Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells

doi: 10.4049/jimmunol.1701039

Figure Lengend Snippet: CD134 and CD137 are both required for induction of Eomes and Runx3 in CD4 T cells. (A–C) WT TEa CD4 T cells (Thy1.1+) transferred into congenic (Thy1.2+) B6 recipients were immunized with Eα peptide along with rat IgG or CD134 or CD137 agonists administered individually or in combination. Spleens were analyzed 5 days post-transfer. Scatter plots show the percentage of specific TEa CD4 T cells that express Eomes (A) and Runx3 (B) as well as the total number of specific CD4 T cells (C). (D–F) A second experiment was performed using a similar setup as in A, except that the TEa CD4 T cells were either WT or CD134−/−. (G–I) A third experiment was performed using a similar setup as in A–F, except that the response of CD134−/− TEa CD4 T cells (Thy1.1/2) to CD137 agonist was compared to WT TEa CD4 T cells (Thy1.1/1) when transferred individually or when cotransferred along with WT TEa CD4 T cells. N = 3–4/group in all three experiments.

Article Snippet: Briefly, mice were treated i.p. with control polyclonal rat IgG (75 μg; Sigma-Aldrich), CD134 agonist (OX86 mAb, 50 μg; Bio X Cell), CD137 agonist (3H3 mAb, 25 μg; Bio X Cell) or CD134 plus CD137 agonists (dual costimulation).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells

doi: 10.4049/jimmunol.1701039

Figure Lengend Snippet: CD134 is not required on CD4 T cells for antigen-induced CD137 expression. WT (Thy1.1/1) and CD134−/− (Thy1.1/2) TEa CD4 T cells were cultured either separately (top) or together at a 1:1 ratio (bottom) in the presence of 1 μg/ml Eα peptide. The percentage of WT and CD134−/− TEa CD4 T cells expressing CD137 (A), CD134 (B) and CD25 (C) that had been cultured for 0, 24 and 48 h are plotted. (D) The percentage of WT and CD134−/− TEa CD4 T cells expressing CD137 in a second independent trial that used a similar experimental setup. In both experiments, the 24 and 48 h values represent the mean ± SEM calculated from triplicate or quadruplicate wells.

Article Snippet: Briefly, mice were treated i.p. with control polyclonal rat IgG (75 μg; Sigma-Aldrich), CD134 agonist (OX86 mAb, 50 μg; Bio X Cell), CD137 agonist (3H3 mAb, 25 μg; Bio X Cell) or CD134 plus CD137 agonists (dual costimulation).

Techniques: Expressing, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells

doi: 10.4049/jimmunol.1701039

Figure Lengend Snippet: CD137 agonist induces Eomes in specific CD4 T cells despite the continued presence of ThPOK and repression of CD8α. CD8-depleted B6 mice were immunized with SEA and treated with rat IgG or CD134 or CD137 agonists administered individually or in combination. SEA-specific Vβ3+ CD4 T cells were analyzed following recovery from spleens 6 days later either directly ex vivo for flow cytometry (gated on CD4+Vβ3+ cells) (A–D) or following FACS-sorting (CD4+CD8negVβ3+ cells, ≥90% purity) for RT-qPCR (E–I). SEA-specific Vβ3+ CD8 T cells recovered from intact mice immunized with SEA and treated with CD137 agonist served as positive control for Eomes and CD8α expression and a negative control for ThPOK. N = 3/group. (A) Representative plots of Eomes versus ThPOK and CD8α versus ThPOK. Scatter plot analyses of specific T cells for % Eomes+ (B), ThPOK MFI (C) and % CD8α+ (D). RT-qPCR scatter plots of FACS-sorted specific T cells for expression of Eomes (E), GzmB (F), Prf1 (G), ThPOK (H) and CD8α (I) mRNAs normalized to β-actin.

Article Snippet: Briefly, mice were treated i.p. with control polyclonal rat IgG (75 μg; Sigma-Aldrich), CD134 agonist (OX86 mAb, 50 μg; Bio X Cell), CD137 agonist (3H3 mAb, 25 μg; Bio X Cell) or CD134 plus CD137 agonists (dual costimulation).

