Tumor rejection in Cblb-/- mice depends on IL-9 and Th9 cells.

BACKGROUND: Casitas B lymphoma-b (Cbl-b) is a central negative regulator of cytotoxic T and natural killer (NK) cells and functions as an intracellular checkpoint in cancer. In particular, Th9 cells support mast cell activation, promote dendritic cell recruitment, enhance the cytolytic function of cytotoxic T lymphocytes and NK cells, and directly kill tumor cells, thereby contributing to tumor immunity. However, the role of Cbl-b in the differentiation and antitumor function of Th9 cells is not sufficiently resolved. METHODS: Using Cblb-/- mice, we investigated the effect of knocking out Cblb on the differentiation process and function of different T helper cell subsets, focusing on regulatory T cell (Treg) and Th9 cells. We applied single-cell RNA (scRNA) sequencing of in vitro differentiated Th9 cells to understand how Cbl-b shapes the transcriptome and regulates the differentiation and function of Th9 cells. We transferred tumor-model antigen-specific Cblb-/- Th9 cells into melanoma-bearing mice and assessed tumor control in vivo. In addition, we blocked interleukin (IL)-9 in melanoma cell-exposed Cblb-/- mice to investigate the role of IL-9 in tumor immunity. RESULTS: Here, we provide experimental evidence that Cbl-b acts as a rheostat favoring Tregs at the expense of Th9 cell differentiation. Cblb-/- Th9 cells exert superior antitumor activity leading to improved melanoma control in vivo. Accordingly, blocking IL-9 in melanoma cell-exposed Cblb-/- mice reversed their tumor rejection phenotype. Furthermore, scRNA sequencing of in vitro differentiated Th9 cells from naïve T cells isolated from wildtype and Cblb-/- animals revealed a transcriptomic basis for increased Th9 cell differentiation. CONCLUSION: We established IL-9 and Th9 cells as key antitumor executers in Cblb-/- animals. This knowledge may be helpful for the future improvement of adoptive T cell therapies in cancer.

Interleukin-9 regulates macrophage activation in the progressive multiple sclerosis brain.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated, chronic inflammatory, and demyelinating disease of the central nervous system (CNS). Several cytokines are thought to be involved in the regulation of MS pathogenesis. We recently identified interleukin (IL)-9 as a cytokine reducing inflammation and protecting from neurodegeneration in relapsing-remitting MS patients. However, the expression of IL-9 in CNS, and the mechanisms underlying the effect of IL-9 on CNS infiltrating immune cells have never been investigated. METHODS: To address this question, we first analyzed the expression levels of IL-9 in post-mortem cerebrospinal fluid of MS patients and the in situ expression of IL-9 in post-mortem MS brain samples by immunohistochemistry. A complementary investigation focused on identifying which immune cells express IL-9 receptor (IL-9R) by flow cytometry, western blot, and immunohistochemistry. Finally, we explored the effect of IL-9 on IL-9-responsive cells, analyzing the induced signaling pathways and functional properties. RESULTS: We found that macrophages, microglia, and CD4 T lymphocytes were the cells expressing the highest levels of IL-9 in the MS brain. Of the immune cells circulating in the blood, monocytes/macrophages were the most responsive to IL-9. We validated the expression of IL-9R by macrophages/microglia in post-mortem brain sections of MS patients. IL-9 induced activation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 and reduced the expression of activation markers, such as CD45, CD14, CD68, and CD11b in inflammatory macrophages stimulated in vitro with lipopolysaccharide and interferon (IFN)-γ. Similarly, in situ the number of activated CD68+ macrophages was significantly reduced in areas with high levels of IL-9. Moreover, in the same conditions, IL-9 increased the secretion of the anti-inflammatory cytokine, transforming growth factor (TGF)-ß. CONCLUSIONS: These results reveal a new cytokine expressed in the CNS, with a role in the context of MS. We have demonstrated that IL-9 and its receptor are both expressed in CNS. Moreover, we found that IL-9 decreases the activation state and promotes the anti-inflammatory properties of human macrophages. This mechanism may contribute to the beneficial effects of IL-9 that are observed in MS, and may be therapeutically potentiated by modulating IL-9 expression in MS.

