recombinant vil 10  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: CD137 Costimulation Enhances the Antitumor Activity of Vγ9Vδ2-T Cells in IL-10-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.3389/fimmu.2022.872122

Figure Lengend Snippet: Antitumor activity of Vγ9Vδ2-T cells was inhibited by IL-10 secreted from EBV-LCL in vitro . (A) The concentration of vIL-10 and total IL-10 in conditioned medium (CM) collected from EBV-LCL established from different donors were detected. (B) Purified Vγ9Vδ2-T cells were pretreated in the IL-10 low CM and IL-10 high CM separately for 24 h, RPMI 1640 with 10% FBS medium (plain medium, PM) as a control. Pretreated Vγ9Vδ2-T cells then cocultured with autologous EBV-LCL at an effector: target (E:T) ratio of 10:1 for 4–6 h in the IL-10 low/high CM and PM, respectively. Cytotoxicity was calculated as the proportion of dead EBV-LCL (CD3 - PI + ). (C) Purified Vγ9Vδ2-T cells were pretreated with IL-10 low CM, IL-10 high CM or PM in the presence of a neutralizing anti-IL-10 mAb (αIL-10, 5μg/ml) or isotype control (mIgG1, 5μg/ml), then cocultured with autologous EBV-LCL at an E:T ratio of 10:1 for 4–6 h in the IL-10 low/high CM and PM respectively. Cytotoxicity was calculated as the proportion of dead EBV-LCL (CD3 - PI + ). (D, E) Purified Vγ9Vδ2-T cells were pretreated with recombinant hIL-10 (D) or vIL-10 (E) at different concentration, then cocultured with autologous EBV-LCL at an E:T ratio of 10:1 for 4–6h. The proportion of dead EBV-LCL (CD3 - PI + ) were detected by flow cytometry. All data are shown as mean ± SEM and representative of three independent experiments. *p < 0.05; **p < 0.01; ns, no significant difference.

Article Snippet: To confirm the suppressive role of IL-10 in the CM, recombinant hIL-10 (Peprotech) or recombinant vIL-10 (R&D systems) was added to culture medium at the indicated concentration.

Techniques: Activity Assay, In Vitro, Concentration Assay, Purification, Control, Recombinant, Flow Cytometry

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Design and characterization of 1-11E/vIL-10 fusion. (A) Schematic representation of the 1-11E/vIL10 fusion protein, as cloned into the pcDNA expression vectors. SP, signal peptide. The scFv and vIL-10 are separated by an MMP cleavage linker, whereas the His-tag and V5-tag were incorporated at the C-terminus. (B) SDS-PAGE analysis of purified 1-11E/vIL-10 is shown in the left panel. Purified 1-11E/vIL-10 fusion protein migrated at approximately 50 kDa, in keeping with the expected molecular weight (~30 kDa scFv plus ~20 kDa vIL-10). Digestion of 1-11E/IL10 by MMPs is shown in the right panel. 1-11E/vIL-10 fusion protein was incubated with the catalytic domains of MMP-1, MMP-3, and MMP-12 overnight and analyzed with Western blot by using anti-tetra-His-HRP antibodies. (C) FPLC profile of 1-11E/IL10. The first major peak at approximately 100 kDa corresponds to the dimer form, and the second minor peak at approximately 50 kDa corresponds to the monomer form.

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Clone Assay, Expressing, SDS Page, Purification, Molecular Weight, Incubation, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH - ; lane 5, peroxynitrate) but not to HEL (lane 6). (C) . IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion ( P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 ( P > 0.05).

