Journal: bioRxiv

Article Title: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

doi: 10.1101/2021.07.12.452090

Figure Lengend Snippet: A. The ability of CTLA-4 mAbs to block binding of CD80 or CD86 was measured using a plate-based ELISA method. CTLA-4 was used to coat the plate, then after the antibody samples were incubated, His-tagged CD80 or CD86 was added, and the amount of ligand able to bind to CTLA-4 was measured. Blocking mAbs that prevent CD80 or CD86 from binding to CTLA-4 reduce the absorbance signal due to lack of CD80 or CD86 binding to CTLA-4. Weak-blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86 compared to ipilimumab and CTLA-4.28, as assessed by ELISA as described in (A). Absorbance values were normalized to an anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. C-D. The key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4, followed by staining and flow cytometry assessment of binding. C. Shown is the crystal structure (Protein Database [PDB] 1I8L) of the complex between CD80 (blue) and CTLA-4 (gray) on which the CTLA-4 epitope residues shared between ipilimumab and GIGA-564 were colored orange and the key differentiating residue R70 was colored red (visualized with Pymol). Additionally, G142 was identified as a secondary residue for the epitope of ipilimumab but not GIGA-564. D. Table showing key amino acids on CTLA-4 of interest for these epitopes; those found by mutational analysis to be important for binding of CTLA-4 to CD80 or CD86 in a cell-based assay are marked in gray to indicate the epitope residues for those proteins .

Article Snippet: Recombinant human His-tagged CD80 (R&D Systems 9050-B1, Minneapolis, MN, USA) or CD86 (R&D Systems 9090-B2, Minneapolis, MN, USA) was then added to the plates at 1 μg/mL.

Techniques: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Mutagenesis, Staining, Flow Cytometry, Residue, Cell Based Assay

Journal: bioRxiv

Article Title: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

doi: 10.1101/2021.07.12.452090

Figure Lengend Snippet: Top panel: Ipilimumab blocks CTLA-4 interaction with CD80/CD86, which allows antigen presenting cells (APCs) to co-stimulate peripheral Tregs enhancing their proliferation. GIGA-564 weakly blocks CTLA-4 interaction with CD80/CD86 and thus induces less Treg proliferation. Bottom panel: Ipilimumab and GIGA-564 bind CTLA-4 on intratumoral Tregs to induce Treg killing via interactions with Fc receptor (FcR) on effector cells. GIGA-564 induces stronger FcR signaling and thus more efficiently depletes intratumoral Tregs than ipilimumab.

Article Snippet: Recombinant human His-tagged CD80 (R&D Systems 9050-B1, Minneapolis, MN, USA) or CD86 (R&D Systems 9090-B2, Minneapolis, MN, USA) was then added to the plates at 1 μg/mL.

Techniques:

Journal: Journal for immunotherapy of cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: Figure 4 In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/ mL solution of recombinant human CD80 or CD86 (9050- B1- 100 or 9090- B2- 100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 1 The distribution of sCD80 levels in 153 apparently healthy individuals. The concentration of sCD80 was evaluated using an ELISA and recombinant CD80-Ig as a standard.

Article Snippet: A standard curve was generated using dilutions of the commercially available recombinant human CD80 ⁄ Fc chimera (R&D Systems).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 2 Alternatively spliced CD80 mRNA. (A) Splice variants of human CD80 mRNA. The markers: *** or ### represent the spliced nucleotides for s1CD80 or s2CD80 respectively. The sequence data are available from GenBank under accession numbers ban- kit512201 and bankit508046. (B) Schematic presentation of the alternative splicing of CD80 mRNA. CD80mRNA consists of four exons: IgV, IgC, transmembrane (TM) and intracellular. The transmembrane domain is spliced out in s1CD80, while both the IgC and TM domains are spliced out in s2CD80. (C) The expression of different CD80 mRNA isoforms in fresh and stimulated B cells, T cells and monocytes is shown. Purified T cells, B cells and monocytes were stimulated for 24 h by ConA, LPS or IFN-c respectively. RNA integrity and cDNA synthesis was veri- fied by amplifying GAPDH cDNA. Mw, molecular weight markers; T, T lymphocytes; Ta, activated T lymphocytes; M, monocytes; Ma, activated monocytes; B, B lymphocytes; Ba, activated B lymphocytes.

Article Snippet: A standard curve was generated using dilutions of the commercially available recombinant human CD80 ⁄ Fc chimera (R&D Systems).

Techniques: Sequencing, Alternative Splicing, Expressing, cDNA Synthesis, Molecular Weight

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 3 Demonstration of Rs1CD80 using Western blot and Coomas- sie staining. Western blot (left part) and Coomassie staining (right part) of purified Rs1CD80 under reducing conditions (lanes 3 and 4). Lanes 1 and 5 represent molecular weight markers (Invitrogen) and lanes 2 and 6 commercially available CD80-Ig (R&D Systems).

