Journal: Scientific Reports

Article Title: An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

doi: 10.1038/s41598-021-93660-4

Figure Lengend Snippet: Characterization of newly identified anti-Tie2 agonistic antibodies. ( a ) Cellular viability as an indicator of agonistic activity of anti-Tie2 antibodies produced by hybridoma clones 2–16 and 2–8 were assessed in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined using a CellTiter-Glo assay, with basal activity without any ligand (PBS) defined as 0%, and the activity of 17.9 nM (1 µg/mL) Ang1 defined as 100%. Data represent mean ± SD. ( b , c ) Fractionation of the antibody solution using AEX. ( b ) shows SDS PAGE of each fraction from AEX, and ( c ) shows cellular viability as an indicator of the agonistic activity of each fraction in human Tie2-expressing Ba/F3. Solid (left) arrow indicates antibody-rich fractions, dashed (right) arrow indicates the fractions with higher agonistic activity. Cellular viability (%) was determined using a CellTiter-Glo assay, with basal activity without any ligand (PBS) defined as 0%, and the activity in fraction 6 defined as 100%. ( d ) Cellular viability as an indicator of the agonistic activity of purified 2–16 monomer, dimer and HMW human monoclonal antibodies, isolated using SEC (Supplementary Fig. and ), in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined as described in ( a ).

Article Snippet: Recombinant human, rat and mouse Tie2-Fc chimeric protein (R&D Systems) and recombinant monkey Tie2-Fc chimeric protein (Sino Biological) were each immobilized on a sensor chip CM5 (GE Healthcare) using an Amine Coupling Kit (GE Healthcare) and HBS-EP + buffer (GE Healthcare) in accordance with the manufacturer’s instructions.

Techniques: Activity Assay, Produced, Clone Assay, Expressing, Glo Assay, Fractionation, SDS Page, Purification, Isolation

Journal: Scientific Reports

Article Title: An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

doi: 10.1038/s41598-021-93660-4

Figure Lengend Snippet: Generation and in vitro evaluation of the tetra-valent anti-human Tie2 antibody. ( a ) Structure of the tetra-valent antibody. ( b ) SDS-PAGE of the tetra-valent antibody under non-reduced (left) and reduced (right) conditions. Molecular weights (kDa) are indicated on the left of each gel. ( c ) Binding activity of ASP4021 to human Tie2 and Tie1 by direct ELISA. ASP4021 binding is shown as the absorbance at OD450. Each value is expressed as mean ± SEM. ( d ) Cellular viability as an indicator of agonistic activity of the tetra-valent and bi-valent antibodies in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined as described Fig. a. ( e ) Anti-apoptotic activity of ASP4021 in human Tie2-expressing Ba/F3 compared to Ang1. Caspase 3/7 activity (%) was calculated using the Caspase-Glo 3/7 assay, with basal activity with IL-3 defined as 0%, and activity without any ligand (PBS) defined as 100%. Data represent mean ± SD. ( f ) Phosphorylation of Tie2 induced by ASP4021 in HUVEC compared to Ang1. HUVEC were treated with ASP4021 or recombinant Ang1 for 30 min. Levels of phosphorylated Tie2 and actin in cell lysates were detected using Western blotting. The assay was performed in duplicate. Full-size versions of Western blots are depicted in Supplementary Fig. .

Article Snippet: Recombinant human, rat and mouse Tie2-Fc chimeric protein (R&D Systems) and recombinant monkey Tie2-Fc chimeric protein (Sino Biological) were each immobilized on a sensor chip CM5 (GE Healthcare) using an Amine Coupling Kit (GE Healthcare) and HBS-EP + buffer (GE Healthcare) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, SDS Page, Binding Assay, Activity Assay, Direct ELISA, Expressing, Caspase-Glo Assay, Recombinant, Western Blot

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A Mice were infected with LCMV, and Il15 transcript levels were measured in the indicated organs 24 h postinfection. Data are represented as fold induction after normalization to levels in corresponding naïve tissue. Data indicate mean ± SEM of n = 3 mice per group and show one representative of two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. B–E Mice were infected with LCMV, and flow cytometry was applied on splenic NK cells from infected animals to assess frequencies of IL‐15Rβ‐positive cells (B), IL‐15Rβ expression levels (C), AKT phosphorylation (D), or S6 phosphorylation (E). For AKT and S6 phosphorylation, representative histograms and cumulative results are depicted. MFI, mean fluorescence intensity; Isotype, isotype‐matched control antibody. Data indicate mean ± SEM of n = 3 (B–D) or n = 4 (E) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05. F, G Splenic NK cells from naïve donors were stimulated in vitro for 1 h with IL‐15, and phosphorylation of AKT (F) or S6 (G) was measured. Data indicate mean ± SEM of n = 3 (F) or n = 4 (G) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. H GZMB expression was measured in splenic NK cells after stimulation with IL‐15 for 20 h. Data indicate mean ± SEM of n = 4 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using one‐way ANOVA with Tukey post‐test. *** P < 0.001; **** P < 0.0001. I WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and GZMB expression was measured in NK cells. Values shown were normalized to unstimulated controls. Data show n = 3 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of mouse TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences, NY, USA) added 1 h prior to activation with IL‐15.

