Fig 2: IL-21+TFH-like cells induced the expression of pro-inflammatory cytokines and matrix metalloproteinases in human gastric epithelial cells. The gastric CD4+T cells were isolated from the gastric mucosa of Helicobacter pylori-positive patients and healthy controls and stimulated with CD3 agonist Ab or recombinant GITRL protein. At 3 days later, the culture supernatant was collected. GES-1 cells were treated with 20% culture supernatant of gastric CD4+T cells with the addition of control IgG or anti-IL-21-neutralizing Ab for 24 h. The mRNA expressions of IL-6 (A), IL-1ββ; (B), MIP-3αα (C), and CCL-25 (D) were determined by real-time PCR. (E) MMP-3, MMP-9, and β-actin expression in the culture supernatants or total extracts of GES-1 cells was detected by western blot. One-way ANOVA was used to compare among three or multiple groups. Data are shown as mean ± SD from at least three independent experiments. ns, P > 0.05; *P < 0.05; ***P < 0.001.

Fig 3: Glucocorticoid-induced tumor necrosis factor receptor (GITR) promoted the polarization of 21+TFH-like cells in Helicobacter pylori-positive gastritis. Biopsy samples of gastric mucosa from H. pylori-positive patients (n = 39) and healthy controls (n = 20) were collected. The GITR expression in (A) CD4+T cells and (B) IL-21+ or IL-21-CD4+T cells was determined by flow cytometry. (C) The sorted gastric CD4+T cells were stimulated with CD3 agonist Ab or recombinant GITRL protein. At 3 days later, IL-21 production by CD4+T cells was determined by flow cytometry. (D) CD4+T cells were isolated from the gastric mucosa of H. pylori-positive patients and treated with CD3 agonist Ab or recombinant GITRL protein. At 3 days later, the IL-21 concentration in the culture supernatant was determined by ELISA. Unpaired Student’s t-test was performed to determine the difference between the two groups. One-way ANOVA was used to compare among three or multiple groups. Data are displayed as mean ± SD from at least three independent experiments. **P < 0.01; ***P < 0.001.

Fig 4: GITRL was upregulated in macrophage and provided ligand signal to TFH-like cells in Helicobacter pylori-positive gastritis. (A) GITRL expression was determined in the mucosal macrophage of H. pylori-positive patients (n = 10) and healthy controls (n = 10) by flow cytometry. (B) Monocyte-derived macrophages (MDM) were infected with H. pylori (multiplicity of infection, MOI = 20) for 24 h. GITRL expression was assessed by flow cytometry. (C) MDM was infected with H. pylori (MOI = 20) and co-cultured with gastric CD4+T cells in the presence of control IgG or anti-GITR-neutralizing Ab. At 3 days later, IL-21 production by CD4+T cells was detected. Unpaired Student’s t-test was performed to determine the difference between the two groups. One-way ANOVA was used to compare among three or multiple groups. Data are shown as mean ± SD from at least three independent experiments. ns, P > 0.05; ***P < 0.001.

Fig 5: IL-21+TFH-like cells induced B cell proliferation in Helicobacter pylori-positive gastritis. (A, B) The correlations of B cell proportion with (A) IL-21 and (B) glucocorticoid-induced tumor necrosis factor receptor expression in mucosal CD4+T cells of H. pylori-positive gastritis patients were analyzed by Spearman correlation analysis (n = 39). (C) The gastric CD4+T cells were respectively isolated from the gastric mucosa of H. pylori-positive patients and healthy controls and treated with CD3 Ab or recombinant GITRL protein for 12 h. Then, the gastric CD4+T cells were co-cultured with carboxyfluorescein succinimidyl ester-labeled autologous CD19+B cells in the presence of IgG or anti-IL-21-neutralizing Ab. At 3 days later, the percentage of proliferated B cells was assessed by flow cytometry. One-way ANOVA was used to compare among three or multiple groups. Data are shown as mean ± SD from at least three independent experiments. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.