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Modification, Western Blot, Positive Control, Recombinant, Negative Control, Glo Assay

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Binding of 1-11E/vIL-10 to arthritic cartilage. (A) Sections of cartilage from mouse model of antigen-induced arthritis (AIA), collagen-induced arthritis (CIA), and surgical destabilization of the medial meniscus (DMM) were incubated with Cy5.5 tagged 1-11E/vIL-10 (left panels) or C7/vIL10 (right panels). Binding to cartilage was diffused in all zones, although AIA staining was mostly in the superficial zone. No staining with C7/vIL10 was seen. Pericellular matrix of the chondrocytes is shown in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red. (B) Staining of human OA cartilage with 1-11E/vIL-10. Binding of the fusion protein was detected with mouse anti-vIL10, followed by anti-mouse-HRP. Binding was strong in the damaged area and as a territorial “halo” around the chondrocyte. Low background staining with C7/vIL-10 was seen.

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Binding Assay, Incubation, Staining, Labeling

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Tracking of 1-11E/vIL-10 to arthritic joints. One day after AIA induction in mice, 1 μg of Cy5.5-labeled fusion proteins was injected i.p. In vivo fluorescence images of the mice were taken to follow the tracking of the fusion proteins to the joint. (A) Representative fluorescence image taken 3 days after injection of 1-11E/vIL-10 (left) and C7/vIL-10 (right). (B) Quantification of the average fluorescence of the region of interest encompassing the knee joint, n = 3. (C) Ex vivo quantification of fluoresce of tissues after dissection of a single mouse 4 days after injection. (D) Representative fluorescence images of cryosections of the excised knee joints, showing the pericellular matrix of the chondrocytes in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red.

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Labeling, Injection, In Vivo, Fluorescence, Ex Vivo, Dissection

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Reduction of oxidative state in inflamed knee by 1-11E/vIL10 fusion proteins. Antigen-induced arthritis was induced in female C57BL/6 mice by injecting mBSA into the knee of each animal 1 day before treatment ( n = 3). On days 1 and 3 after mBSA rechallenge, animals were injected i.p. with 30 μg of 1-11E/vIL10 or C7/vIL10. As a control, PBS was injected. Level of inflammation and redox state were determined by monitoring the luminescence 15 minutes after i.p. administration of luminol, a redox-sensitive compound that glows when mixed with oxidizing agents present in the inflamed knee and thus correlates with degree of inflammation. (A) Representative luminescence images taken at days 1, 2, and 5 after injection of 1-11E/vIL-10, C7/vIL-10, and control nontreated mice are shown. (B) The reduction in oxidation level was faster in the 1-11E/vIL-10-treated animal than in C7/vIL-10 and nontreated mice, which correlated with reduction in swelling (C) .

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Injection

Journal: Arthritis Research & Therapy

Article Title: Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

doi: 10.1186/ar4613

Figure Lengend Snippet: Treatment efficacy of 1-11E/vIL-10. A . AIA was induced in female C57BL/6-mice 1 day before treatment ( n = 10-12). On days 1 and 3 after mBSA rechallenge, animals were injected i.p. with 30 μg of 1-11E/vIL10 or C7/vIL10 or PBS. Significant reduction in knee swelling in 1-11E/vIL-10 treated mice compared with mice treated with C7/vIL-10 or PBS controls was observed ( P = 0.0048). (B) Safranin O staining of cartilage from nontreated AIA knee displayed weak staining. The cartilage from healthy control animals was smooth, with clear strong staining with safranin O. Cartilage from C7/vIL-10-treated mouse had weak safranin O staining compared with healthy controls. 1-11E/vIL-10-treated knees displayed histology similar to that of the healthy joints. (C) Serum cytokine levels in treated mice were obtained from mice treated with 1-11E/vIL10 or C7/vIL10 as well as from healthy control animals (HC) on day 3. Cytokines levels were measured with ELISA by using the 7-plex mouse Pro-Inflammatory Cytokine Kit. Except for IFN-γ, levels of pro-inflammatory cytokines were significantly lower in the 1-11E/vIL-10-treated animal ( P = 0.0175) and similar to levels of healthy control animals ( P > 0.05).

Article Snippet: In brief, on day 1, 5 × 10 4 cells were treated with fusion proteins (with or without MMP-1 digestion) at concentrations of 1,000 ng/ml, 100 ng/ml, or 10 ng/ml, as well as control recombinant vIL-10 (R&D Systems, Abingdon UK).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay