Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/mL solution of recombinant human CD80 or CD86 (9050-B1-100 or 9090-B2-100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: CD80 binding-mediated CD28 activation induces sustained expression of the NKG2D receptor on mouse CD8+ T cells. (A) CD28 deficiency abolished the induction of NKG2D expression on CD8+ T cells. LLC tumor bearing C57bl/6 mice (N = 3) and CD28−/− mice were subjected to twice administrations (10 d apart) with IL-12 DNA (10 μg/mouse) plus doxorubicin (1 mg/kg). Four days after the second administration, splenocytes were isolated from C57bl/6 and CD28−/− mice, respectively, and stained with anti-mouse CD3, CD8+, and NKG2D antibodies to evaluate the median fluorescence intensity (MFI) and percentage of CD8+ T cells expressing NKG2D. (B) Induction of NKG2D expression upon CD80 binding on CD28+/+ CD8+ T cells but not CD28−/−CD8+ T cells. Splenocytes obtained from CD28+/+ or CD28−/− mice were treated with anti-CD3 microbeads and control Fc or CD80-Fc (1 μg/mL). After 24 h of incubation, cells were stained with anti-CD8+ and anti-NKG2D antibodies to evaluate the MFI and percentage of CD8+ T cells expressing NKG2D. (C) Induction of sustained NKG2D expression on CD8+ T cells by CD28 activation resulting from CD80 binding. Splenocytes obtained from CD28+/+ C57BL/6 mice were stimulated with anti-CD3 microbeads and treated with control Fc or CD80-Fc. The CD8+ T cells were stained for CD8+ and NKG2D 1, 2, 3, 4, and 5 d after incubation for flow cytometric analysis. The bar graphs show the mean (± standard error of the mean [SEM]). The data are representative of three repeated experiments.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Binding Assay, Activation Assay, Expressing, Isolation, Staining, Fluorescence, Control, Incubation

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: CD80 binding-mediated CD28 activation induces sustained human NKG2D receptor expression on CD8+ T cells. (A) Induction of NKG2D expression on human CD8+ T cells after CD28 activation. PBMCs isolated from healthy donors were stimulated with anti-CD3 microbeads and treated with control Fc or human CD80-Fc (1 μg/mL) for 24 h. The median (± SEM) of NKG2D expression on CD8+ T cells is shown. (B) Induction of sustained NKG2D expression on CD8+ T cells by binding of CD80-Fc to CD28. PBMCs obtained from healthy donors were treated as described in (A) for 1, 2, 3, 4 and 5 d, and stained for CD8+ and NKG2D for flow cytometric analysis. The bar graphs show the mean MFI of NKG2D (± SEM) on CD8+ T cells (n = 3). The results represent those for five different healthy donors.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Binding Assay, Activation Assay, Expressing, Isolation, Control, Staining

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: CD80 binding-mediated CD28 activation upregulates pSTAT3 expression on mouse and human CD8+ T cells. (A, B) Mouse splenocytes (A) and human PBMCs (B) were stimulated with anti-CD3 microbeads, treated with control Fc or CD80-Fc for 15 min, 30 min, 1 h or 2 h, and stained for CD8+ and intracellular pSTAT3 for flow cytometric analysis. The median (± SEM) MFI of pSTAT3 expression is shown for the CD8+ T population (n = 3). The data are representative of three repeated experiments.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Binding Assay, Activation Assay, Expressing, Control, Staining

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: Elevated STAT3 phosphorylation resulting from CD80 binding-mediated CD28 activation via the tyrosine kinase Lck/JAK/STAT3 signaling pathway. The effects of CD28 activation and treatment with pharmacologic inhibitors on the expression of pSTAT, total STAT, and β-actin according to immunoblot assay. Mouse and human CD8+ T cells were stimulated with anti-CD3 microbeads and treated with ctrl Fc or CD80-Fc in the presence or absence of the pharmacologic inhibitor JSI-124 (0.1 μM), PP2 (1 nM), or AG-490 (50 μM) for 24 h. Vehicle control is included. The intensity quantification shown (intensity of Rae-1/intensity of β-actin) represents the mean intensity from three repeated experiments.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Phospho-proteomics, Binding Assay, Activation Assay, Expressing, Western Blot, Control

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: Blockade of Lck/JAK/STAT3 signaling abolishes CD28 activation-mediated induction of NKG2D expression on mouse and human CD8+ T cells. (A, B) Mouse (A) and human (B) CD8+ T cells were stimulated with anti-CD3 microbeads and treated with control Fc or CD80-FC in the presence or absence of the pharmacologic inhibitor JSI-124, PP2, or AG-490 for 24 h. NKG2D expression on the surface of CD8+ T cells was measured using flow cytometry. The bar graphs show the median (± SEM) MFI of NKG2D (n = 3). The data are representative of three repeated experiments.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Activation Assay, Expressing, Control, Flow Cytometry

