Abstract 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 + CD33 low CD11b dim MDSCs in the peripheral blood of GC patients compared to healthy individuals. CD45 + CD33 low CD11b dim MDSCs morphologically resembled neutrophils and expressed high levels of the neutrophil marker CD66b. Circulating CD45 + CD33 low CD11b dim 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 + CD33 low CD11b dim 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 + CD33 low CD11b dim 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 + CD33 low CD11b dim 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 + CD33 low CD11b dim MDSCs is functionally immunosuppressive and activated via the IL-6/IL-8-arginase I axis in GC patients.
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.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.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.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.
Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3-/- mice but increased in protease-activated receptor 1-/- (Par1-/-) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori-associated gastritis.
Abstract Adrenomedullin (ADM) is a multifunctional peptide that is expressed by many surface epithelial cells, but its relevance to H. pylori -induced gastritis is unknown. Here, we found that gastric ADM expression was elevated in gastric mucosa of H. pylori -infected patients and mice. In H. pylori -infected human gastric mucosa, ADM expression was positively correlated with the degree of gastritis, accordingly, blockade of ADM resulted in decreased inflammation within the gastric mucosa of H. pylori -infected mice. During H. pylori infection, ADM production was promoted via PI3K-AKT signaling pathway activation by gastric epithelial cells in a cagA -dependent manner, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterized by the increased IFN-γ-producing T cells, whose differentiation was induced via the phosphorylation of AKT and STAT3 by ADM derived from gastric epithelial cells. ADM also induced macrophages to produce IL-12, which promoted the IFN-γ-producing T-cell responses, thereby contributing to the development of H. pylori -associated gastritis. Accordingly, blockade of IFN-γ or knockout of IFN-γ decreased inflammation within the gastric mucosa of H. pylori -infected mice. This study identifies a novel regulatory network involving H. pylori , gastric epithelial cells, ADM, macrophages, T cells, and IFN-γ, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Author summary H. pylori infect almost half the world’s population. Once infected, most of people carry the bacteria lifelong if left untreated, so that persistent H. pylori infection can lead to chronic gastritis, peptic ulceration and ultimately gastric cancer. H. pylori infection is accompanied with increased inflammation in gastric mucosa, but the mechanisms of chronic gastritis induced by H. pylori infection remains poorly understood. We studied a multifunctional peptide known as adrenomedullin (ADM) in gastric epithelial cells, which was known as a key factor of regulating gastrointestinal physiology and pathology. Here, we found that gastric ADM expression was elevated in gastric mucosa of H. pylori -infected patients and mice, and was positively correlated with the degree of gastritis. ADM production was promoted via PI3K-AKT signaling pathway activation by gastric epithelial cells in a cagA -dependent manner. Blockade of ADM during H. pylori infection resulted in decreased gastric inflammation that was characterized by the increased IFN-γ-producing T cells which was induced via the phosphorylation of AKT and STAT3 by ADM derived from gastric epithelial cells. ADM also induced macrophages to produce IL-12, which promoted the IFN-γ-producing T-cell responses. These data demonstrate that H. pylori -induced ADM modulates FN-γ-producing T-cell responses and contribute to gastritis.
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.
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.
Mast cells are prominent components of solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the nature, regulation, function, and clinical relevance of mast cells in human gastric cancer (GC) are presently unknown.
Methods
Flow cytometry analyses were performed to examine level and phenotype of mast cells in samples from 114 patients with GC. Multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Kaplan-Meier plots for patient survival were performed using the log-rank test. Mast cells, T cells and tumor cells were isolated or generated, stimulated and/or cultured for in vitro and in vivo function assays.
