].

Dominique and colleagues reported that IL-9, an adaptive immune response inhibitor, can prevent immune memory formation that promotes tumor growth. IL-9 deficiency can cause host T cells to become sensitive to tumors and develop immune memory, thereby protecting mice from the recurrence of certain tumors [38]. During the study, researchers injected tumors in both wild-type (WT) mice and mice lacking the IL-9 gene (IL-9KO). The WT mice developed tumors at a rapid rate, but the majority of IL-9KO animals did not develop any tumors [38]. Furthermore, compelling data indicates that IL-9 can enhance the immunosuppressive capabilities of mouse Tregs cells and shield tumor cells from immune cell attacks [41]. However, we found that human Tregs cells do not express IL-9R based on the Human Protein Atlas (Fig. 4A), which suggests that the expression of IL-9R in the human lineage may differ from those indicated in studies involving mice.

To further elucidate the role of IL-9R in the human setting, we performed an RNA-seq analysis using data from the Human Protein Atlas database to evaluate the RNA expression of IL-9R in different cancer tissues. However, our findings indicate that only urothelial cancer exhibits very low IL-9R expression, while other cancer tissues do not express IL-9R, as illustrated in Fig. 4B. The RNA-seq data was presented as the average FPKM (number of Fragments per Kilobase of exon per Million reads), calculated by the TCGA. Therefore, IL-9R is unlikely to contribute to tumorigenesis in solid tumors directly.

Currently, no study has elucidated whether and how IL-9 may play an immunosuppressive role or promote tumor progression in humans. To investigate this, we analyzed the potential association of tumor progression in patients with IL9 levels using Tumor Immune Estimation Resource (TIMER; cistrome.shinyapps.io/timer). As shown in Fig. 5A, IL-9 does not promote patient tumor progression. In addition, we also analyzed the association between IL9 expression in tumors and CD8⁺ T cell infiltration to determine if IL-9 has an immunosuppressive role in patients. As shown in Fig. 5 B, we found no evidence that IL-9 suppresses CD8⁺ T cell infiltration. Thus, further research is necessary to validate these roles.

Fig. 5. Potential association of tumor progression in patients with IL9 levels from Tumor Immune Estimation Resource.

A. IL9 does not promote patient tumor progression. Tumors in the top 20th percentile of IL9 expression were compared with those in the bottom 20th percentile from Tumor Immune Estimation Resource (TIMER; cistrome.shinyapps.io/timer). B. The correlations between the IL9 expression and infiltration of CD8⁺ T cells in patient tumors from TIMER. LUAD: Lung squamous cell carcinoma; PAAD: Pancreatic adenocarcinoma; OV: ovarian serous cystadenocarcinoma cancer; BRCA: Bladder Urothelial Carcinoma.

2.2.3. Unmet needs to develop human Th9 cell biomarkers

Despite ample evidence suggesting a role for IL-9 and IL-9-producing T cells in mouse tumor models, the significance of human Th9 cells in cancer is not yet clear. In individuals with lung cancer, the presence of Th9 cells in cancerous pleural effusion is associated with an elevated risk of death [42]. While some studies have explored the relationship between Th9 cells and IL-9 with melanoma, their impact on patient survival remains uncertain [43]. However, correlation studies between patient outcome and IL-9 as a biomarker for “Th9 cells” may reflect the effects of Tregs or Th2 cells due to the absence of a specific marker for Th9 cells. Therefore, conducting thorough research on cancer patients is crucial to fully understand the function of IL-9 in humans and its potential significance in cancer immunotherapy. IL-9 is a cytokine that can accelerate the development of blood cancers by promoting the growth of lymphocytes. On the other hand, in solid cancers, IL-9 may inhibit tumor growth by triggering immune responses. Nonetheless, these roles are not absolute, and in some solid tumors, IL-9 could contribute to tumor growth [12]. Therefore, the function of IL-9 and IL-9-producing cells, particularly in humans, requires further research to elucidate its role in tumor therapy and tumorigenic response.

