Journal: bioRxiv

Article Title: Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity

doi: 10.1101/2023.11.02.565371

Figure Lengend Snippet: ( a ) Schematic representation of the experimental design deployed to investigate the functionality of engineered EVs ex vivo using human PBMCs. ( b ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IFNγ in human CD4 + T cells and CD8 + T cells treated with hCD80 EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=6 PBMC samples. ( c ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IL-2 in human CD4 + T cells, and activation markers (CD25 and CD69), IFNγ, and cytolytic marker (Granzyme B), in human CD8 + T cells treated with hOX40L EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=3 PBMC samples. ( d ) Representative histograms of activation markers (CD25 and CD69), proliferation marker (Ki67), and IFNγ in human CD8 + T cells treated with hPD-L1 EVs. Accompanying bar graphs show mean +/- s.e.m. of MFI from n=8 PBMC samples. Statistical significance was determined using ordinary one-way ANOVA, p-values are shown. Statistical significance defined as p < 0.05.

Article Snippet: To generate overexpressing cells, plasmids for hCD80 (Sino Biological HG10698-UT)), hOX40L (Sino Biological HG13127-UT), hPD-L1 (Origene RC213071L4), mCD80 (Sino Biological MG50446-UT), mOX40L (Sino Biological MG53582-UT), and mPD-L1 (Origene MR203953L4) were transfected using lipofectamine 3000 (Invitrogen) in HEK293T/17 cells according to manufacturer’s instructions and selected using hygromycin (CD80 and OX40L plasmids) or puromycin (PD-L1 plasmids).

Techniques: Ex Vivo, Activation Assay, Marker

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: ILC2s Are the Main OX40L-Expressing Cell in Response to IL-33 (A) TNFSF gene expression was analyzed in the indicated immune cell populations using IMMGEN and naive lung ILC2 microarray data. (B) OX40L expression was measured on lung DCs or ILC2s in WT mice on day 2 after treatment with PBS or IL-33 (i.n., day 0 and 1). (C) The percentage of OX40L + ILC2s (top) and DCs (bottom) was measured on day 2 in response to the indicated stimuli (i.n., day 0 and 1). (D) The percent of Ki67 + ILC2s and Treg cells was measured on day 2 after treatment with PBS or IL-33 (day 0 and 1). (E) OX40L expression was measured on ILC2s and ILC3s in the lung or mLN on day 2 after PBS or IL-33 treatment (i.n., day 0 and 1). Numbers in (B) and (E) indicate percent (mean ± SD) gated populations. (A), two independent datasets per group; (B), three repeat experiments; (C), ANOVA, two repeat experiments; (D), ANOVA, two repeat experiments; (E), two-tailed Student’s t test, two repeat experiments. ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S4 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Expressing, Microarray, Two Tailed Test

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L Expression by ILC2s Is Essential for Tissue-Specific Adaptive Immune Response to IL-33 (A) OX40L expression on lung ILC2s in the specified genotypes on day 2 after IL-33 administration (i.n., day 0 and 1). (B–D) Lung GATA3 + and GATA3 − Treg and Th2 cell numbers were quantified on day 5 after treatment with PBS or IL-33 (i.n., days 0 and 1). (E–G) OX40L expression on ILC2s was measured in the indicated tissues (L. int., large intestine) after treatment with PBS or IL-33 (days 0 and 1) on day 2 (E), while the number of ILC2s (F) and GATA3 + Treg cells was quantified on day 5 (G). Numbers in (A) and (E) indicate percent (mean ± SD) gated populations. Bar graphs indicate mean (±SEM). (A), three repeat experiments; (B)–(D), ANOVA, two repeat experiments; (E)–(G), ANOVA, two repeat experiments. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. See also Figure S5 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Expressing

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L-Driven Response to IL-33 Is Restricted to ILC2s (A–C) Lung GATA3 + and GATA3 − Treg cells and Th2 cells were quantified on day 5 in the specified genotypes, treated with PBS or IL-33 (i.n., days 0 and 1). (D and E) Bone marrow and mixed-bone marrow chimeric mice were created with the indicated genotypes. 6 to 7 months after bone marrow transfer, mice received IL-33 (i.n., days 0 and 1) followed by quantification of lung GATA3 + Treg (D) and GATA3 − Treg (E) cells on day 5. Bar graphs indicate mean (±SEM). ANOVA, two repeat experiments. ns = not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗ p ≤ 0.0001. See also Figure S6 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques:

