Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Binding of BTLA agonist antibodies to a panel of BTLA mutants by ELISA as described in Methods. Lighter color indicates loss of binding signal to particular mutants. (B) Flow cytometric binding of BTLA agonist antibodies to BTLA mutants expressed in 293T cells. 293T cells transfected with various BTLA mutant-plasmids were used for the flow cytometry-based binding analysis. Cells were incubated with anti-BTLA clones (22B3, 6F4, and MIH26) for 40 min at 4°C. Appropriate secondary antibodies were used for detecting anti-BTLA agonist antibodies bound to mutant BTLA-expressing 293T cells. (C) Crystal structure of the human BTLA-HVEM complex: BTLA (brown) and HVEM (yellow) (2AW2.pdb). Residues that are important for HVEM and 22B3 binding are indicated. (D) Mimicry of HVEM binding by 22B3. Alignment of the 22B3:BTLA complex (green and cyan blue : brown) with the HVEM:BTLA complex (yellow : brown) shows that the CDR H3 (green sticks) is highly similar to a loop located on the CRD1 region of HVEM (yellow sticks).

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Flow Cytometry, Incubation, Clone Assay, Expressing

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Competitive binding of BTLA agonist mAbs 22B3, 25F7, and 23C8. BTLA-expressing 293T cells were used for flow cytometry-based analysis of competitive mAb binding. (Left) Competitive binding between 22B3 and 23C8. Cells were co-incubated with a fixed concentration of fluorescently labeled 22B3 (22B3-AF647) and graded concentrations of 23C8 mAb for 40 min at 4°C. (Middle) Competitive binding between 22B3 and 25F7. (Right) Competitive binding between 25F7 and 23C8. (B) Equilibrium binding analysis of 22B3, 25F7, and 23C8 to BTLA in 293T cells. BTLA-expressing 293T cells (293T-BTLA; magenta) and BTLA-HVEM co-expressing 293T cells (293T-BTLA-HVEM; green) were used for the flow cytometry-based binding analysis. Cells were incubated with graded concentrations of anti-BTLA mAb for 40 min at 4°C. and detected with an appropriate secondary antibody.

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Concentration Assay, Labeling

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines. The relative number of NF-κB reporter was determined in 293T-NFκB-HVEM and 293T-NFκB-HVEM-BTLA cells activated overnight with PMA (100 ng/ml) with the luciferase assay as described in the Materials and Methods section. NF-κB mediated luciferase activity was measured relative to the PMA activated positive control 293T-NFκB reporter cells. Each data point is the mean of 2 technical replicates. (B) Measurements of HVEM-mediated NF-κB activities were determined in the unstimulated reporter cells. Each data point is the mean of 2 technical replicates. (C) Antagonist activity of BTLA antibody 22B3 on HVEM signaling was determined in 293T-NFκB-HVEM-BTLA reporter cells. Each data point is the mean of 3 technical replicates.

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Expressing, Flow Cytometry, Luciferase, Activity Assay, Positive Control

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Proposed structural model of interaction between the HVEM-BTLA complex and anti-BTLA antibody 22B3. HVEM (salmon); BTLA 1 (magenta); BTLA 2 (yellow); 22B3 (green and blue). (B) Schematic illustration of the HVEM-BTLA cis -complex in the absence of 22B3. (C) Effect of 22B3 on HVEM-BTLA signaling. Diagrams show a top view of (B). Absence of 22B3 (left); bivalent binding of 22B3 (middle); monovalent binding of 22B3 (right).

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay

Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: Expression of PRRs by mouse and human skeletal muscle cells. Expression of PRR mRNA transcripts in mouse satellite cells (SC), FAPs (FP), endothelial cells (EC) and monocyte/macrophages (MΦ) sorted from skeletal muscles. Whole mouse bone marrow (BM) cells and splenocytes (SP) were used as positive controls for these transcripts. Human PDGFRα + FAPs were sorted from muscles surrounding NHO biopsies, and CD14 + monocytes were isolated from peripheral blood from healthy donors. A TLR1, B TLR2, C TLR3, D TLR4, E TLR5, F TLR6, G TLR7, H TLR8, I TLR9, J STING1, K RIGI, L MDA5 (IFIH1), M PRK, N NOD1, O NOD2, P Dectin-1 (CLEC7A), Q Dectin-2 (Clec4n/CLEC6A), R Mincle (CLEC4E). For each gene, left hand histogram represents expression of the mouse gene in mouse cells and the right-hand histogram represents expression of the human gene in human cells. For mouse cells, relative mRNA expression was quantified relative to house-keeping gene Hprt . For human cells, values were normalized using three references genes HPRT , RPLP0 and PPIA for FAPs and ACTB , GAPDH and PPIA for CD14 + cells. Each dot represents a mouse or a human donor, bars represent mean ± SD

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Expressing, Muscles, Isolation

Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: OSM and IL-1 neutralization in monocyte-conditioned media strongly inhibit hFAPs mineralization. A OSM, IL-1α and IL-1β concentrations quantified in conditioned media from human CD14 + macrophages stimulated with 200 ng/ml Pam2CSK4 (CM Pam2CSK4 ), 200 ng/ml Pam3CSK4 (CM Pam3CSK4 ) or non-stimulated (CM ∅ ). B OSM, IL-1α and IL-1β concentrations in CM Pam2CSK4 , CM Pam3CSK4 and CM ∅ were correlated with hFAP calcium mineralization measured by Alizarin Red staining after 2 weeks of culture in osteogenic conditions in the presence of the conditioned media. Each dot represents a different conditioned medium sample. C–F Mouse anti-human OSM antibody, isotype control antibody and IL-1RA were used to neutralize OSM and IL-1 in human CD14 + monocyte conditioned media as indicated below each chart. Calcium mineralization of hFAPs cultured for 2 weeks in osteogenic conditions with C CM Pam2CSK4 or D CM Pam3CSK4 was measured using Alizarin Red staining and quantified by spectrophotometry. E, F RUNX2 protein quantification by Western blot using Stain-Free normalization for hFAPs cultured for 2 weeks in osteogenic conditions with E CM Pam2CSK4 or F CM Pam3CSK4 . In C-F, each dot represents a hFAP individual donor. Bars represent mean ± SD, One-way ANOVA Dunnett’s multiple comparison test versus CM ∅ in ( A ) and ( B ), versus CM Pam2CSK4 in ( C ) and ( E ), or versus CM Pam3CSK4 in ( D ) and ( F ), * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Neutralization, Staining, Control, Cell Culture, Spectrophotometry, Western Blot, Comparison