Techniques: Infection, Two Tailed Test, Flow Cytometry, Expressing, Fluorescence, In Vitro, Cell Culture

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A NK cells were MACS‐purified from single‐splenocyte suspensions from naïve donors, stimulated in vitro for 1 h with IL‐15, and phosphorylation of S6 was measured by flow cytometry. One representative of two independent experiments is depicted ( n = 1 mouse per group). B AKT and S6 phosphorylation were measured in naive splenic NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. C Splenocytes were cultured with IL‐15 ± wortmannin (WRM)/LY294002, and GZMB expression was measured in NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using one‐way ANOVA with Dunn's post‐test. * P < 0.05; ** P < 0.01. D, E WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and AKT phosphorylation (D) or S6 phosphorylation (E) was measured in NK cells. Values shown were normalized to unstimulated control. Data show n = 5 mice per group, pooled from two independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. F–H Splenocytes from naïve WT and Trail −/− mice were cultured with the indicated cytokines, and frequencies of IFNγ + NK cells (F), or IFNγ‐expression levels in NK cells (G) were measured after 5 h. Alternatively, naïve splenocytes were cultured with IL‐18/IL‐12, and IFNγ was measured in the supernatant at the indicated time points (H). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of two (F, G) or three (H) independent experiments. Unpaired two‐tailed t ‐test was used. * P < 0.05. I, J MACS‐purified DX5 + cells were cultured in wells coated with an anti‐NK1.1 antibody, and frequencies of IFNγ + NK cells were measured after 5 h (I) or GZMB expression was measured after 24 h (J). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of three (I) or one (J) independent experiments. Unpaired two‐tailed t ‐test was used. ** P < 0.01. K–O Flow cytometry was applied on NK‐92 cells to assess expression of TRAIL (K) and the TRAIL receptors DR4 (L) and DR5 (M). NK‐92 cells were stimulated with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation (N) or GZMB expression (O) was measured by flow cytometry ( n = 1 per condition). (N, O) Data show one representative of 2 independent experiments.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of mouse TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences, NY, USA) added 1 h prior to activation with IL‐15.

Techniques: Purification, In Vitro, Flow Cytometry, Two Tailed Test, Cell Culture, Expressing

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: Human peripheral blood mononuclear cells were cultured with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation was measured in CD56 bright NK cells. Data were pooled from three independent experiments ( n = 9 donors per condition). Values shown were normalized to unstimulated controls. Representative histograms are also shown (right‐hand side). Statistical analyses were performed using Wilcoxon matched‐pairs signed rank test. ** P < 0.01.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of mouse TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences, NY, USA) added 1 h prior to activation with IL‐15.

Techniques: Cell Culture

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A Mice were infected with LCMV, and Il15 transcript levels were measured in the indicated organs 24 h postinfection. Data are represented as fold induction after normalization to levels in corresponding naïve tissue. Data indicate mean ± SEM of n = 3 mice per group and show one representative of two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. B–E Mice were infected with LCMV, and flow cytometry was applied on splenic NK cells from infected animals to assess frequencies of IL‐15Rβ‐positive cells (B), IL‐15Rβ expression levels (C), AKT phosphorylation (D), or S6 phosphorylation (E). For AKT and S6 phosphorylation, representative histograms and cumulative results are depicted. MFI, mean fluorescence intensity; Isotype, isotype‐matched control antibody. Data indicate mean ± SEM of n = 3 (B–D) or n = 4 (E) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05. F, G Splenic NK cells from naïve donors were stimulated in vitro for 1 h with IL‐15, and phosphorylation of AKT (F) or S6 (G) was measured. Data indicate mean ± SEM of n = 3 (F) or n = 4 (G) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. H GZMB expression was measured in splenic NK cells after stimulation with IL‐15 for 20 h. Data indicate mean ± SEM of n = 4 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using one‐way ANOVA with Tukey post‐test. *** P < 0.001; **** P < 0.0001. I WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and GZMB expression was measured in NK cells. Values shown were normalized to unstimulated controls. Data show n = 3 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Infection, Two Tailed Test, Flow Cytometry, Expressing, Fluorescence, In Vitro, Cell Culture

