Deleterious Role of Th9 Cells in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology. Immune disorders play an important role in IPF pathogenesis. Here, we show that Th9 cells differentiate and activate in the lung tissue of patients with IPF and bleomycin (BLM)-induced lung fibrosis mice. Moreover, we found that Th9 cells promote pulmonary fibrosis in two ways. On the one hand, Th9 cells promote fibroblast differentiation, activation, and collagen secretion by secreting IL-9. On the other hand, they promote differentiation of Th0 cells into Th2 cells by secreting IL-4. Th9 cells and Th2 cells can promote each other, accelerating the Th1/Th2 imbalance and eventually forming a positive feedback of pulmonary fibrosis. In addition, we found that neutralizing IL-9 in both preventive and therapeutic settings ameliorates bleomycin-induced pulmonary fibrosis. Furthermore, we identified several critical signaling pathways involved in the effect of neutralizing IL-9 on pulmonary fibrosis by proteomics study. From an immunological perspective, we elucidated the novel role and underlying mechanism of Th9 cells in pulmonary fibrosis. Our study suggested that Th9-based immunotherapy may be employed as a treatment strategy for IPF.

P02.04 Tissue-infiltrating Th9 cells in human endometrial cancer

BackgroundEndometrial cancer (EC) is a hormone-related carcinoma with increased morbidity among female patients of all backgrounds. The immune microenvironment of EC is uncertain.Materials and Methods102 patients were recruited in the present study. 90 postoperative specimens from the patients were analyzed by immunohistochemistry. The leukocyte landscape of endometrial cancer was mapped using high-dimensional single-cell profiling (CyTOF) for 12 patients.ResultsNK cells, MDMs, and neutrophils were enriched in adjacent normal tissue. CCR5+CD38+ PD1+Th9 cells were enriched in the invasive margin. Additionally, PD1+ESRneg T cells and Siglec1+CCR5+CD40+ESRhi macrophage were infiltrated in the tumors.Abstract P02.04 Figure 1ConclusionsImmunological landscape of EC might shed light on new immunotherapuetic approach.Disclosure InformationH. Tong: None. H. Feng: None. X. Wan: None.

TGF-β1 drives Th9 but not Treg cells upon allergen exposure

TGF-β1 is known to have a pro-inflammatory impact by inducing Th9 cells, while it also induces anti-inflammatory Treg cells (Tregs). In the context of allergic airway inflammation (AAI) its dual role can be of critical importance in influencing the outcome of the disease. Here we demonstrate that TGF-β acts in AAI by driving effector T cells into Th9 cells, while Tregs differentiate independently. Induction of experimental AAI and airway hyperreactivity in a mouse model with inducible genetic ablation of the TGFβ-receptor 2 (TGFBR2) on CD4+T cells significantly reduced the disease phenotype. Further, it blocked the induction of Th9 cell frequencies, but increased Treg cells. To translate these findings into a human clinically relevant context, Th9 and Treg cells were quantified both locally in induced sputum and systemically in blood of allergic rhinitis and asthma patients with or without allergen-specific immunotherapy (AIT). Natural allergen exposure induced local and systemic Th2, Th9 cell and reduced Tregs, while therapeutic allergen exposure by AIT suppressed Th2 and Th9 cell frequencies along with TGF-β and IL-9 secretion. Altogether, these findings support that neutralization of TGF-β represents a viable therapeutic option in allergy and asthma, not posing the risk of immune dysregulation by impacting Tregs.

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

BackgroundCasitas 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.MethodsUsing 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.ResultsHere, 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.ConclusionWe 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-35 Suppresses Interleukin-9-Secreting CD4+ T Cell Activity in Patients With Hepatitis B-Related Hepatocellular Carcinoma

Chronic hepatitis B virus (HBV) infection induces dysfunction of immune response and chronic liver damage. However, the mechanisms that account for HBV-related hepatocellular carcinoma (HCC) are poorly understood. The aim of present study was to investigate the modulatory role of interleukin (IL)-35, an immunosuppressive cytokine, to IL-9-secreting T cells in hepatitis B-related HCC. Twenty-two HBV-related HCC patients, twenty-seven chronic hepatitis B (CHB) patients, and eleven controls were enrolled. Serum IL-35 and IL-9 concentration was measured by ELISA. Peripheral and liver-infiltrating non-specific and HBV-specific Th9 and Tc9 cells were assessed by flow cytometry. The regulatory activity of IL-35 to peripheral and liver-infiltrating Th9 cells was assessed in co-culture system between CD8+ T cells and HepG2.2.15 cells. Serum IL-35 was up-regulated, while IL-9 was down-regulated in HBV-related HCC patients compared with in CHB patients and controls. Peripheral non-specific and HBV-specific Th9 cells, but not Tc9 cells, were decreased in HBV-related HCC patients. Liver-infiltrating non-specific and HBV-specific Th9 cells were also reduced in HCC tumor sites. CD8+ T cells from CHB and HBV-related HCC patients revealed decreased cytotoxicity compared with those from controls. Autologous Th9 cells mediated the elevation of CD8+ T cell cytotoxicity, and this process was depending on IL-9 secretion. Recombinant IL-35 stimulation inhibited IL-9 secretion and PU.1 mRNA expression in non-specific and HBV-specific Th9 cells, leading to the suppression of Th9-mediated CD8+ T cell cytotoxicity in CHB and HBV-related HCC patients. Our current data indicated that IL-35 might dampen non-specific and HBV-specific Th9 cells activity in HBV-related HCC patients.