Techniques: Ex Vivo, Flow Cytometry, Quantitative RT-PCR, Positive Control, Expressing, Negative Control

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: OX40 expression by CD4+Foxp3+ Tregs and CD4+Foxp3— T effector cells. (A) Spleen cells from naive foxp3gfpKI mice were stained with cychrome-anti-CD4 and PE-anti-OX40. CD4+GFP(Foxp3)+ cells and CD4+GFP(Foxp3)− cells were selectively gated and expression of OX40 on the Foxp3+ and Foxp3− subsets was analyzed. A representative plot of 5 experiments is shown. (B) FACS sorted CD4+ GFP(Foxp3)− T effector cells were stimulated in vitro with anti-CD3 and anti-CD28, and expression OX40 on the activated T cells was analyzed and shown. Naive CD4+ T effector cells were included as a control (the solid histogram). (C) The sorted CD4+ GFP(Foxp3)− T effector cells were stimulated with anti-CD3/anti-CD28 plus TGF-β for 4 days, and the induction of GFP(Foxp3)+ cells was determined. Expression of OX40 on the GFP(Foxp3)− T effector cells and the converted GFP(Foxp3)+ Tregs was shown. (D) CD4+GFP(Foxp3)− T effector cells were cocultured with the natural CD4+GFP(Foxp3)+ Tregs or the converted CD4+GFP(Foxp3)+ Tregs. The cell mixture was stimulated with anti-CD3 plus APCs, and suppression of T effector cell proliferation was shown as mean (CPM ± SD) of triplicate assays. Representative data of 3 individual experiments are shown.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: Expressing, Staining, In Vitro, Control

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Role of OX40 in the genesis and suppressor functions of CD4+Foxp3+ Tregs. (A). Real-time RT-PCR analysis of Foxp3 gene transcripts in CD4+CD25+ Tregs sorted from wt C57BL/6 and OX40KO mice. Data shown are representative of 3 individual experiments. (B) Spleen (SP) and lymph node (LN) cells from wt foxp3gfpKI mice and OX40KO foxp3gfpKI mice were stained with cychrome–anti-CD4 and then compared for the presence of CD4+GFP(Foxp3)+ T cells by FACS. The data shown are representative data of 4 individual experiments. Numbers in quadrants are the relative percentage of cells in each region given by the flow cytometer. (C) CD4+GFP− T effector cells sorted from foxp3gfpKI mice were mixed with CD4+GFP(Foxp3)+ Tregs from wt and OX40KO foxp3gfpKI mice at different ratios, and suppression of T effector cell proliferation was shown. The data shown are representative of 4 individual experiments. Error bars represent SD of triplicate assays.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: Quantitative RT-PCR, Staining, Flow Cytometry

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Effect of OX40 costimulation on the induction of new CD4+Foxp3+ Tregs from T effector cells. (A) CD4+GFP(Foxp3)− T effector cells were sorted from wt foxp3gfpKI mice and OX40KO foxp3gfpKI mice, The T effector cells were stimulated with anti-CD3 plus APCs in the presence or absence of TGF-β for 2 to 5 days. Induction of new GFP(Foxp3)+ T cells in the CD4+ fraction was determined by FACS. The dot plot shown is one of 3 individual experiments 4 days after the culture. Numbers in quadrants are the relative percentage of cells in each region given by the flow cytometer. (B) Induction of new GFP(Foxp3)+ Tregs calculated from 3 individual experiments. The conversion shown is the mean ± SD of 3 independent experiments at different time points. (C) Suppression of T effector cell proliferation by CD4+GFP(Foxp3)+ Tregs converted from either the wt or the OX40KO T effector cells. Data shown are representative of 3 independent experiments. Error bars represent SD of triplicate assays.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: Flow Cytometry