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A NK cells were MACS‐purified from single‐splenocyte suspensions from naïve donors, stimulated in vitro for 1 h with IL‐15, and phosphorylation of S6 was measured by flow cytometry. One representative of two independent experiments is depicted ( n = 1 mouse per group). B AKT and S6 phosphorylation were measured in naive splenic NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. C Splenocytes were cultured with IL‐15 ± wortmannin (WRM)/LY294002, and GZMB expression was measured in NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using one‐way ANOVA with Dunn's post‐test. * P < 0.05; ** P < 0.01. D, E WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and AKT phosphorylation (D) or S6 phosphorylation (E) was measured in NK cells. Values shown were normalized to unstimulated control. Data show n = 5 mice per group, pooled from two independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. F–H Splenocytes from naïve WT and Trail −/− mice were cultured with the indicated cytokines, and frequencies of IFNγ + NK cells (F), or IFNγ‐expression levels in NK cells (G) were measured after 5 h. Alternatively, naïve splenocytes were cultured with IL‐18/IL‐12, and IFNγ was measured in the supernatant at the indicated time points (H). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of two (F, G) or three (H) independent experiments. Unpaired two‐tailed t ‐test was used. * P < 0.05. I, J MACS‐purified DX5 + cells were cultured in wells coated with an anti‐NK1.1 antibody, and frequencies of IFNγ + NK cells were measured after 5 h (I) or GZMB expression was measured after 24 h (J). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of three (I) or one (J) independent experiments. Unpaired two‐tailed t ‐test was used. ** P < 0.01. K–O Flow cytometry was applied on NK‐92 cells to assess expression of TRAIL (K) and the TRAIL receptors DR4 (L) and DR5 (M). NK‐92 cells were stimulated with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation (N) or GZMB expression (O) was measured by flow cytometry ( n = 1 per condition). (N, O) Data show one representative of 2 independent experiments.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Purification, In Vitro, Flow Cytometry, Two Tailed Test, Cell Culture, Expressing

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: Human peripheral blood mononuclear cells were cultured with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation was measured in CD56 bright NK cells. Data were pooled from three independent experiments ( n = 9 donors per condition). Values shown were normalized to unstimulated controls. Representative histograms are also shown (right‐hand side). Statistical analyses were performed using Wilcoxon matched‐pairs signed rank test. ** P < 0.01.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Cell Culture

Journal: Nature Communications

Article Title: Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

doi: 10.1038/s41467-021-22985-5

Figure Lengend Snippet: a Super-resolution STED imaging used to generate 3D views of T cells on ICAM1-PAAG nanotopographies ( G ’ = 50 kPa in this example) to characterize T-cell interactions with nanotopography. Views: 1, stereometric; 2, cross-section; 3, interface from atop (arrowheads, “in-groove” invasions); and 4, F-actin (phalloidin). Colors: white, ICAM1; fire, F-actin; blue, nuclei. b Projection schematics of nanotexture–T cell interactions, showing simultaneous “on-ridge” and “in-groove” dynamics. The nano-“ridge/groove” configuration supports both (1) unconfined, multidirectional “on-ridge” lamellipodium (LP), and (2) “in-groove” sterically interactive pseudopodium. “In-groove” T-cell dentations are sterically confined and are guided along the nanogrooves, driving T-cell contact guidance. c T-cell motility in 3D extracellular matrix where extension of invasive pseudopodium and rear retraction propel T cells through the 3D environment. Although 3D pseudopodia may not be confined to singular motility direction, they remain analogous to directed “in-groove” dentations. d Our model for the mechanomodulated “in-groove” (i.e., more amoeboid-like phenotype with actin-rich pseudopodia) and “on-ridge” (i.e., more mesenchymal-like phenotype, which for T cells is defined as lamellipodia driven protrusions during migration, particular on flat environments) dynamics can be competitive and regulate the amoeboid-mesenchymal plasticity balance. Our model predicts “on-ridge” mesenchymal spreading enhancement on rigid nanotopography outbalances and antagonizes “in-groove” amoeboid invasiveness. Alternatively, soft nanotextures shift the balance towards amoeboid “in-groove” steric cell invasiveness. e Metrics (left) and measurement (right) of T-cell “in-groove” relative invasiveness as a ratio between area of invasive regions and entire T-cell area at the topography interface (see Eq. in the “Methods” section). Invasiveness is a function of nanotopography rigidity G ’ and microtubule stability. G ’ range: 16–50 kPa (PAAG) to ≫1000 kPa (pU: polyurethane plastic). Remarkably, at ICAM1-PAAG nanotextures of G ’ < 16 kPa hCD4+ T cells fail to attach to the nanotopographies, indicating G ’ ≈ 16 kPa may be the lower mechanosensing limit for hCD4+ T cells on ICAM1. Individual dots correspond to the individual cells. Box plots depict the 25th percentile, median, and 75th percentile, and whiskers depict the 95% confidence intervals. Statistical tests are one-way ANOVA, Tukey’s multiple comparisons tests. All n - and p -values are shown on the plots. Number of replicates per condition: 3. Source data are provided as a Source Data file.