3. IL-9-producing T (T9) cells in cancer immunotherapy

IL-9-producing T (T9) cells have emerged as a promising focus in cancer immunotherapy, attributed to their dual capacity to enhance anti-tumor immune responses and directly mediate tumor cell cytotoxicity. Th9 cells play a crucial role in potentiating the immune system’s ability to target and eliminate cancer cells, thereby augmenting the efficacy of immune checkpoint inhibitor (ICI) therapy and improving therapeutic outcomes. In the context of adoptive cell therapy, Th9-polarized CAR-T cells exhibit robust anti-tumor activity, characterized by enhanced proliferative capacity, reduced exhaustion, and the maintenance of a central memory phenotype. Additionally, T9 CAR-T cells demonstrate efficient tumor targeting, differentiate into effector memory T cells that secrete granzyme-B and persist as hyperproliferative, long-lived T cells in vivo. These advancements underscore the pivotal role of IL-9-producing T cells in advancing cancer immunotherapy strategies.

3.1. Th9 cells in ICI therapy

The existence of Th9 cells in the solid tumor microenvironment (TME) is linked to a significant immune response to cancer through both innate and adaptive immune mechanisms, as discussed previously [12]. For immune checkpoint inhibitor (ICI) therapy, a study of 46 melanoma patients treated with Nivolumab (anti-PD-1 antibody) discovered a significant increase in the presence of Th9 cells in the blood of patients who responded positively to the treatment, while other Th subsets showed no difference between responders and non-responders [44]. To investigate the involvement of Th9 cells in CRC, Chenfei Wang and colleagues obtained resected tumor samples from 20 patients diagnosed with CRC [67]. Within TILs, they identified the presence of IL-9⁺ IL-4⁺ CD4⁺ T cells in greater quantities. This indicated that the portion of IL-9-producing TILs were indeed authentic Th9 cells. Notably, these IL-9-secreting TILs exhibited elevated PD-1 expression when analyzed directly ex-vivo. Furthermore, IL-9 expression was significantly diminished due to PD-L1 mediated inhibition, which could be counteracted by applying anti-PD-1 blocking agents. The findings demonstrated that Th9 cells infiltrate CRC tumors, can be modulated via the PD-1/PD-L1 pathway, and play a role in the expression of CD8⁺ T cells. Other in vitro findings indicate that Th9 cells may be a useful predictor of the efficacy of anti-PD-1 therapy in bladder cancer patients, but further clinical research is needed to corroborate this notion [45]. Additionally, higher level of IL-9 in the blood of mice were observed by Yiming Jiang and colleagues, following PD-1 blockade in an orthotropic model of liver cancer [46]. They used nanosecond pulsed electric field (NsPEF) ablation, which has proven to be an effective method for treating early-stage HCC through localized ablation. Additionally, it showed promise for advanced HCC by triggering a significant and lasting immune response. The researchers investigated how NsPEF ablation and PD-1 blockade activate the immune system in a mouse model of orthotropic xenografts HCC. Their findings revealed that both treatments increased immune cell presence in the local tumors and altered cytokine levels in the peripheral blood. However, the specific changes varied between the two treatment modalities. These data suggest that Th9 cells may enhance the response of immune checkpoint inhibitor therapy in solid cancer, such as melanoma, live cancer, and bladder cancer.

Furthermore, Th9 cells exhibit a considerable ability to alter the tumor microenvironment, as the infusion of Th9 cells markedly reduced immunosuppressive cell populations, including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSCs) [47]. It is important to recognize that the predominance of an immunosuppressive microenvironment is a principal factor contributing to the ineffectiveness of ICI therapies and ICI resistance [48–50], such as environment leads to poor immune cell infiltration and T-cell exhaustion. The ability of Th9 cells to target MDSCs and TAMs within solid tumors may be highly valuable when integrated with other cancer immunotherapy approaches. For example, the synergistic potential of combining Th9 cell-based adoptive cell therapy with ICI therapy could enhance the overall anti-tumor response.

