Journal: Frontiers in Immunology

Article Title: A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes

doi: 10.3389/fimmu.2020.01704

Figure Lengend Snippet: In vitro comparison of T cells transduced with TSPAN8 and CD66c CAR constructs, incorporating different spacer domains. (A) Structure of the TSPAN8 and CD66c CAR constructs with the Siglec spacers and extracellular domain comparison of the CAR constructs and target molecules. (B) Cytolytic kinetics and specific endpoint killing of AsPC1 target cells incubated with CAR T cells and Mock T cells from three different donors in effector to target ratios of 2:1. n = 6. (C) Frequency of 4-1BB, LAG3 and PD1 positive CAR T cells was analyzed at the end of the cytolytic evaluation with AsPC1 cells by flow cytometry. (D) GM-CSF, IFN-γ, IL-2, IL-6, and TNF-α production after 24 h of co-culture of TSPAN8 or CD66c CAR T cells with AsPC1 cells from one donor assessed by flow cytometry. n = 2. Data from (B–D) were taken from the same experiment. Shown is the mean ± SD. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 [one-way ANOVA, multiple comparisons].

Article Snippet: The CD20-specific scFv was derived from the murine monoclonal antibody Leu16 as originally described by Jensen and colleagues , while the CD66c- and TSPAN8-targeting scFv sequences were derived from the antibody clones REA414 (CD66c) and REA443 (TSPAN8) (Miltenyi Biotec).

Techniques: In Vitro, Comparison, Transduction, Construct, Incubation, Flow Cytometry, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes

doi: 10.3389/fimmu.2020.01704

Figure Lengend Snippet: The TSPAN8 specific Siglec-4 spacer CAR T cells exhibit the same anti-tumor efficacy as the CD8α spacer CAR T cells, while retaining a more memory-like phenotype. (A) Overview of the study workflow. (B) Tumor burden and change in tumor size over time after TSPAN8 CAR T cell infusion. Untreated and Mock T cell treated animals served as controls, T cells from one donor were used. IgG4: n = 5; Sig4 and CD8α: n = 4. PSM p < 0.05 (green) [one-way ANOVA, multiple comparisons]. (C) Total number of CAR positive T cells recovered from spleens of TSPAN8 CAR-treated animals at the end of the experiment calculated after flow cytometric analysis. IgG4: n = 5; Sig4 and CD8α: n = 4. (D) CD4 and CD8 CAR+ T cell phenotypes in the spleens of TSPAN8 CAR-treated animals analyzed at the end of the experiment by flow cytometry. n = 4.

Article Snippet: The CD20-specific scFv was derived from the murine monoclonal antibody Leu16 as originally described by Jensen and colleagues , while the CD66c- and TSPAN8-targeting scFv sequences were derived from the antibody clones REA414 (CD66c) and REA443 (TSPAN8) (Miltenyi Biotec).

Techniques: Flow Cytometry

Journal: Frontiers in Oncology

Article Title: COL10A1-DDR2 axis promotes the progression of pancreatic cancer by regulating MEK/ERK signal transduction

doi: 10.3389/fonc.2022.1049345

Figure Lengend Snippet: COL10A1-DDR2 axis promotes EMT in PDAC cells through activation of MEK/ERK pathway. (A) GSEA validated EMT-related pathways in high COL10A1 expression PDAC cohorts of GSE15471 dataset. (B) The effect of COL10A1 on the expressions of EMT signaling proteins by Immunofluorescence. (C) The effect of COL10A1 and DDR2 on the expressions of EMT signaling proteins by Western blot. (D) Western blot detection of the effect of overexpression of DDR2 on protein expression changes in the EMT pathway induced by interference with COL10A1. (E) Western blot analyses of MEK/ERK pathway proteins in the indicated cells. (F) The protein expression of EMT pathway was detected by western blot in PDAC cells after treated with ERK inhibitor SCH772984. (G) H&E staining and IHC staining of COL10A1, DDR2, P-ERK, E-cadherin, N-cadherin and Vimentin in PDAC orthotopic models. Scale bars, 20 μm.

Article Snippet: To probe the biological mechanisms using GSEA software v4.2.3 (Broad Institute, MIT, Cambridge, MA, USA), a 54675 (gene)×36 (samples) expression matrix was employed.

Techniques: Activation Assay, Expressing, Immunofluorescence, Western Blot, Over Expression, Staining, Immunohistochemistry

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: 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 anti-human 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, Flow 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: 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 anti-human 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, Flow 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: 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 anti-human 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, Flow 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: 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 anti-human 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, Flow 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: 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.

Article Snippet: The anti-human 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