Article Snippet: The resultant fluorescent PAA nanopatterns of Fab anti-Fc antibody fragment protein were incubated overnight with 1 mg/mL ICAM1-Fc chimeric protein (Sino Biological, China) in cold PBS (Gibco, USA) at 4 °C, rinsed, and used for experiments.

Techniques: Imaging, Migration

Journal: Nature Communications

Article Title: Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

doi: 10.1038/s41467-021-22985-5

Figure Lengend Snippet: a Left to right: schematic, 3D micrograph, and “in-groove” cross-section and side views of human T cells on ICAM1 nanotextures (grooves/ridges widths = 800 nm). Top to bottom: control (+DMSO), destabilized MTs (+Nocodazole), and stabilized MTs (+Taxol) showing increased “in-groove” actin-rich pseudopodia in cells with destabilized MTs, in contrast to more mesenchymal-like behavior with Taxol. Nocodazole-induced MT disassembly and Taxol-induced MT stabilization are shown on immunofluorescence panels (green). Arrowheads indicate “in-groove” protrusions. Colors: light blue, polyurethane nanotexture; green, microtubules; red, F-actin; dark blue, nuclei. b T cells migration tracks on compliant ( G ’ = 16 kPa), intermediate ( G ’ = 50 kPa), or rigid ( G ’ ≫ 1000 kPa) ICAM1 nanotextures, where compliant nanotopographies enhance contact guidance. Top to bottom: T-cell migration under control (+DMSO), Nocodazole, or Taxol treatment conditions, where MT disassembly results in the enhanced directed migration across all rigidities. All n -values are shown on the plots. Number of replicates for per case: 3. Source data are provided as a Source Data file. c T-cell averaged per cell speeds on ICAM1 nanotextures of various rigidities (16, 50, and ≫1000 kPa) for control (+DMSO, top), Nocodazole (middle), and Taxol (bottom) conditions. Individual dots correspond to the individual cells. Statistical tests are one-way ANOVA, Tukey’s multiple comparisons test. All n - and p -values are depicted on the plots. Number of replicates for each condition: 3. Source data are provided as a Source Data file. d T-cell phenotype transitions from the mixed “in-groove” and “on-ridge” phenotype to the more amoeboid “in-groove” dominant phenotype after MT disassembly from Nocodazole treatment. Micrographs are generated with human CD4+ T cells on rigid polyacrylamide gels ( G ’ = 50 kPa), functionalized with human ICAM1. Sequence 1 capture (see Supplementary Movies and ) displays a well-developed lamellipodium and non-aligned motility during vehicle treatment as a control condition, whereas Nocodazole treatment induces amoeboid dynamics, pseudopodia, and migration along the nanogrooves (Sequence 2 capture; Supplementary Movies and ). Corresponding STED super-resolution images show detailed T cell–nanogrooves interactions. Arrowheads indicate lamellipodia in DMSO (left) or pseudopodia in Nocodazole (right). e Left, schematics of our model for T cell–ICAM1 nanotexture interactions. Right, mechanosensitive “in-groove” steric invasion and “on-ridge” mesenchymal-like (lamellipodial-based) interactions as a function of the mechanical rigidity of the microenvironment and cell contractility. f Quantification of directionality of human T cells migration during contact guidance as a function of substrate mechanical rigidity and the state of microtubules. Corresponding T-cell migration tracks are shown in the same matrix order in b . Measurements represent frequency distributions of cell-to-nanogroove angles every 10 s step. All n -values are indicated on the plot. Number of replicates for each condition: 3. Source data are provided as a Source Data file.

Article Snippet: The resultant fluorescent PAA nanopatterns of Fab anti-Fc antibody fragment protein were incubated overnight with 1 mg/mL ICAM1-Fc chimeric protein (Sino Biological, China) in cold PBS (Gibco, USA) at 4 °C, rinsed, and used for experiments.

Techniques: Immunofluorescence, Migration, Generated, Sequencing