IL-33/ST2/IL-9/IL-9R signaling disrupts ocular surface barrier in allergic inflammation.

This study was to explore a novel IL-33/ST2/IL-9/IL-9R signaling pathway that disrupts ocular surface barrier and amplifies allergic inflammation. Two murine models of experimental allergic conjunctivitis (EAC) and IL-9 topical challenge in wild type Balb/c and ST2-/- mice, and two culture models of primarily human corneal epithelial cells (HCECs) and mouse CD4+ T cells were performed. Clinical manifestations, Oregon-Green Dextran (OGD) staining, the apical junction complexes (AJCs), IL-33/ST2 and IL-9/IL-9R signaling molecules were evaluated in ocular surface and its draining cervical lymph nodes (CLNs) by RT-qPCR, immunostaining and ELISA. The typical allergic signs, enhanced OGD staining intensity, disrupted morphology of AJCs, including ZO-1, claudin 1, occludin, and E-cadherin, and the stimulated signaling of IL-33/ST2 and IL-9/IL-9R were observed in ocular mucosa and draining CLNs in EAC-Balb/c mice, but significantly reduced or eliminated in EAC-ST2-/- mice. Topical challenge of IL-9 resulted in the obvious OGD staining and disrupted ocular surface AJCs in Balb/c mice and in HCECs in vitro. IL-9 production was found to be stimulated by IL-33 in CD4+ cells from Balb/c mice in vitro. Our findings uncovered a novel phenomenon and mechanism by which ocular surface barrier integrity is disrupted in allergic conjunctivitis by IL-33/ST2/IL-9/IL-9R signaling pathway, which may amplify the allergic inflammation.

IL-33 induced airways inflammation is partially dependent on IL-9.

Asthma is an inflammatory disease of the airways and numerous cytokines contribute to this pathogenesis. It is shown that challenge of airways with IL-33 induces asthma-like pathological changes in mice, but the possible downstream cytokines in this process remain to be characterised. To explore this, we compared changes in the airways of wildtype (WT) and IL-9 deficient mice challenged with IL-33. In line with previous report, per-nasal challenge of WT mice with IL-33 significantly increased the responsiveness of the airways along with infiltration of inflammatory cells, goblet cell hyperplasia, collagen deposition and smooth muscle hypertrophy, and the expression of cytokines compared with control group. Surprisingly, all of these pathological changes were significantly attenuated in IL-9 deficient mice following identical IL-33 challenge. These data suggest that IL-9 is one downstream cytokine relevant to the effects of IL-33 in asthmatic airways and consequently a potential therapeutic target for the treatment of asthma.

Decreased placental IL9 and IL9R in preeclampsia impair trophoblast cell proliferation, invasion, and angiogenesis.

OBJECTIVE: Our study aimed to investigate IL9 and IL9R expression in preeclampsia and assess their effects on trophoblast biological behaviors. METHODS: IL9 and IL9R expression of placenta tissue were evaluated by immunochemistry and q-PCR. Using transwell, CCK-8, and tubule formation assays measured invasion, proliferation and angiogenesis of trophoblast with adding IL9 or anti-IL9R antibody. RESULTS: IL9 and IL9R levels were significantly decreased in preeclampsia. IL9 improved trophoblast activities. Blocking IL9/IL9R resulted in decreased proliferation, invasion, and tube-formation capability of trophoblast. CONCLUSIONS: IL9 and IL9R contribute to the pathogenesis of preeclampsia. IL9/IL9R signaling provides a new potential therapeutic target for preventing preeclampsia.

Th9 cells are subjected to PD-1/PD-L1-mediated inhibition and are capable of promoting CD8 T cell expansion through IL-9R in colorectal cancer.

