Ting-ting Wang

Increased Circulating Th22 and Th17 Cells are Associated with Tumor Progression and Patient Survival in Human Gastric Cancer

Although Th22 and Th17 cells have been reported to play critical roles during autoimmunity and inflammation, information on their role in cancer-immunity is limited. In this study, we investigated clinical relevance of circulating Th22 and Th17 cells in patients with gastric cancer (GC). Using multi-color flow cytometry and PMA stimulation, we determined the levels of Th22, Th17 and Th1 cells in the peripheral blood of 32 GC patients and 19 healthy donors, and evaluated their correlations with tumor stage and overall survival. Compared with healthy donors, the frequencies of circulating CD4+IL-22+ T cells, CD4+IL-17+ T cells, Th22 (CD4+IL-22+IL-17-INF-γ−) cells, Th17 (CD4+IL-17+INF-γ−) cells were increased in patients with GC, but there was no significant differences in the frequencies of CD4+IFN-γ+ T cells and Th1 (CD4+IL-17−INF-γ+) cells. Th22 cells showed positive correlation with Th17 cells and CD4+IL-17+ T cells in patients with GC. Furthermore, the frequencies of Th22 and Th17 cells were significantly higher in stage III–IV GC patients versus stage I–II and correlated with patients’ overall survival. These data suggest that circulating Th22 cells as well as Th17 cells are increased in the peripheral blood of GC patients with tumor progression, and that these cells may be promising novel clinical markers for GC.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis

Objective Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. Design Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. Results Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. Conclusions This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.

Increased tumor-infiltrating CD8+Foxp3+ T lymphocytes are associated with tumor progression in human gastric cancer

BackgroundCD8+Foxp3+ T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.MethodsThe levels of CD8+Foxp3+ T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8+Foxp3− T cells was investigated in vitro. The suppressive function of CD8+Foxp3+ T lymphocytes was analyzed by their effect on CD4+ T-cell proliferation and IFN-γ production. The percentages of CD8+Foxp3+ T lymphocytes were evaluated for the association with tumor stage.ResultsThe frequency of CD8+Foxp3+ T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8+Foxp3+ T lymphocytes were activated effector cells (CD45RA−CD27−). TGF-β1 levels were positively correlated with the frequency of CD8+Foxp3+ T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8+Foxp3+ T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8+Foxp3+ T lymphocytes suppressed the proliferation and IFN-γ production of CD4+ T cells. Finally, intratumoral CD8+Foxp3+ T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.ConclusionsOur data have shown that increased intratumoral CD8+Foxp3+ T lymphocytes are associated with tumor stage and potentially influence CD4+ T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.

Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway

Objective Neutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments. However, the nature, regulation, function and clinical relevance of neutrophils in human gastric cancer (GC) are presently unknown. Design Flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 105 patients with GC. Kaplan-Meier plots for overall survival were performed using the log-rank test. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro and in vivo regulation and function assays. Results Patients with GC showed a significantly higher neutrophil infiltration in tumours. These tumour-infiltrating neutrophils showed an activated CD54+ phenotype and expressed high level immunosuppressive molecule programmed death-ligand 1 (PD-L1). Neutrophils activated by tumours prolonged their lifespan and strongly expressed PD-L1 proteins with similar phenotype to their status in GC, and significant correlations were found between the levels of PD-L1 and CD54 on tumour-infiltrating neutrophils. Moreover, these PD-L1+ neutrophils in tumours were associated with disease progression and reduced GC patient survival. Tumour-derived GM-CSF activated neutrophils and induced neutrophil PD-L1 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway. The activated PD-L1+ neutrophils effectively suppressed normal T-cell immunity in vitro and contributed to the growth and progression of human GC in vivo; the effect could be reversed by blocking PD-L1 on these neutrophils. Conclusions Our results illuminate a novel mechanism of PD-L1 expression on tumour-activated neutrophils in GC, and also provide functional evidence for these novel GM-CSF-PD-L1 pathways to prevent, and to treat this immune tolerance feature of GC.