3.2. Tumor-specific Th9 cells in adoptive cell therapy

Adoptive T-cell therapy (ACT) is suggested as a novel therapeutic approach that has exhibited promise in preliminary clinical studies [51,52]. Although evidence suggests that tumor-specific Th9 cells possess strong antitumor capabilities, their use in ACT remains under investigation. Studies in pre-clinical models of melanoma and lung adenocarcinoma have demonstrated that transferring antigen-specific Th9 cells can generate a powerful antitumor response [21,46]. This response, driven by Th9 cells, includes the attraction and sustenance of dendritic cells (DCs) at the tumor site [53]. Furthermore, in a leukemia mouse model, in vitro polarized Th9 cells for allogenic bone marrow transplantation significantly eliminated tumors without triggering graft versus host disease [54]. Following the successful adoptive transfer of tumor-specific Th9 cells for cancer treatment, our group sought to induce this phenotype in CAR-T cells. The authors demonstrated that engineering CAR-T cells to release IL-9 enhanced their antitumor activity compared to regular Th1-polarized CAR-T cells. Th9-polarized CAR-T cells, exhibited increased proliferative ability, lower exhaustion and displayed a central memory phenotype. Further research demonstrated that CAR-T cells receiving an IL-9 signal develop a unique T cell phenotype that combines beneficial characteristics of both memory stem cells and effector T cells, resulting in enhanced antitumor activity in vivo [26]. Tao Chen and colleagues outlined an immune cell surface engineering approach aimed at substantially augmenting the anti-tumor efficacy of Th9-mediated ACT. This approach involves rapidly identifying tumor-specific binding ligands and enhancing the infiltration of administered cells into solid tumors [47]. The authors found that the non-genetic modification of Th9 cells with tumor-targeting peptides, via phage display, enabled precise targeting of highly heterogeneous solid tumors and markedly improved the infiltration of CD8⁺ T cells, resulting in enhanced antitumor efficacy. From this mechanistic perspective, the authors found that the infusion of Th9 cells modified with tumor-specific binding ligands improved the distribution of tumor-eradicating cells and restructured the immunosuppressive microenvironment of solid tumors through IL-9 mediated immunomodulation [47]. These findings suggest that Th9 cells are promising candidates for ACT in human cancers.

Tumor heterogeneity and antigen loss can lead to tumor relapse or progression following cancer-adoptive cell immunotherapy. This heterogeneity can be observed among cancer cells within a single tumor, between tumors of the same type in different individuals, or between primary and metastatic tumors. It includes variations in gene expression, cell structure, metabolism, growth, and metastasis, which are common in many cancer patients and contribute to developing resistance to cancer therapies. Patients may develop resistance to ACT if antigen-loss-variant (ALV) cancer cells expand [55]. Recently, our group discovered that tumor-specific Th9 cells can not only target and eliminate antigen-positive cancer cells but also kill antigen-negative cancer cells within tumors. In mouse models of melanoma, we compared the effectiveness of Th1, Th17, and Th9 cells in combination with tumor antigen-loaded dendritic cells for ACT. Mice were treated with cyclophosphamide to induce temporary lymphopenia before cell transfer. Th9 cells were the only group to significantly reduce established tumors, promote long-term survival, and protect against cancer re-challenge. In contrast, Th1 and Th17 cells only caused temporary tumor-shrinkage, followed by rapid regrowth. Further analysis revealed that Th9 cells expressed more co-stimulatory molecules and fewer inhibitory receptors compared to Th1 cells [56]. These findings highlight that tumor-specific Th9 cells are a unique subset of T cells capable of effectively eliminating ALVs to achieve therapeutic effects.

3.3. Tumor-specific Tc9 cells in adoptive cell therapy

CD8⁺ T cells play a crucial role in the adaptive immune response against tumors and can be classified into different subtypes, such as Tc1, Tc2, Tc17, and Tc9 cells [57,58]. Tc9 cells can develop from CD8⁺ T cells in the presence of TGFβ1 and IL-4 [59]. Tc9 cells can provoke more robust antitumor responses against established tumors than type-I CD8⁺ cytotoxic T cells presently utilized in clinical treatment. Unlike typical cytotoxic T lymphocytes (CTLs), it was reported that tumor-specific Tc9 cells have lower levels of granzyme B, Eomes, T-bet, and IFN-γ but produce high amounts of IL-9 [15].

Our original study showed that when tumor-specific Tc9 cells are used in combination with in vivo therapies like IL-2 injections, tumor antigen vaccination, and lymphodepletion, they can successfully target and treat melanoma tumors [60]. In addition, it was observed that transferring tumor-reactive Tc9 cells resulted in stronger antitumor responses against large established tumors compared to conventional type-I CD8⁺ cytotoxic T cells commonly used in clinical settings. Tc9 cells also demonstrated a higher potential for persistence and the ability to maintain their effector function after transfer. The study also highlighted the importance of IL-9 production by Tc9 cells for their therapeutic effect in vivo and found that prolonged antitumor responses mediated by Tc9 cells have the potential to advance cancer immunotherapy significantly [15]. Additionally, Tc9 cells demonstrated superiority over Tc1 cells in adoptive cell transfer due to their ability to develop into effector cells that can remain active for a longer period in vivo, show resistance to exhaustion and apoptosis, and thus exhibit better control over tumor growth [15]. In addition, we reported that polarized and expanded human CAR-T cells grown in a Th9 culture condition (T9 CAR-T) showed increased anticancer activity against established tumors [35]. T9 CAR-T cells expressed central memory phenotype, exhibited robust proliferative potential, and released IL-9 but limited IFN-γ compared to IL2-polarized (T1) cells. Consequently, in vivo studies, researchers found that T9 CAR-T cells exhibited greater antitumor efficacy against established hematologic and solid tumors than T1 CAR-T cells. Following the transfer, T9 CAR-T cells target tumors efficiently, develop into effector memory T cells that secrete granzyme-B and IFN-γ, and subsequently persist as hyperproliferative, long-lived T cells [35].