Th9 cells are named after their expression of IL-9. Studies in recent years demonstrated that Th9 cells could contribute to antitumor immunity by enhancing the recruitment and activation of mast cells, natural killer cells, CD8 T cells, and dendritic cells in the tumor microenvironment. To determine whether Th9 cells participate in colorectal cancer (CRC), we collected resected tumor samples from 20 CRC patients. In the tumor-infiltrating lymphocytes (TILs), IL-9+IL-4- CD4+ T cells could be observed and were present at higher frequencies than the IL-9+IL-4+ and the IL-9-IL-4+ cells, suggesting that the majority of IL-9-producing TILs were bona fide Th9 cells. IL-9-secreting TILs presented particularly high PD-1 expression directly ex vivo. The expression of IL-9 was significantly reduced with PD-L1-mediated inhibition, which in turn was suppressed by anti-PD-1 blocking. Interestingly, the circulating CD4+ T cell compartment in CRC patients also presented Th9 enrichment, characterized by higher IL-9+IL-4- and IL-9+IL-4+ cell frequencies in the CXCR3-CCR6- compartment as compared to that in non-cancer controls. Using exogenous TGF-ß and IL-4, we were capable of enriching Th9 cells without concurrent enrichment of Th2 cells. Th9-enriched CD4+ T cells, but not Th9-non-enriched cells, significantly increased the expansion of activated CD8+ T cells, in a manner that was dependent on the expression of IL-9R. In addition, the frequencies of Th9 cells in the tumor were positively correlated with the frequencies of CD8+ TILs. Together, we demonstrated that Th9 cells infiltrated CRC tumor, could be regulated via the PD-1/PD-L1 pathway, and could contribute the CD8+ T cell expansion.

Interleukin 9 prevents immune thrombocytopenia in mice via JAK/STAT5 signaling.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoimmune-mediated platelet destruction and impaired platelet production, which can lead to an increased risk of bleeding. The clinical management of ITP currently remains a challenge for hematologists. We explored the role of interleukin-9 (IL-9) in the treatment of CD41-induced ITP, and investigated its underlying mechanisms in a CD41-induced ITP mouse model. IL-9 treatment increased the numbers of mature megakaryocytes (CD41+CD42d+) and CD41+Sca-1+ cells in the bone marrow in these model mice, while IL-9 receptor (IL-9R) small interfering RNA (siRNA) inhibited the process. Moreover, phosphorylated signal transducer and activator of transcription 5 (STAT5), as a downstream molecule of IL-9R, was increased after IL-9 treatment. We next investigated the source of IL-9 in bone marrow, osteoblasts produced the highest level of IL-9. These results confirmed that IL-9 could prevent CD41-induced ITP in BALB/c mice by regulating osteoblasts and activating IL-9R/STAT5 signaling in megakaryocytes, thus providing further evidence for IL-9 as a promising therapeutic agent for the treatment of ITP.

Th9 cytokines curb cervical cancer progression and immune evasion.

Cervical cancer is one of the most common cancers among women in developing countries. Persistent infection with high-risk human papillomavirus (HPV) is the major determinant for the development of cervical cancer. Role of newly discovered T helper 9 (Th9) cells in cervical cancer pathogenesis is yet unfolded. In this study, we observed a huge infiltration of PU.1+ cells and overrepresentation of IL-9R in tissue biopsy specimens of CIN patients in cervical cancer cases. Treatment with Th9 signatory cytokines, IL-9 and IL-21, suppressed proliferation, enhanced apoptosis and stimulated the expression of MHC I and e-cadherin on HeLa cell lines. Th9 thus seems enhance antitumor immune response through T cell cytotoxicity and play crucial role in a controlling malignant cell transformation. Therefore, this study helps in firmer understanding of relevance of Th9 in cervical cancer immunity.

An Update on Interleukin-9: From Its Cellular Source and Signal Transduction to Its Role in Immunopathogenesis.

Interleukin-9 (IL-9) is a pleiotropic cytokine and was primarily studied in the context of T helper 2 (TH2)-associated immuno-pathological conditions such as asthma and parasitic infections. There was a paradigm shift in the biology of IL-9 after the recent discovery of TH9 cells, a new subtype of TH cells which secrete IL-9 in copious amounts. This has resulted in renewed interest in this cytokine, which was neglected since discovery because it was considered it to be just another TH2 cytokine. Recent studies have shown that it has multiple cellular sources and is critically involved in the immune-pathogenesis of inflammatory diseases and in guarding immune tolerance. In this review, we will discuss its discovery, gene organization, cellular sources, and signaling pathways. Especially, we will give an update on the recent development regarding its relevance in the immune pathogenesis of human diseases.

IL-9 Blockade Suppresses Silica-induced Lung Inflammation and Fibrosis in Mice.

Recapitulative animal models of idiopathic pulmonary fibrosis (IPF) and related diseases are lacking, which inhibits our ability to fully clarify the pathogenesis of these diseases. Although lung fibrosis in mouse models is often induced by bleomycin, silica-induced lung fibrosis is more sustainable and more progressive. Therefore, in this study, we sought to elucidate the mediator(s) responsible for the pathogenesis of lung fibrosis, through the use of a mouse model of silica-induced lung fibrosis. With a single nasal administration of 16 mg of silica, lung inflammation (assessed by elevated cellular components in the BAL fluids [BALFs]) and lung fibrosis (assessed by lung histology and lung hydroxyproline levels) were induced and sustained for as long as 24 weeks. Of the mediators measured in the BALFs, IL-9 was characteristically elevated gradually, and peaked at 24 weeks after silica administration. Treatment of silica-challenged mice with anti-IL-9-neutralizing antibody inhibited lung fibrosis, as assessed by lung hydroxyproline level, and suppressed the levels of major mediators, including IL-1ß, IL-6, IL-12, CCL2, CXCL1, and TNF-α in BALFs. Moreover, human lung specimens from patients with IPF have shown high expression of IL-9 in alveolar macrophages, CD4-positive cells, and receptors for IL-9 in airway epithelial cells. Collectively, these data suggest that IL-9 plays an important role in the pathogenesis of lung fibrosis in diseases such as IPF.

Interleukin 9 Alters Epithelial Barrier and E-cadherin in Eosinophilic Esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic TH2-assocated inflammatory condition accompanied by substantial impairments in epithelial barrier function and increased numbers of interleukin 9 (IL-9) expressing inflammatory cells. While IL-9 is known to affect barrier function in the intestine, the functional effects of IL-9 on the esophagus are unclear. Herein we aimed to understand the expression of the IL-9 receptor and effects of IL-9 on the epithelium in EoE. METHODS: We used esophageal biopsies from pediatric EoE patients with active and inactive disease to analyze the expression of the IL-9 receptor, the adherens junction protein E-cadherin and the tight junction protein claudin-1. We treated primary human esophageal epithelial cells with IL-9 to understand its effects on E-cadherin expression and function. RESULTS: Active EoE subjects had increased epithelial expression of IL-9 receptor mRNA and protein (P < 0.05) and decreased membrane bound E-cadherin (P < 0.01) and claudin-1 (P < 0.05) expression. IL-9 receptor expression and mislocalized claudin-1 positively correlated and while membrane bound E-cadherin expression negatively correlated with the degree of histologic epithelial remodeling (P < 0.05). IL-9 decreased epithelial resistance in stratified primary human esophageal epithelial cells (P < 0.01) and membrane bound E-cadherin in epithelial cell monolayers (P < 0.01). CONCLUSIONS: These data suggest that IL-9, its receptor, and its effects on E-cadherin may be important mechanisms for epithelial barrier disruption in EoE.

IL-9 Promotes the Development of Deep Venous Thrombosis by Facilitating Platelet Function.

The development of deep venous thrombosis (DVT) is a sterile inflammatory process related to cytokines, such as interleukin (IL)-6 or IL-17. IL-9 is a cytokine involved in many inflammatory diseases, including cystic fibrosis, ulcerative colitis, psoriasis and psoriatic arthritis. However, it remains unknown whether IL-9 is related to DVT. In this study, we characterized the role and mechanism of IL-9 in DVT. Analysis of the data of patients with and without DVT revealed that stasis, venous surgery as well as elevated IL-9 and sP-selectin levels were related to the development of DVT. We also showed for the first time that IL-9 receptor was expressed in mouse platelets, and it dramatically promoted the aggregation rate and expression of P-selectin (CD62P) in the presence of adenosine diphosphate, but otherwise exhibited no effect on platelets. This study also revealed that Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway, not phosphoinositide 3-kinase/AKT pathway, was involved in the process. We also showed in a mouse model of stasis that the thrombus size (weight and length) and CD62P expression in the thrombus were higher and lower in the IL-9 group and IL-9 antibody group, respectively, than in the control group. All these findings indicated that IL-9 facilitated platelet function through the JAK2/STAT3 pathway, thus promoting the development of DVT.

IL-9 receptor signaling in memory B cells regulates humoral recall responses.

Memory B cells (Bmem cells) are the basis of long-lasting humoral immunity. They respond to re-encountered antigens by rapidly producing specific antibodies and forming germinal centers (GCs), a recall response that has been known for decades but remains poorly understood. We found that the receptor for the cytokine IL-9 (IL-9R) was induced selectively on Bmem cells after primary immunization and that IL-9R-deficient mice exhibited a normal primary antibody response but impaired recall antibody responses, with attenuated population expansion and plasma-cell differentiation of Bmem cells. In contrast, there was augmented GC formation, possibly due to defective downregulation of the ligand for the co-stimulatory receptor ICOS on Bmem cells. A fraction of Bmem cells produced IL-9. These findings indicate that IL-9R signaling in Bmem cells regulates humoral recall responses.

IL-9 and Mast Cells Are Key Players of Candida albicans Commensalism and Pathogenesis in the Gut.

Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-ß in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut.

Interleukin-9 over-expression and T helper 9 polarization in systemic sclerosis patients.

T helper 9 (Th9) cells and interleukin (IL)-9 are involved in the pathogenesis of several autoimmune diseases. The exact role of IL-9 and Th9 cells in patients with systemic sclerosis (SSc) have not yet been studied adequately. IL-9, IL-9R, transcription factor PU.1 (PU.1), IL-4, thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-ß expression were assessed in skin and kidney biopsies of SSc patients and healthy controls (HC) by immunohistochemistry (IHC). The cellular source of IL-9 was also analysed by confocal microscopy analysis. Peripheral IL-9-producing cells were also studied by flow cytometry. The functional relevance of IL-9 increased expression in SSc was also investigated. Our results demonstrated a strong expression of IL-9, IL-9R, IL-4, TSLP and TGF-ß in skin tissues of patients with both limited and diffuse SSc. IL-9 expression was observed mainly in the context of skin infiltrating mononuclear cells and keratinizing squamous epithelium. IL-9 over-expression was also observed in renal biopsies of patients with SSc. IL-9 producing cells in the skin were identified as Th9 cells. Similarly, Th9 cells were expanded and were the major source of IL-9 among SSc peripheral blood mononuclear cells (PBMC), their percentage being correlated directly with the modified Rodnan skin score. Infiltrating mononuclear cells, mast cells and neutrophils expressed IL-9R. In in-vitro studies stimulation with rIL-9 significantly induced NET (neutrophil extracellular traps) release by dying cells (NETosis) in neutrophils, expansion of mast cells and increase of anti-systemic scleroderma 70 (Scl70) production by B cells. Our findings suggest that Th9 cells and IL-9 could be implicated in the pathogenesis of SSc.

Osteoblasts support megakaryopoiesis through production of interleukin-9.

Severe thrombocytopenia is a significant challenge in patients undergoing myelosuppressive chemotherapy for malignancies. Understanding the biology of platelet-producing megakaryocytes development in the bone marrow microenvironment may facilitate the development of novel therapies to stimulate platelet production and prevent thrombocytopenia. We report here that osteoblasts supported megakaryopoiesis by secreting interleukin-9 (IL-9), which stimulated IL-9 receptor (IL-9R)/Stat3 signaling in promoting megakaryopoiesis. IL-9 production in osteoblasts was negatively regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signaling in a NF-κB-dependent manner. Constitutive activation of mTORC1 inhibited IL-9 production in osteoblasts and suppressed megakaryocytic cells expansion, whereas mTORC1 inactivation increased IL-9 production and enhanced megakaryocyte and platelet numbers in mice. In mouse models, we showed that IL-9 administration stimulated megakaryopoiesis, whereas neutralizing endogenous IL-9 or IL-9R depletion inhibited the process. Importantly, we found that low doses of IL-9 efficiently prevented chemotherapy-induced thrombocytopenia (CIT) and accelerated platelet recovery after CIT. These data indicate that IL-9 is an essential regulator of megakaryopoiesis and a promising therapeutic agent for treatment of thrombocytopenia such as CIT.

Th9 and other IL-9-producing cells in allergic asthma.

Allergic asthma is a worldwide increasing chronic disease of the airways which affects more than 300 million people. It is associated with increased IgE, mast cell activation, airway hyperresponsiveness (AHR), mucus overproduction and remodeling of the airways. Previously, this pathological trait has been associated with T helper type 2 (Th2) cells. Recently, different CD4+ T cell subsets (Th17, Th9) as well as cells of innate immunity, like mast cells and innate lymphoid cells type 2 (ILC2s), which are all capable of producing the rediscovered cytokine IL-9, are known to contribute to this disease. Regarding Th9 cells, it is known that naïve T cells develop into IL-9-producing cells in the presence of interleukin-4 (IL-4) and transforming growth factor beta (TGFß). Downstream of IL-4, several transcription factors like signal transducer and activator of transcription 6 (STAT6), interferon regulatory factor 4 (IRF4), GATA binding protein 3 (GATA3), basic leucine zipper transcription factor, ATF-like (BATF) and nuclear factor of activated T cells (NFAT) are activated. Additionally, the transcription factor PU.1, which is downstream of TGFß signaling, also seems to be crucial in the development of Th9 cells. IL-9 is a pleiotropic cytokine that influences various distinct functions of different target cells such as T cells, B cells, mast cells and airway epithelial cells by activating STAT1, STAT3 and STAT5. Because of its pleiotropic functions, IL-9 has been demonstrated to be involved in several diseases, such as cancer, autoimmunity and other pathogen-mediated immune-regulated diseases. In this review, we focus on the role of Th9 and IL-9-producing cells in allergic asthma.

Interleukin (IL)-9/IL-9R axis drives γδ T cells activation in psoriatic arthritis patients.

Cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-12, interferon (IFN)-γ, IL-23 and, more recently, IL-9, have been implicated in the initiation/maintenance of inflammation in psoriasis and psoriatic arthritis (PsA). In the present study we aimed to characterize the role of γδ T cells in peripheral blood and synovial fluid of PsA patients and to investigate their response to in-vitro stimulation with antigen or cytokines (IL-9 and IL-23). γδ T cells isolated from peripheral blood mononuclear cells and synovial fluid were analysed by flow cytometry to evaluate the phenotype and cytokine production. IL-23R and IL-9R gene expression were also evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood mononuclear cells (PBMC), sorted γδ T cells and γδ cell lines were also stimulated in vitro with isopentenyl pyrophosphate (IPP), recombinant IL-9 or recombinant IL-23. Our results show an expansion of γδ T cells with a predominant effector memory phenotype in peripheral blood and synovium of untreated PsA patients, which reverses significantly after treatment with anti-TNF-α or anti-IL-12/IL-23R monoclonal antibodies (mAbs). Moreover, in PsA patients γδ T cells activation is driven prevalently by IL-9/IL-9R interaction, and not only by IL-23/IL-23R. Together these findings indicate γδ T cells and IL-9 as new players in the pathogenesis of PsA.