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L on ILC2s Orchestrates Adaptive Type 2 Immunity to Allergens (A–D) Mice of the specified genotypes were treated with PBS or papain (Pap) (i.n., day 0 and 1), followed by quantification of lung Th2, GATA3 + Treg, and GATA3 − Treg cells on day 5 (A–C). Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of IL-13 + Th2 cells by intracellular staining (D). (E–G) Mice were treated with papain on days 0, 1, 14, and 21, followed by quantification on day 24 of lung eosinophils (E), and detection (F) and quantification (G) of RELMα + M2 alveolar (CD45 + SiglecF + CD11c + CD11b − F4/80 + ) macrophages (MΦ). (H and I) Mice were treated with A. alternata ( A.alt ) (i.n., days 0 and 1), followed by quantification on day 9 of lung Th2, GATA3 + Treg, and GATA3 − Treg cells (H) and serum IgE concentration (I). Bar graphs indicate mean (±SEM). (A)–(D), ANOVA, two repeat experiments; (E), ANOVA, three repeat experiments; (F) and (G), ANOVA, two repeat experiments, representative gate shown in (F); (H) and (I), ANOVA, two repeat experiments. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Staining, Concentration Assay

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: ILC2-Expressed OX40L Is Essential for Airway Adaptive Type 2 Immune Response to Helminth Infection Mice of the specified genotypes were infected with Nippostrongylus brasiliensis ( N.b. ) on day 0, followed by analysis on day 28 (or day 5) of: (A–C) Lung Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (D) Representative lung histology (Mason’s trichrome). (E and F) Lung (E) and bronchoalveolar lavage (F) eosinophil numbers. (G) Lung RELMα + M2 macrophage (MΦ) numbers. (H) Bronchoalveolar lavage IL-4 and IL-5 cytokine concentrations. (I) Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of lung IL-13 + Th2 cell numbers by intracellular staining. (J–L) Mediastinal lymph node Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (M) Concentration of IgE present in lung homogenate, normalized for total protein content. (N) Intestinal worm burden of indicated mouse genotypes 5 days post infection. Bar graphs indicate mean (±SEM). (A)–(C), ANOVA, three repeat experiments; (D), two repeat experiments; (E)–(I), ANOVA, two repeat experiments; (J)–(L), ANOVA, three repeat experiments; (M), ANOVA, two repeat experiments; (N), two-tailed Student’s t test, two pooled experiments. ns = not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Infection, Staining, Concentration Assay, Two Tailed Test

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet:

Article Snippet: Anti-human OX40L , Biolegend , 326307; RRID: AB_2207272.

Techniques: Recombinant, Staining, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Bioconjugate Chemistry

Article Title: Adapting Ferritin, a Naturally Occurring Protein Cage, to Modulate Intrinsic Agonism of OX40

doi: 10.1021/acs.bioconjchem.4c00020

Figure Lengend Snippet: OX40+ Jurkat reporter assay. RLU denotes relative luminescence. Molecule concentrations (nM) were calculated as follows: rFer 24 -Fab conjugate and aOX40 Fab concentrations were based on Fab molar mass, OX40L-Fc XL concentration was based on OX40L-Fc XL molar mass, aOX40 IgG1 concentration was based on the antibody molar mass, and rFer 24 concentration was based on the molar mass of the assembled ferritin cage.

Article Snippet: OX40L protein extracellular domain (ECD) fused to the fragment crystallizable (Fc) region of human IgG1 (OX40L-Fc) was obtained from Sino Biological (13127-H01H).

Techniques: Reporter Assay, Concentration Assay

Journal: Molecular Therapy Oncolytics

Article Title: Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

doi: 10.1016/j.omto.2021.02.006

Figure Lengend Snippet: Novel VALO-D102 oncolytic adenovirus produces high levels of biologically active human CD40 ligand (CD40L) and OX40 ligand (OX40L) (A) Schematic representation of genetic modifications in VALO-D102. The virus has a 24-base pair deletion in E1A; the CR1-alpha and gp19K genes in the E3A region have been replaced with human OX40L and CD40L genes; the 14.7K gene in the E3B region has been deleted; and finally, the adenovirus 5 knob domain has been replaced with the knob domain from adenovirus serotype 3. (B) A549 cells were infected with VALO-D102 at a MOI of 10. 72 h postinfection, supernatant was collected and added to a culture of Ramos-Blue reporter cells, and CD40 receptor activation by functional virus-produced CD40L was measured. (C) A549 cells were infected with VALO-D102 at a MOI of 10. 48 h postinfection, HEK293-OX40/NF-κB reporter cells were added to the infected A549 cells for 6 h, and OX40 activation by virus-expressed, functional membrane-bound OX40L was measured.