However, murine Tc9 cells reportedly do not maintain their marker cytokine IL-9 in two disease models. In an allergic airway disease model, murine Tc9 cells exhibit a Tc1/Tc2 hybrid profile, producing both IL-13 and IFN-γ simultaneously [61]. Our results revealed decreased IL-9 production from murine Tc9 cells over time following in vivo transfer. Nevertheless, these cells consistently maintain low levels of IL-9 expression in vivo [15]. Other studies revealed that Tc9 cells demonstrate a degree of instability in vivo, showing flexibility toward becoming Tc1 or Tc1/Tc2 cells. However, most of the current research on Tc9 cells pertains to murine models. Further investigation into human Tc9 cells is needed to elucidate their characteristics and apply findings to the clinic.

4. Strategies to enhance T9 cells’ anti-tumor effects

The anti-tumor efficacy of T9 cells can be precisely enhanced using cytokines, co-stimulation molecules, and other strategies, as shown in Fig. 6. Cytokines such as IL-1β and TGF-β promote T9 cell differentiation and increase IL-9 production, thereby enhancing their anti-tumor activity. Co-stimulatory molecules like GITR improve T9 cell activation, survival, and cytokine secretion, leading to more effective anti-tumor responses. Additionally, other strategies in genetic and metabolic modifications, such as knocking out Id3, are also utilized to further amplify Th9 cells’ anti-tumor effects by producing IL-9. These approaches offer a highly specific means to optimize T9 cell-mediated anti-tumor effects in cancer immunotherapy.

Fig. 6. Strategies to enhance T9 cells’ anti-tumor effects.

This figure depicts various methods to improve the anti-tumor activity of T9 cells. Key strategies include: 1. Cytokine Enhancement: utilization of cytokines such as IL-1β in combination with IL-4 and TGF-β, or IL-1β with IL-4, IL-7, or IL-33, to bolster T9 cells’ anti-tumor activity. 2. Co-Stimulatory Pathways: Engagement of co-stimulatory signaling pathways like OX40-OX40L or GTTR-GITRL to support T9 cells’ anticancer activity. 3. Other strategies include genetic modifications, including IRF8 overexpression or Atg5 and Id3 knockout, to modify T9 cells’ differentiation and enhance their antitumor effects and metabolic modifications, such as inhibition of fatty acid biosynthesis to improve T9 cell performance against tumors. NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; STAT, signal transducer and activator of transcription; TGF-β, transforming growth factor-β; GATA-3, GATA-binding protein 3; IRF, transcription factors interferon (IFN)-regulatory factor; OX40, tumor necrosis factor receptor superfamily member 4; GITR, glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein; PU.1, purine-Rich Box 1; Id3, inhibitor of DNA-binding 3; ETV6, ETS variant transcription factor 6; Atg5, Autophagy-Related 5; TRAF6, TNF receptor-associated factor 6.