Elevated interleukin-32 expression is associated with Helicobacter pylori-related gastritis.

Background Interleukin-32 (IL-32) is a recently discovered proinflammatory cytokine involved in inflammatory diseases. We investigated the expression of IL-32 and its regulation mechanism in the inflammatory response of patients with Helicobacter pylori (H. pylori) infection. Design and Methods IL-32 mRNA and protein expression in gastric tissues was detected by quantitative real-time PCR and immunohistochemistry. The regulation of IL-32 in human gastric epithelia cell line AGS was investigated by different cytokine stimulation and different H. pylori strain infection. Results Gastric IL-32 mRNA and protein expression were elevated in patients with H. pylori infection and positively correlated with gastritis. In H. pylori-infected patients, the mRNA level of IL-32 was also correlated with that of proinflammatory cytokines IL-1β and TNF-α. In vitro IL-1β and TNF-α could upregulate IL-32 mRNA and protein level in AGS cells, which was dependent on NF-κB signal pathway. The regulation of IL-32 expression in response to H. pylori-infection could be weakened by using neutralizing antibodies to block IL-1β and TNF-α. Moreover, H. pylori-infected AGS cells also induced IL-32 mRNA and protein expression, which was dependent on CagA. Conclusions IL-32 level is elevated in patients with H. pylori infection and its expression is regulated by proinflammatory stimuli, suggesting that IL-32 may play a role in the pathogenesis of H. pylori-related gastritis.

Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFβ1 in Human Gastric Cancer

Patients with gastric cancer had few NK cells infiltrating their tumors, a condition associated with tumor progression and poor survival. These intratumoral NK cells were functionally impaired, due to the presence of TGFβ1 derived from tumor-associated monocytes/macrophages. Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ+ and TNFα+ NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFβ1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248–56. ©2017 AACR.

Altered phenotypic and functional characteristics of CD3 + CD56 + NKT-like cells in human gastric cancer

CD3+CD56+ natural killer T (NKT)-like cells are a group of CD3+ T cells sharing characteristics of NK and T cells and constitute a major component of host anti-tumor immune response in human cancer. However, the nature, function and clinical relevance of CD3+CD56+ NKT-like cells in human gastric cancer (GC) remain unclear. In this study, we showed that the frequencies of CD3+CD56+NKT-like cells in GC tumors were significantly decreased and low levels of tumor-infiltrating CD3+CD56+ NKT-like cells were positively correlated with poor survival and disease progression. Most CD3+CD56+NKT-like cells in GC tumors were CD45RA−CD27+/− central/effector-memory cells with decreased activity and lower expression levels of CD69, NKG2D and DNAM-1 than those in non-tumor tissues. We further observed that tumor-infiltrating CD3+CD56+ NKT-like cells had impaired effector function as shown by decreased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Moreover, in vitro studies showed that soluble factors released from GC tumors could induce the functional impairment of CD3+CD56+ NKT-like cells. Collectively, our data indicate that decreased tumor-infiltrating CD3+CD56+ NKT-like cells with impaired effector function are associated with tumor progression and poor survival of GC patients, which may contribute to immune escape of GC.

Modulation of CD8+ memory stem T cell activity and glycogen synthase kinase 3β inhibition enhances anti-tumoral immunity in gastric cancer.

ABSTRACT The potential contributions of CD8+ memory stem T cells to anti-tumor immunity and immunotherapy responses in gastric cancer has not been demonstrated. We found that CD8+ memory stem T cell frequencies were increased in the peripheral blood of gastric cancer patients compared to healthy donors and declined in frequency with disease progression. Despite minimal in vitro cytotoxic activity, the adoptive transfer of CD8+ memory stem T cells into Rag1−/− tumor bearing mice enhanced tumor regression compared to CD8+ central or effector memory T cell counterparts. This effect was associated with an increase in splenic, draining lymph node and tumor infiltrating CD8+ T cell numbers and the development of an altered CD8+ T cell phenotype not seen during homeostasis. GSK-3β inhibition is known to promote memory stem T cell accumulation by arresting effector T cell differentiation in vivo. Surprisingly however, GSK-3β inhibition conversely increased the cytotoxic capacity of CD8+ memory stem T cells in vitro, and this was associated with the induction of effector T cell-associated effector proteins including FasL. Finally, FasL neutralization following GSK-3β inhibition directly attenuated the anti-tumoral capacity of CD8+ memory stem T cells both in vitro and in vivo. Altogether, our findings identify the therapeutic potential of modulating CD8+ memory stem T cells for improved anti-tumoral responses against gastric cancer.