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: Augmentation of CD8+ T-cell antitumor cytolytic activity by treatment with CD80-Fc. (A–C) Induction of antitumor cytolytic activity by mouse CD8+ T cells after exposure to Rae-1+ LLC cells. Splenocytes were collected from LLC-bearing mice on day 14 after tumor-cell inoculation. CD8+ T cells were isolated from splenocytes and treated with control Fc or CD80-Fc in the presence or absence of an anti-NKG2D antibody or JSI-124 for 24 h. (A) CD8+ T cells were co-incubated with CFSE-labeled LLC cells at a ratio of 25:1 (E:T) for 5 h. The cell culture medium was subjected to ELISA analysis of perforin. The bar graphs show the median (± SEM) normalized concentration of perforin (n = 3). (B) Flow cytometry analysis of NKG2D ligand Rae-1 expression on LLC tumor cells. (C) CD8+ T cells were co-incubated with CFSE-labeled LLC cells at ratios of 5:1, 10:1, and 25:1 (E:T) for 5 h. After incubation, the cells were stained with PI (1 mg/mL). Live target cells were identified according to light-scatter parameters and PI negativity. Survival of the target cells was measured as the percentage of normalized target cells that remained after incubation with CD8+ T cells. The data are representative of three repeated experiments. (D) Induction of human CD8+ T-cell degranulation by CD80-Fc binding after exposure to target cells. Human CD8+ T cells were enriched from PBMCs, incubated with anti-CD3 microbeads, and treated with control IgG or CD80-Fc in the presence or absence of the STAT3 inhibitor JSI-124 for 24 h. After stimulation, human CD8+ T cells were exposed to CFSE-labeled target K562 cells at a ratio of 1:1 and co-incubated with an anti-CD107a antibody or isotype control antibody for 4 h. Cells were then stained with CD8+ and NKG2D for flow cytometric analysis. The bar graphs show the mean (± SEM) percentage of CD107a+CD8+ T cells before and after exposure to the target cells (n = 3). The results represent those for three different healthy donors.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Activity Assay, Isolation, Control, Incubation, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Expressing, Staining, Binding Assay

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: Adoptive transfer of CD80 pre-treated CD8+ T cells improved the antitumor therapeutic effects in LLC tumor model. CD8+ T cells were isolated from the spleens of LLC tumor bearing mice, and stimulated with anti-CD3 plus control Fc (1μg/mL) and control IgG (10 μg/mL), CD80 Fc (1 μg/mL) and control IgG (10 μg/mL), or CD80 Fc (1 μg/mL) and anti-NKG2D antibody (10 μg/mL) for 48 h. 5 × 106 stimulated CD8+ T cells were adoptively transferred to LLC tumor bearing mice (n = 6) weekly via intravenous injection. (A) NKG2D expression on isolated CD8+ T cells after 48 h treatment with control Fc plus control IgG, CD80 Fc plus control IgG, or CD80 Fc plus NKG2D blocking antibody. (B) Tumors were dissociated and stained with anti-mouse CD45, CD8+, and NKG2D antibodies to assess NKG2D expression on tumor infiltrating CD8+ T cells. The bar graphs show the median (± SEM). (C) Individual tumor volume (left panel) and survival time (right panel) were monitored twice weekly. The data are representative of three repeated experiments.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Adoptive Transfer Assay, Isolation, Control, Injection, Expressing, Blocking Assay, Staining

Journal: Oncoimmunology

Article Title: Regulation of NKG2D + CD8 + T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

doi: 10.1080/2162402X.2016.1252012

Figure Lengend Snippet: A hypothetical illustration of CD80-Fc-induced sustained NKG2D expression on CD8+ T cells. CD80-Fc binding to CD28 can co-stimulate sustained activation of the tyrosine kinase receptor Lck, which triggers a cascade that recruits ZAP70 to amplify the activated Lck-induced signal. JAK/STAT3 is downstream from and activated by ZAP70 and pSTAT3 translocates to the nucleus to induce NKG2D expression.

Article Snippet: Mouse CD80-Fc (Asp37-Lys245-mIgG2a Fc) recombinant protein was synthesized by SYD Labs. Immunoblotting Mouse and human CD8 + T cells were lysed with RIPA buffer.

Techniques: Expressing, Binding Assay, Activation Assay