Results
Patients with GC showed a significantly higher mast cell infiltration in tumors. Mast cell levels increased with tumor progression and independently predicted reduced overall survival. These tumor-infiltrating mast cells accumulated in tumors by CXCL12-CXCR4 chemotaxis. Intratumoral mast cells expressed higher immunosuppressive molecule programmed death-ligand 1 (PD-L1), and mast cells induced by tumors strongly express PD-L1 proteins in both time-dependent and dose-dependent manners. Significant correlations were found between the levels of PD-L1+ mast cells and pro-inflammatory cytokine TNF-α in GC tumors, and tumor-derived TNF-α activated NF-κB signaling pathway to induce mast cell expression of PD-L1. The tumor-infiltrating and tumor-conditioned mast cells effectively suppressed normal T-cell immunity through PD-L1 in vitro, and tumor-conditioned mast cells contributed to the suppression of T-cell immunity and the growth of human GC tumors in vivo; the effect could be reversed by blocking PD-L1 on these mast cells.
Conclusion
Thus, our results illuminate novel immunosuppressive and protumorigenic roles of mast cells in GC, and also present a novel mechanism in which PD-L1 expressing mast cells link the proinflammatory response to immune tolerance in the GC tumor milieu.
Background Overexpression of programmed cell death protein 1 (PD-1) is linked to CD8+ T cell dysfunction and contributes to tumor immune escape. However, the prevalence and functional regulations of PD-1 expression on CD8+ T cells in human gastric cancer (GC) remain largely unknown. Methods Flow cytometry was performed to analyze the level, phenotype, functional and clinical relevance of PD-1+CD8+ T cells in GC patients. Peripheral blood CD8+ T cells were purified and subsequently exposed to culture supernatants from digested primary GC tumor tissues (TSN) in vitro for PD-1 expression and functional assays. Tumor responses to adoptively transferred TSN-stimulated CD8+ T cells or to the TSN-stimulated CD8+ T cell transfer combined with an anti-PD-1 antibody injection were measured in an in vivo xenograft mouse model. Results GC patients’ tumors showed a significantly increased PD-1+CD8+ T cell infiltration. However, these GC-infiltrating PD-1+CD8+ T cells showed equivalent function to their PD-1−CD8+ counterparts and they did not predict tumor progression. High level of transforming growth factor-β1 (TGF-β1) in tumors was positively correlated with PD-1+CD8+ T cell infiltration, and in vitro GC-derived TGF-β1 induced PD-1 expression on CD8+ T cells via Smad3 signaling, whereas Smad2 signaling was involved in GC-derived TGF-β1-mediated CD8+ T cell dysfunction. Furthermore, GC-derived TGF-β1-mediated CD8+ T cell dysfunction contributed to tumor growth in vivo that could not be attenuated by PD-1 blockade. Conclusions Our data highlight that GC-derived TGF-β1 promotes PD-1 independent CD8+ T cell dysfunction. Therefore, restoring CD8+ T cell function by a combinational PD-1 and TGF-β1 blockade might benefit future GC immunotherapy.
Heat shock proteins (HSPs) are crucial proteins in maintaining the homeostasis of human gastric epithelial cells. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a member of the HSP90 family, has been shown to be involved in various crucial physiological processes, particularly against apoptosis. However, the regulation and function of TRAP1 in Helicobacter pylori infection is still unknown. Here, we found that TRAP1 expression was downregulated on human gastric epithelial cells during H. pylori infection by real-time polymerase chain reaction (PCR) and western blot analysis. Through virulence factors mutant H. pylori strains infection and inhibitors screening, we found that H. pylori vacuolating cytotoxin A ( vacA), but not cytotoxin-associated gene A ( cagA) protein, induced human gastric epithelial cells to downregulate TRAP1 via P38MAPK pathway by real-time PCR and western blot analysis. Furthermore, downregulation of TRAP1 with lentivirus carrying TRAP1 short hairpin RNA constructs impairs mitochondrial function, and increases apoptosis of gastric epithelial cells. The results indicate that H. pylori vacA downregulated TRAP1 is involved in the regulation of gastric epithelial cell apoptosis.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)