Altered NKp30, NKp46, NKG2D, and DNAM-1 Expression on Circulating NK Cells Is Associated with Tumor Progression in Human Gastric Cancer

Natural killer (NK) cell activity is influenced by a complex integration of signaling pathways activated downstream of both activating and inhibitory surface receptors. The tumor microenvironment can suppress NK cell activity, and there is a great clinical interest in understanding whether modulating tumor-mediated NK cell suppression and/or boosting preexisting NK cell numbers in cancer patients is therapeutically viable. To this light, we characterized the surface receptor phenotypes of peripheral blood NK cells and examined their clinical relevance to human gastric cancer (GC). We found that the proportion of peripheral blood NK cells which expressed the activating receptors NKp30, NKp46, NKG2D, and DNAM-1 was significantly decreased in GC patients compared to healthy donors, and that this decrease was positively associated with tumor progression. At the same time, plasma TGF-β1 concentrations were significantly increased in GC patients and negatively correlated with the proportion of NKp30, NKp46, NKG2D, and DNAM-1 expressing NK cells. Furthermore, TGF-β1 significantly downregulated the expression of NKp30, NKp46, NKG2D, and DNAM-1 on NK cells in vitro, and the addition of galunisertib, an inhibitor of the TGF-β receptor subunit I, reversed this downregulation. Altogether, our data suggest that the decreased expression of activating receptors NKp30, NKp46, NKG2D, and DNAM-1 on peripheral blood NK cells is positively associated with GC progression, and that TGF-β1-mediated NK cell suppression may be a therapeutically targetable characteristic of GC.

CD45 + CD33 low CD11b dim myeloid-derived suppressor cells suppress CD8 + T cell activity via the IL-6/IL-8-arginase I axis in human gastric cancer

Myeloid-derived suppressor cells (MDSCs) are a prominent component of the pro-tumoral response. The phenotype of and mechanisms used by MDSCs is heterogeneous and requires more precise characterization in gastric cancer (GC) patients. Here, we have identified a novel subset of CD45+CD33lowCD11bdim MDSCs in the peripheral blood of GC patients compared to healthy individuals. CD45+CD33lowCD11bdim MDSCs morphologically resembled neutrophils and expressed high levels of the neutrophil marker CD66b. Circulating CD45+CD33lowCD11bdim MDSCs effectively suppressed CD8+ T cells activity through the inhibition of CD8+ T cell proliferation and interferon-γ (IFN-γ) and granzyme B (GrB) production. The proportion of CD45+CD33lowCD11bdim MDSCs also negatively correlated with the proportion of IFN-γ+CD8+ T cell in the peripheral blood of GC patients. GC patient serum-derived IL-6 and IL-8 activated and induced CD45+CD33lowCD11bdim MDSCs to express arginase I via the PI3K-AKT signaling pathway. This pathway contributed to CD8+ T cell suppression as it was partially rescued by the blockade of the IL-6/IL-8-arginase I axis. Peripheral blood CD45+CD33lowCD11bdim MDSCs, as well as IL-6, IL-8, and arginase I serum levels, positively correlated with GC progression and negatively correlated with overall patient survival. Altogether, our results highlight that a subset of neutrophilic CD45+CD33lowCD11bdim MDSCs is functionally immunosuppressive and activated via the IL-6/IL-8-arginase I axis in GC patients.