4.1. Cytokines enhancing T9 cells’ anti-tumor effects

The anti-tumor activity of T9 cells can be enhanced through the involvement of several cytokines (Fig. 6). Initially, IL-4 and TGF-β have been recognized as indispensable for Th9 cell differentiation [62,63]. However, neither alone is sufficient to produce IL-4 nor TGF-β. TGF-β can generate the Th9 cell transcriptional profile or induce high levels of IL-9 expression in T cells [62,63]. Végran et al. demonstrated that the adoptive transfer of Th9 cells induced by a combination of IL-4, TGF-β, and IL-1β into mice bearing melanoma or lung adenocarcinoma tumors resulted in potent anticancer effects, which were dependent on the expression of IRF1, and IL-21 produced by Th9 cells [64]. Mechanically, under Th9-skewing conditions, IL-1β induced phosphorylation of the transcription factor STAT1, leading to the IRF1 expression. IRF1 subsequently binds to the promoters of Il9 and Il21 and enhances the secretion of the cytokines IL-9 and IL-21 from Th9 cells, exhibiting potent anticancer functions [64]. However, our group’s findings suggested that TGF-β is not essential for the formation and anti-tumor activity of Th9 cells; IL-4 combined with IL-1β can stimulate IL-9 synthesis even in the absence of TGF-β signaling [65]. Additionally, our previous study showed that IL-9 production by Th9^{IL−4+IL−1β} cells relies on the NF-κB pathway, specifically, RelA. Tumor-specific Th9^{IL−4+IL−1β} cells exhibit potent anti-cancer activity and cytolytic effector functions [65]. Qing Yi’s group demonstrated that IL-7 further enhances the differentiation of naïve CD4⁺ T cells into Th9 cells and augmented their antitumor activity. IL-7 markedly increased the abundance of the histone acetyltransferase p300 by activating the STAT5 and PI3K-AKT mTOR signaling pathways and promoting histone acetylation at the Il9 promoter. Consequently, the transcriptional regulator forkhead box protein O1 (Foxo1) was dephosphorylated and translocated to the nucleus, binds to the Il9 promoter, and induces the production of IL-9 protein. In contrast, forkhead box protein P1 (Foxp1), which binds to the Il9 promoter in naïve CD4⁺ T cells and inhibits Il9 expression, is outcompeted by Foxo1 for Il9 promoter binding and translocating to the cytoplasm. Additionally, Ramadan and colleagues demonstrated that the differentiation of human T9 cells in the presence of IL-33 enhances IL-9 production by CD4⁺ T cells [66]. Th9^IL−33 cells also upregulated the expression of the cytolytic molecule granzymes A and B compared to Th9 cells, and exhibited higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations [66].

4.2. Co-stimulation molecules enhancing T9 cells’ anti-tumor effects

The co-stimulation signaling pathway plays a critical role in generating T9 cells and their antitumor activity (Fig. 6). We have reported that dendritic cells (DCs) stimulated by dectin-1 agonists can upregulate TNFSF15 and OX40L, which can promote Th9 differentiation and enhance antitumor response [67]. Mechanistically, dectin-1 activates Syk, Raf1, and NF-κB signaling pathways, leading to increased nuclear translocation of p50 and RelB and expression of TNFSF15 and OX40L, which are essential for dectin-1-activated DC-induced Th9 cell priming. These findings demonstrate that TNFSF15 and OX40L-mediated co-stimulation signaling can amplify the Th9 cells’ anti-tumor response. In addition, Il-Kyu Kim et al. reported that GITR co-stimulation profoundly augments Th9 cell differentiation and promotes tumor-specific Tc9 cell responses and DC activation in an IL-9-dependent fashion [68]. They further found that GITR signaling enhances IL-9–producing CD4⁺ Th9 cell differentiation through a TNFR-associated factor 6 (TRAF6)- and NF-κB–dependent manner and inhibited the generation of induced regulatory T cells in vitro.

4.3. Other strategies to enhance T9 cells’ anti-tumor effects

Recent advancements in enhancing the anti-tumor effects of T9 cells include genetic and metabolic modifications. Id3-deficient Th9 cells have shown stronger anti-tumor activity than naïve Th9 cells [69]. Specifically, the authors adoptively transferred TGF-β1-treated Th9 cells from Id3^f/fCd4-Cre ⁺ mice or Id3^+/+Cd4-Cre ⁺ mice into Rag1^−/−mice, which were then challenged with melanoma cancer cells. Mice receiving TGF-β1-treated naïve Id3^f/fCd4-Cre⁺ T cells showed substantial suppression of tumor growth and development compared to those receiving TGF-β1-treated naïve Id3^+/+Cd4-Cre⁺ T cells [69]. Mechanically, reduced Id3 expression enhanced the binding of the transcription factors E2A and GATA-3 to the Il9 promoter, increasing Il9 transcription and thus boosting anticancer activity [69].

Another study demonstrated that IRF8 is essential for the anti-tumor effect of Th9 cells in the melanoma mouse model. IRF8 functions within a transcription factor complex consisting of IRF8, IRF4, PU.1, and BATF, which binds to DNA and boosts Il9. transcription. In contrast, IRF8 deficiency leads to increased expression of other genes, such as Il4, as IRF8 dimerizes with the transcriptional repressor ETV6, which inhibits Il4 expression [70].