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Stimulation of OX40 on CD4+GFP(Foxp3)+ Tregs abrogates their suppressor functions. (A) CD4+GFP(Foxp3)− T effector cells sorted from wt and OX40KO foxp3gfpKI mice were stimulated with anti-CD3 plus wt APCs. T effector cell proliferation in the presence or absence of wt CD4+GFP(Foxp3)+ Tregs at different Tregs to T effector ratios was shown. Data shown are mean (CPM ± SD) of triplicate assays. (B) OX40 deficient CD4+GFP(Foxp3)− T effector cells were stimulated with anti-CD3 plus wt APCs or OX40Ltg APCs in the presence or absence of wt CD4+GFP(Foxp3)+ Tregs. Cell proliferation was determined 3 days later by 3H-TdR uptake. Data shown are mean (CPM ± SD) of triplicate assays. (C) OX40 deficient CD4+GFP(Foxp3)− T effector cells were stimulated with anti-CD3 plus OX40Ltg APCs. In these cultures, graded numbers of wt or OX40KO CD4+GFP(Foxp3)+ Tregs were added, and cell proliferation was determined 3 days later by 3H-TdR uptake. Data shown are mean (CPM ± SD) of triplicate assays. In all the suppression assays, representative data of 3 independent experiments are shown.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques:

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Suppression of IFN-γ production by CD4+GFP(Foxp3)+ Tregs with or without OX40 stimulation. (A) CD4+GFP(Foxp3)− T effector cells sorted from OX40KO foxp3gfpKI mice were stimulated with anti-CD3 plus wt APCs or OX40Ltg APCs, IFN-γ production by the T effector cells in the presence or absence of wt CD4+GFP(Foxp3)+ Tregs was analyzed by ELISPOT assay. Data shown are mean (± SD) of 3 experiments. (B) OX40 deficient CD4+GFP(Foxp3)− T effector cells were stimulated with anti-CD3 plus OX40Ltg APCs. In these cultures, wt or OX40 deficient CD4+GFP(Foxp3)+ Tregs were added as indicated, and suppression of IFN-γ production was shown. Data shown are mean (± SD) of 3 independent experiments. (C) CD4+GFP(Foxp3)− T effector cells from OX40KO foxp3gfpKI mice were transferred into syngeneic Rag−/− hosts (5 × 105 cells/mouse), groups of host mice were also transferred with equal number of wt CD4+GFP(Foxp3)+ Tregs. The host mice were then grafted with DBA/2 skin grafts and treated with an agonist anti-OX40 mAb, and skin allograft survival was shown. (*) P < .05

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: Enzyme-linked Immunospot

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Effect OX40 stimulation on survival and Foxp3 expression of CD4+GFP(Foxp3)+ Tregs. (A) Sorted CD4+GFP(Foxp3)+ Tregs were stimulated with anti-CD3 plus wt and OX40Ltg APCs. Cells were stained with PE-annexin V 24 hours later and analyzed by FACS. Freshly prepared Foxp3+ Tregs stained with PE-annexin V were included as a control. The annexin V profile in the GFP+ fraction was shown. (B) Sorted CD4+GFP(Foxp3)+ Tregs were stimulated with anti-CD3 and wt or OX40Ltg APCs. Foxp3 gene transcripts were analyzed 4 days later by real-time PCR. Data shown are mean A.U. of 4 experiments in each group. Error bars represent the SD of triplicate assays. (C) Sorted CD4+GFP(Foxp3)+ Tregs were stimulated with anti-CD3 and wt or OX40Ltg APCs, levels of GFP(Foxp3) expression in the CD4+ fraction were determined by FACS 4 days later and shown. The plot shown is one of 3 experiments. (A,C) Numbers are the relative percentage of cells in each region given by the flow cytometer.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: Expressing, Staining, Control, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal:

