Journal: Frontiers in immunology

Article Title: GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori -Positive Gastritis.

doi: 10.3389/fimmu.2021.736269

Figure Lengend Snippet: FIGURE 2 | 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.

Article Snippet: The antihuman antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Techniques: Expressing, Cytometry, Recombinant, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori -Positive Gastritis.

doi: 10.3389/fimmu.2021.736269

Figure Lengend Snippet: FIGURE 3 | 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.

Article Snippet: The antihuman antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Techniques: Expressing, Cytometry, Derivative Assay, Infection, Cell Culture, Control

Journal: Frontiers in immunology

Article Title: GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori -Positive Gastritis.

doi: 10.3389/fimmu.2021.736269

Figure Lengend Snippet: FIGURE 4 | Glucocorticoid-induced tumor necrosis factor receptor (GITR) promoted IL21+TFH-like cell polarization dependent on the STAT3 signal pathway in Helicobacter pylori infection. (A) Gastric CD4+T cells were isolated from the gastric mucosa of H. pylori-positive patients and treated with CD3 agonist Ab or recombinant GITRL protein for 12 h. Western blot was performed to detect the expression of phosphorylated STAT3 (Ser727) and total STAT3. (B) Monocyte- derived macrophages were infected with H. pylori (multiplicity of infection = 20) and co-cultured with gastric CD4+T cells with the addition of control IgG or anti-GITR- neutralizing Ab. At 12 h later, the phosphorylated level of STAT3 (Ser727) in CD4+T cells was determined by flow cytometry. (C, D) Sorted gastric CD4+T cells were pretreated with stattic or dimethyl sulfoxide for 1 h and stimulated with CD3 agonist Ab or recombinant GITRL protein for 3 days. (C) The BCL6 expression in CD4+T cells was detected by flow cytometry. (D) The IL-21 concentration in the culture supernatant was determined by ELISA. One-way ANOVA was used to compare among three or multiple groups. Data represent mean ± SD from at least three independent experiments. ns, P > 0.05; *P < 0.05; ***P < 0.001.

Article Snippet: The antihuman antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Techniques: Infection, Isolation, Recombinant, Western Blot, Expressing, Derivative Assay, Cell Culture, Control, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori -Positive Gastritis.

doi: 10.3389/fimmu.2021.736269

Figure Lengend Snippet: FIGURE 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.

Article Snippet: The antihuman antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Techniques: Expressing, Isolation, Recombinant, Cell Culture, Labeling, Cytometry

Journal: Frontiers in immunology

Article Title: GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori -Positive Gastritis.

doi: 10.3389/fimmu.2021.736269

Figure Lengend Snippet: FIGURE 6 | 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-1bb; (B), MIP-3aa (C), and CCL-25 (D) were determined by real-time PCR. (E) MMP-3, MMP-9, and b-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.

Article Snippet: The antihuman antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Techniques: Expressing, Isolation, Recombinant, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Death Discovery

Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment

doi: 10.1038/s41420-026-03053-7

Figure Lengend Snippet: A RUNX1 expression is significantly elevated in CRC tissues compared to normal tissues, as analyzed using the TCGA dataset via the GEPIA online platform. B Analysis of DFS data from GEPIA indicates that patients with low RUNX1 expression show a higher DFS rate compared to those with high RUNX1 expression. C GO-Biological Process enrichment analysis reveals that RUNX1 overexpression in HCT116 cells significantly enriches immune-related signaling pathways. D A heatmap illustrates distinct expression patterns of immune-related genes between RUNX1-overexpressing cells and wild-type control cells. E Quantitative RT-PCR confirms differential mRNA expression levels of selected immune-related genes in RUNX1-overexpressing cells vs control cells. F qRT-PCR and western blot analyses demonstrate efficient knockdown of RUNX1 and corresponding upregulation of GITRL at both mRNA and protein levels in HCT116 and RKO cells. Data are presented as mean ± SD from three independent experiments. * P < 0.05; *** P < 0.001. Statistical analysis: Student’s t test (two-sided). COAD colorectal adenocarcinoma, READ rectum adenocarcinoma, T tumor, N normal, HR hazard ratio, NC negative control, sh shRNA, OE overexpression, n number, KD kilodalton.

Article Snippet: Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene).

Techniques: Expressing, Over Expression, Protein-Protein interactions, Control, Quantitative RT-PCR, Western Blot, Knockdown, Negative Control, shRNA

Journal: Cell Death Discovery

Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment

doi: 10.1038/s41420-026-03053-7

Figure Lengend Snippet: A Representative images of subcutaneous tumors derived from MC38 cells transduced with RUNX1-overexpressing or empty vector. B , C Proportions of Treg cells (FOXP3⁺CD4⁺) among CD45⁺ lymphocytes isolated from dissociated tumors, as determined by flow cytometry. D , E Proportions of IFN-γ⁺CD8⁺ T cells among CD45⁺ lymphocytes isolated from dissociated tumors, as determined by flow cytometry. F , G Expression of GITR on Treg cells analyzed by flow cytometry. H , I Expression of GITR on CD8⁺ T cells analyzed by flow cytometry. J , K Proportions of Treg cells (FOXP3⁺CD4⁺) among CD45⁺ lymphocytes isolated from dissociated tumors with GITRL silencing, as determined by flow cytometry. Animal experiments were performed twice with consistent results, and data are presented as mean ± SD from one representative experiment. Statistical analysis: Student’s t test (two-sided). NC negative control, sh shRNA, OE overexpression, n number, KD kilodalton. Vector: the empty vector as a negative control.