Recently, Takahiro et al. reported that inhibiting de novo fatty acid biosynthesis and the depleting environmental lipids enhanced Th9 differentiation and IL-9 production, improving the anti-tumor activity of Th9 cells in both mice and humans [71]. Their mechanistic studies revealed that these effects were mediated by the retinoic acid receptor and the TGF-β–SMAD signaling pathways. When adoptively transferred, acetyl-CoA carboxylase 1 (ACC1)-inhibited Th9 cells suppressed tumor growth in murine models of melanoma and adenocarcinoma. Together, these findings suggest a novel strategy to enhance the anti-tumor response of Th9 cells in vivo by inhibiting fatty acid biosynthesis [71]. However, most existing strategies to improve the anticancer efficacy of T9 cells have been developed primarily in murine models. Further research is urgently needed to investigate human T9 cells and translate these insights into effective clinical applications.

5. Conclusion and prospectives

IL-9 can influence various cell types in proinflammatory and anti-inflammatory ways, resulting in its dual impact on cancer development. Despite notable progress in treatments in recent years, the lack of direct evidence from animal and clinical studies on the role of IL-9 in cancer immunology remains a challenge. The most convincing evidence for IL-9’s direct effects on cells highlights mast cells, ILCs, and macrophages as key contributors to cancer development outcomes [72]. However, this is not a comprehensive list and further research will likely reveal more about how IL-9 affects different cell types and their role in cancer immunology. This will enable the development of more targeted strategies to modify IL-9-mediated immune responses. A deeper understanding of IL-9 signaling pathways and their specific effects on different cell types will provide a more comprehensive view of how IL-9 functions in proinflammatory or anti-inflammatory contexts in cancer.

Th9 cells, a recently identified subset of CD4⁺ T cells, can stimulate both innate and adaptive immune responses through IL-9 and IL-21 secretion. Numerous previous studies have shown that Th9 cells possess anti-tumor properties by releasing granzyme B to directly target and eliminate blood and solid tumor cells, although only one study has observed a tumorigenic role for Th9 cells in human HCC. Notably, most of these studies have focused on mouse models. Recent research has shown that human T9 CAR-T cells exhibited greater antitumor efficacy against established hematologic and solid tumors than T1 CAR-T cells both in vitro and in vivo [35]. Specifically, T9 CAR-T cells display central memory phenotype, exhibited robust proliferative potential, and released IL-9 but limited IFN-γ comparison to T1 CAR-T cells. However, further investigations are highly necessary to fully understand the function of human Th9 or Tc9 cells in cancer immunology and to assess the safety and effectiveness of using Th9 or Tc9 cells in adoptive cell therapy.

Highlights.

• The roles of IL-9 and T9 cells in cancer progression are discussed.

• T9 cells are promising in cancer immunotherapy.

• Strategies to enhance T9 cells’ anti-tumor effects in immunotherapy are explored.

Abbreviations

ACT

Adoptive cell therapy

ALV

Antigen loss variant

BATF

Basic leucine zipper ATF-like transcription factor

CAC

Colitis-associated cancer

CAR-T

Chimeric antigen receptor T cells

CCL20

Chemokine (C-C motif) ligand 20

CD4

Cluster of differentiation 4

CD8

Cluster of differentiation 8

CTCL

Cutaneous T cell lymphoma

DC

Dendritic cell

DLBCL

Diffuse large B cell lymphoma

GITR

Glucocorticoid-induced TNFR-related

GM-CSF

Granulocyte macrophage colony stimulating factor

ICI

Immune checkpoint inhibitor

IL-9

Interleukin-9

ILC2s

Type 2 innate lymphoid cells

IRF4

Interferon regulatory factor-4

IRS

Insulin receptor substrate

JAK

Janus kinase

MAPK

Mitogen activated protein kinase

NK cell

Natural killer cell

PD-1

Programmed cell death protein 1

PUVA

Psoralen and ultraviolet A irradiation

STAT

Signal transducer and activation of transcription

Tc9

Cytotoxic T cell 9

TCGF-III

T cell growth factor-III

TGF-β

Transforming growth factor-Beta

Th9

T helper cell 9

TME

Tumor microenvironment

TNFR

Tumor necrosis factor receptor

TNFSF

Tumor necrosis factor superfamily

VEGF

Vascular endothelial growth factor

WT

Wild type