Article Title: OX40 costimulation turns off Foxp3 + Tregs

doi: 10.1182/blood-2007-01-070748

Figure Lengend Snippet: Effect of OX40 stimulation to T effector cells on the induction of new Foxp3+ Tregs.(A) CD4+GFP(Foxp3)− T effector cells were sorted from foxp3gfpKI mice and stimulated in vitro with anti-CD3 plus wt APCs, OX40Ltg APCs or OX40L−/− APCs in the presence or absence of TGF-β, and induction of GFP(Foxp3)+ cells was determined 4 days later by gating onto the CD4+ fraction. The FACS plot shown is the representative data of 4 individual experiments. (B) The experiments were set up as described in panel A, and the image shown was captured by confocal microscopy 4 days after the culture using a Nikon Eclipse 80i system equipped with E-max software (Nikon Instruments, Melville, NY) (40×/0.75 NA oil immersion lens). Cells were labeled with PE-antimouse CD4. (C) CD4+CD25− T effector cells were sorted from congenic CD90.1 mice and adoptively transferred into wt C57BL/6 and OX40Ltg mice (CD90.2). The host mice were treated with DST and anti-CD154. Induction of Foxp3 expression in the CD90.1+ fraction in the host spleen was determined by intracellular staining for the Foxp3 protein 6 days later. Data shown are representative of 3 experiments. (A,C) Numbers are the relative percentage of cells in each region given by the flow cytometer.

Article Snippet: An agonist antimouse OX40 mAb (clone OX86) and the antimouse CD154 (clone MR1) were produced from hybridoma cell lines by BioExpress (West Lebanon, NH) and were used in some in vitro and in vivo studies.

Techniques: In Vitro, Confocal Microscopy, Software, Labeling, Expressing, Staining, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Enhanced systemic tumor suppression by in situ vaccine combining radiation and OX40 agonist with CpG therapy

doi: 10.1186/s12967-023-04504-w

Figure Lengend Snippet: CpG and radiotherapy upregulated the expression of OX40 on tumor infiltrating CD4+ T cells in the tumor microenvironment. A The correlation between OX40 expression and TLR9 or TLR9 downstream elements in SKCM was analyzed by an online tool GEPIA 2. B , C 48 h after treated with PBS, CpG intratumoral injection or 10 Gy irradiation followed by CpG intratumoral injection, tumors were excised and OX40 expression on the CD45+CD4+ T cell subset was analyzed by flow cytometry (OX40+ subset gate on CD45+CD4+ cells). Data are represented as mean ± s.e.m. n = 4. Student’s t test was used for statistical analysis. * p < 0.05

Article Snippet: Anti-OX40 agonistic antibody (αOX40, Clone OX-86) was provided by GenScript USA Inc. Murine anti-PD-1 antibody (G4C2) was provided by Shanghai Junshi Biosciences Co.,Ltd (Suzhou, China).Monoclonal antibodies (mAbs) used for flow cytometry were listed as flow: FITC anti-mouse CD45 (Biolegend, USA), PE/DazzleTM anti-mouse CD3 (Biolegend, USA), FITC anti-mouse CD8 (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, USA), FITC anti-mouse CD11c (Biolegend, USA), PE anti-mouse CD86 (Biolegend, USA), PE/Cyanine7 anti-mouse CD80 (Biolegend, USA), PE anti-mouse CD44 (Biolegend, USA), PE/Cyanine7 anti-mouse CD62L (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse PD-1 (Biolegend, USA), PE/Cyanine7 anti-mouse OX40 (Biolegend, USA), FITC anti-mouse CD11b (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse F4/80 (Biolegend, USA), APC anti-mouse CD206 (Biolegend, USA), Mouse Regulatory T cell staining kit (eBioscience, USA).

Techniques: Expressing, Injection, Irradiation, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Enhanced systemic tumor suppression by in situ vaccine combining radiation and OX40 agonist with CpG therapy