Article Snippet: Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene).

Techniques: Derivative Assay, Transduction, Plasmid Preparation, Isolation, Flow Cytometry, Expressing, Negative Control, shRNA, Over Expression

Journal: Cell Death Discovery

Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment

doi: 10.1038/s41420-026-03053-7

Figure Lengend Snippet: A Representative immunoblot analysis of GITRL expression in RUNX1-overexpressing MC38 cells. B Representative immunoblots of RUNX1-Flag or GITRL-Flag in MC38 cells. C , D Representative photograph ( C ) and growth curve ( D ) of subcutaneous tumors. E , F Flow-cytometric analysis of the proportion of Treg cells (FOXP3⁺CD4⁺) among CD4⁺ T cells within dissociated tumors. G , H Flow-cytometric analysis of the proportion of IFN-γ⁺CD8⁺ T cells among CD8⁺ T cells within dissociated tumors. I , J UMAP visualization of single-cell RNA-seq data from 6 CRC tissues, showing all cells ( I ) or T cells only ( J ). K Mean GITRL expression levels in RUNX1-high vs RUNX1-low groups. L Proportion of Treg cells among total tumor-infiltrating cells. M Percentage of GITR-expressing Treg cells among total Treg cells. N Percentage of GITR-expressing CD8⁺ T cells among total cytotoxic CD8⁺ T cells. Animal experiments were performed twice with consistent results, and data are presented as mean ± SD from one independent experiment. Statistical analysis: Student’s t -test (two-sided). OE overexpression. Vector: the empty vector as a negative control. T tissue, KD kilodalton.

Article Snippet: Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene).

Techniques: Western Blot, Expressing, Single Cell, RNA Sequencing, Over Expression, Plasmid Preparation, Negative Control

Journal: Cell Death Discovery

Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment

doi: 10.1038/s41420-026-03053-7

Figure Lengend Snippet: A , B Correlation analyses between RUNX1 and GITRL ( A ) or FOXP3 ( B ) expression based on the TCGA database. C Representative immunohistochemical staining of RUNX1, GITRL, and FOXP3 in CRC tissues. RUNX1 localizes to both the nucleus and cytoplasm of tumor cells, as well as of infiltrating lymphocytes. GITRL is primarily expressed in the cytoplasm of tumor cells, whereas FOXP3 is specifically expressed in regulatory T cells (Tregs). Scale bars: 200 μm. D – F Scatter plots showing the distribution of 36 CRC patients according to immunohistochemistry scores for RUNX1 or GITRL and the corresponding mean numbers of infiltrating FOXP3⁺ cells. FOXP3⁺ cells were counted in each 20× field of view, and the average number from five randomly selected fields was calculated for each sample. G Representative multiplex immunofluorescence images of three CRC tissue cases. Scale bars: 50 μm. Data presented are mean ± SEM. Statistical analysis: Student’s t -test (two-sided).

Article Snippet: Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry, Multiplex Assay, Immunofluorescence

Journal: Cell Death Discovery

Article Title: RUNX1 restrains STAT1-GITRL signaling to shape an immunosuppressive CRC microenvironment

doi: 10.1038/s41420-026-03053-7

Figure Lengend Snippet: A CUT&Tag density heat map of RUNX1 enrichment in HCT116 cells stably expressing RUNX1-flag, within 3 kb around TSS. Gene order is arranged from highest to lowest density. B CUT&Tag-seq data show that there are no RUNX1 peaks at the promoter region of GITRL. C Identification of STAT1 in the RUNX1-flag overexpressing HCT116 cells by MS. D Endogenous STAT1 was immunoprecipitated with exogenous RUNX1-flag in HCT116 and RKO cells. E , F Western blotting ( E ) and qPCR ( F ) analyses for the expression of STAT1, RUNX1, and GITRL in HCT116 cells with the silencing of RUNX1 or STAT1 in the presence or absence of 100 U mL –1 IFN-γ for 72 h. G A proposed model illustrating the regulation of GITRL’s luciferase activity by RUNX1 and STAT1. H HCT116 cells were cotransfected with the GITRL-Luc reporter and pRL-TK plasmid without IFN-γ stimulation, then subjected to a luciferase activity assay after 48 h. I ChIP-PCR of STAT1 abundance at the GITRL’s promoter in HCT116 cells after treatment with 100 U mL –1 IFN-γ for 24 h. J The amplicons of the three primers used for ChIP-qPCR at the GITRL promoter region. K ChIP-qPCR analysis of STAT1 abundance at the GITRL promoter in HCT116 cells after treatment with 100 U mL –1 IFN-γ for 24 h. HCT116 cells transduced with an empty vector are the negative control. Data presented are mean ± SD of three independent experiments. Statistical analysis: Student’s t test (two-sided). Kb kilobases, bp base pair, chr chromosome, TSS transcription start site, IP immunoprecipitation, ns not statistically, Luc luciferase, F forward primer, R reverse primer, sh shRNA, si siRNA, GAS gamma-activated sequence, n number, KD kilodalton.

Article Snippet: Subsequently, the slides were incubated overnight at 4 °C in a humidified chamber with primary antibodies against RUNX1 (1:400, 25315-1-AP, Proteintech), GITRL (1:1500, 23899-1-AP, Proteintech), or FOXP3 (1:1500, UM870140, Origene).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, ChIP-qPCR, Transduction, Negative Control, shRNA, Sequencing