Interleukin-9 promotes cell survival and drug resistance in diffuse large B-cell lymphoma.

BACKGROUND: Interleukin-9 (IL-9) was discovered as a helper T cell growth factor. It has long been recognized as an important regulator in allergic inflammation. Recent years it was discovered to induce cell growth and differentiation of multiple transformed cells. However, its oncogenic activities in B-cell lymphomas have not been reported in detail. METHODS: Serum levels of IL-9 in DLBCL patients were quantified by ELISA, and its clinical significance was analysed. The expression of IL-9 receptor (IL-9R) was investigated in lymphoma cell lines by RT-PCR and western blot, respectively. In DLBCL cell lines LY1 and LY8, IL-9R genes were knocked down by RNA interference and stable transfected cells were selected with puromycin. Normal and final siIL-9R (and siControl) LY1 and LY8 cells were treated with IL-9 alone and in synergy with chemotherapeutic drugs. Cell proliferation and apoptosis were assessed by Brdu incorporation and flow cytometric analysis. The mRNA of apoptosis regulation genes were measured with real-time PCR. RESULTS: Elevated serum levels of IL-9 were detected in DLBCL patients (24/30) compared to healthy controls (0/15). Positive expression of IL-9 (defined as a serum level ≥1 pg/ml) was correlated with lower serum albumin levels and high international prognostic index (IPI) scores. IL-9R was expressed in both mRNA and protein levels in the five lymphoma cell lines, including LY1, LY8, MINO, SP53 and Jurkat. In vitro studies showed that IL-9 directly induced proliferation and inhibited apoptosis in LY1 and LY8 cells. It protects LY1 and LY8 cells from prednisolone induced apoptosis, and promotes their proliferation that were inhibited by rituximab, vincristine and prednisolone. Its molecular mechanism may be concerned with upregulating expression of p21CIP1 gene. Knock-down of IL-9R gene could reverse the effects of IL-9 on LY1 and LY8 cells. CONCLUSIONS: IL-9 is associated with clinical features of DLBCL patients. It promotes survival of DLBCL cells and reduces the sensitivities of tumor cells to chemotherapeutic drugs via upregulation of p21CIP1 genes.

IL-9 promotes the survival and function of human melanoma-infiltrating CD4(+) CD8(+) double-positive T cells.

We previously demonstrated an accumulation of tumor-reactive CD4(+) CD8(+) double positive (DP) T cells within melanoma-infiltrating lymphocytes, supporting their role in the regulation of anti-tumor immune responses. Similarly to their CD8(+) counterparts, intra-tumor DP T cells are MHC class-I restricted but differed by a limited lytic activity against autologous melanoma cells. Based on these observations and to further characterize DP T cells, both populations were compared at the transcriptional level. Our results revealed the overexpression of the IL-9 receptor (IL-9R) by DP T cells and prompted us to investigate the impact of IL-9 on their biology. We show that IL-9 favors DP T-cell survival by protecting them from apoptosis and by promoting their proliferation. In addition, IL-9 enhances their ability to produce cytokines and increased their levels of granzyme B/perforin as well as degranulation capacity, leading to a strengthened cytotoxic activity against melanoma cells. Taken together, the IL-9R(high) DP T-cell population could be a new preferential target for IL-9, which could take part in their retention within the melanoma infiltrate while also favoring their anti-tumor activity. More generally, our results extend the pleiotropic effects of IL-9 to IL-9R-expressing intra-tumor T cells, which could further potentiate anti-tumor immune responses.