doi: 10.1186/s12967-023-04504-w

Figure Lengend Snippet: Systemic immune response induced by triple combining in situ vaccine. A , B Proportion of effector memory T cells (TEMs, CD44+CD62L– gate on CD3+CD8+ cells) in A abscopal tumors and B spleens of C57BL/6 mice in each group a week after the last administration. Data are represented as mean ± s.e.m. n = 5–6. Student’s t test was used for statistical analysis. * p < 0.05, ** p < 0.01. C Spleen cells of mice in control group and RT + CpG + αOX40 group were incubated with CFSE labeled (indicating live cells) B16F10 melanoma cells at effector-to-target ratio (E:T) of 5:1, 10:1, 20:1 and 30:1. PI was added after 5 h-incubation and the percentage of dead cells was analyzed by flow cytometry. Data are represented as mean ± s.e.m. n = 4. Student’s t test was used for statistical analysis. * p < 0.05. D Representative flow cytometry plots indicating proportions of dead cells of B16F10 melanoma cells (CFSE + PI + gate on CFSE + cells). E , F Proportion of E PD-1+ CTLs (CD8+PD-1+ gate on CD3+ cells) and F regulatory T cells (Tregs, CD25+FoxP3+ gate on CD3+CD4+ cells) in abscopal tumors of C57BL/6 mice in each group a week after the last administration. Data are represented as mean ± s.e.m. n = 3–5. Student’s t test or corrected Student’s t test was used for statistical analysis. * p < 0.05

Article Snippet: Anti-OX40 agonistic antibody (αOX40, Clone OX-86) was provided by GenScript USA Inc. Murine anti-PD-1 antibody (G4C2) was provided by Shanghai Junshi Biosciences Co.,Ltd (Suzhou, China).Monoclonal antibodies (mAbs) used for flow cytometry were listed as flow: FITC anti-mouse CD45 (Biolegend, USA), PE/DazzleTM anti-mouse CD3 (Biolegend, USA), FITC anti-mouse CD8 (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, USA), FITC anti-mouse CD11c (Biolegend, USA), PE anti-mouse CD86 (Biolegend, USA), PE/Cyanine7 anti-mouse CD80 (Biolegend, USA), PE anti-mouse CD44 (Biolegend, USA), PE/Cyanine7 anti-mouse CD62L (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse PD-1 (Biolegend, USA), PE/Cyanine7 anti-mouse OX40 (Biolegend, USA), FITC anti-mouse CD11b (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse F4/80 (Biolegend, USA), APC anti-mouse CD206 (Biolegend, USA), Mouse Regulatory T cell staining kit (eBioscience, USA).

Techniques: In Situ, Control, Incubation, Labeling, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Enhanced systemic tumor suppression by in situ vaccine combining radiation and OX40 agonist with CpG therapy

doi: 10.1186/s12967-023-04504-w

Figure Lengend Snippet: RNA-seq analysis of control tumors and tumors receiving in situ triple vaccine. A Heat map revealing in situ vaccine-induced changes in the gene-expression profile of the tumor microenvironment. n = 3 for both control and RT + CpG + αOX40 group. Color bars indicate normalized expression, the color from blue to red indicates that the gene expression from low to high. B GO enrichment analysis of up- and downregulated genes. Gene ratio (shown in green) and − log10(Q value) of all GO terms are shown. C KEGG enrichment analysis of up- and downregulated genes. Gene ratio (shown in green) and − log10(Q value) of all KEGG signaling pathway are shown. D Representative GSEA plot showing down-regulated and up-regulated enriched gene sets of all detected genes in tumors receiving in situ triple vaccine

Article Snippet: Anti-OX40 agonistic antibody (αOX40, Clone OX-86) was provided by GenScript USA Inc. Murine anti-PD-1 antibody (G4C2) was provided by Shanghai Junshi Biosciences Co.,Ltd (Suzhou, China).Monoclonal antibodies (mAbs) used for flow cytometry were listed as flow: FITC anti-mouse CD45 (Biolegend, USA), PE/DazzleTM anti-mouse CD3 (Biolegend, USA), FITC anti-mouse CD8 (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, USA), FITC anti-mouse CD11c (Biolegend, USA), PE anti-mouse CD86 (Biolegend, USA), PE/Cyanine7 anti-mouse CD80 (Biolegend, USA), PE anti-mouse CD44 (Biolegend, USA), PE/Cyanine7 anti-mouse CD62L (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse PD-1 (Biolegend, USA), PE/Cyanine7 anti-mouse OX40 (Biolegend, USA), FITC anti-mouse CD11b (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse F4/80 (Biolegend, USA), APC anti-mouse CD206 (Biolegend, USA), Mouse Regulatory T cell staining kit (eBioscience, USA).

Techniques: RNA Sequencing, Control, In Situ, Gene Expression, Expressing