Article Snippet: Recombinant human OX40L (BPS Bioscience) was used as a standard.

Techniques: Infection, Activation Assay, Functional Assay, Produced

Journal: Molecular Therapy Oncolytics

Article Title: Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

doi: 10.1016/j.omto.2021.02.006

Figure Lengend Snippet: Virus-encoded OX40L and CD40L improve anti-tumor efficacy and induce robust infiltration of tumor-specific CD8 + T cells into the tumor in a syngeneic mouse model of B16.OVA melanoma (A) 1 × 10 9 VP of PeptiCRAd Ad5/3-D24-OVA or PeptiCRAd VALO-mD901-OVA was given intratumorally 6, 8, and 20 days post-tumor implantation. Average tumor growth curves for all treatment groups are shown. (B) Immunological analysis of tumors and tumor-draining lymph nodes of treated mice. Lymph nodes from all mice from each treatment group were pooled in order to get enough cells for the flow cytometric analysis. The number of mice in the mock group was 7 and in both PeptiCRAd groups, was 10. Statistical analysis was performed with one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Recombinant human OX40L (BPS Bioscience) was used as a standard.

Techniques: Tumor Implantation

Journal: Journal for Immunotherapy of Cancer

Article Title: FcγRIIB engagement drives agonistic activity of Fc-engineered αOX40 antibody to stimulate human tumor-infiltrating T cells

doi: 10.1136/jitc-2020-000816

Figure Lengend Snippet: Multimeric OX40 ligation enhances TIL expansion, and activation. Tumor tissues from patients with LM-CRC and HCC were collected; immune cells were isolated and cultured in vitro in the presence of αCD3/CD28 activation beads (ctrl) and additionally (B, C, E) 20 µg/mL or (C) 2 µg/mL OX40L or (B, D, E) 10 µg/mL monomeric αOX40 IgG2 or (F) multimeric αOX40 IgG2 for a period of 8–10 days. (A) Schematic outline of hexameric OX40L, monomeric αOX40 IgG2 antibody and αOX40 IgG2 antibodies conjugated to magnetic beads. (B, C, F) TIL numbers after in vitro cultures were acquired by flow cytometry and normalized to counting beads. (B, C) Relative changes over cell numbers in ctrl are shown (n=11). (D, E) Cytokine levels in culture supernatants were acquired by cytokine multiplex assay; (D) n=12, (E) n=15). (F) Cell numbers are depicted as relative to soluble and bead-conjugated IgG2 isotypes, respectively (n=9). (B) One-way non-parametric Kruskal-Wallis, including Dunn’s post-testing and (C–F) non-parametric Wilcoxon test, was performed. *P≤0.05, **P≤0.01, ***P≤0.001. HCC, hepatocellular carcinoma; IFN, interferon; LM-CRC, liver metastasis colorectal cancer; ns, not significant; OX40L, OX40 ligand; pCRC, primary colorectal cancer; TIL, tumor-infiltrating lymphocyte; TNF-α, tumor necrosis factor alpha.

Article Snippet: Cells were treated with 2–20 μg/mL hexameric human OX40L (Pfizer), 10 μg/mL soluble human αOX40 IgG1, human isotype HA-IgG1, human αOX40 IgG2 (PF-04518600), human isotype HA-IgG2 or human αOX40IgG1_v12 (all Pfizer). αOX40 IgG2 and isotype IgG2 antibody coupling to Dynabeads was performed by using Dynabeads Antibody coupling Kit according to the manufacturer’s instructions (14 311D), whereby 6 μg antibody was coupled per 1 mg of beads.

Techniques: Ligation, Activation Assay, Isolation, Cell Culture, In Vitro, Magnetic Beads, Flow Cytometry, Multiplex Assay