Article Snippet: A standard curve was generated using dilutions of the commercially available recombinant human CD80 ⁄ Fc chimera (R&D Systems).

Techniques: Western Blot, Staining, Molecular Weight

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 1 Pre-engagement of Ipilimumab and Tremelimumab with soluble CTLA-4-Ig prevents CTLA4 binding to cell bound CD80 or CD86 ligand. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was incubated on ice with a titration of soluble anti- CTLA-4 Abs to pre-engage CTLA-4 with anti-CTLA-4. This was added to DG75 B cells expressing either CD80 or CD86-GFP and incubated on ice for 30 minutes. Cells were washed and analysed for Abatacept-ligand interaction by flow cytometry. All reagents were chilled on ice before use. (B) Representative concatenated FACS plots show impact of Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab(Ipi) (Black line) and tremelimumab (Treme) (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 2 Ipilimumab and Tremelimumab outcompete cell bound CD80 or CD86 for engagement with soluble CTLA-4-Ig. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were mixed with a titration of soluble anti-CTLA-4 Abs. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was added to assess direct competition between soluble anti-CTLA-4 and cell bound ligand for Abatacept binding. Cells were incubated on ice, washed and Abatacept-ligand engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti- CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Binding Assay, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 3 Ipilimumab and Tremelimumab are unable to displace pre-engaged CTLA-4-Ig from cell bound CD80 or CD86. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were pre-incubated on ice with a fixed dose (2µg/ml) of soluble APC conjugated Abatacept to pre-engage soluble CTLA-4 with ligand. Cells were washed, treated with a titration of soluble anti-CTLA-4 Abs and incubated on ice or at 37°C. Cells were washed and analysed for Abatacept-ligand engagement by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show Abatacept-ligand binding at different doses of anti-CTLA-4 Abs when incubated on ice or when incubated at 37°C. (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI CTLA-4-Ig binding from data shown in (B) Data presented are mean +/- SD from 3-6 independent experiments. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Incubation, Titration, Cytometry, Ligand Binding Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 4 Pre-engagement of cell bound CTLA-4 with soluble CD80 and CD86Ig inhibits Ipilimumab and Tremelimumab from binding. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-incubated on ice with either soluble CD80 or CD86Ig to pre-engage CTLA-4 with ligand. Cells were washed, incubated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs and incubated on ice. Cells were washed and analysed for anti-CTLA-4-CTLA-4 binding by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. CD80 or CD86Ig doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Expressing, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 5 Soluble CD80 and CD86Ig outcompete Ipilimumab and Tremelimumab for engagement with cell bound CTLA4. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs was pre-incubated with a titration of either soluble CD80 or CD86Ig on ice. Jurkat expressing CTLA-4 cells were added to facilitate direct competition between anti-CTLA4 Abs and CD80 or CD86Ig for cell bound CTLA-4 occupancy. Cells were incubated on ice, washed and anti-CTLA4-CTLA4 engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 6 Soluble CD80 and CD86Ig are unable to displace pre-engaged Ipilimumab and Tremelimumab from cell bound CTLA-4. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-treated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA-4 Abs on ice to pre-engage anti-CTLA-4-CTLA-4. Cells were washed and a titration of either soluble CD80 or CD86Ig was added. Cells expressing WT CTLA-4 were incubated on ice and cells expressing a non-internalising CTLA-4 mutant (Del 36) were incubated at either on ice or at 37°C. Cells were washed and analysed for anti-CTLA-4-CTLA-4 staining by flow cytometry. (B) Concatenated FACS plots showinganti-CTLA-4 binding at various doses of CD80 or CD86Ig (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI anti- CTLA-4 binding from data in (B) Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Incubation, Mutagenesis, Staining, Cytometry, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 7 Ipilimumab and Tremelimumab block CD80 and CD86 ligand loss for APC by transendocytosis. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 5 hours at 37°C. A titration of soluble ipilimumab (Black line) or tremelimumab (Blue line) were added at 0h. Percentage of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI of CTV+ CHO cells relative to a negative control where no CTLA-4 was present. (B) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log EC50 for Iipilimumab (Black line) and tremelimumab (Blue line). (C) Graphs show the mean EC50 with 95% confidence interval calculated using Prism v6. (D-F) As in A, B & C except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates, data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Blocking Assay, Staining, Expressing, Cell Culture, Titration, Negative Control, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 8 Ipilimumab and Tremelimumab interfere with pre-established transendocytosis of CD80 and CD86 to prevent further ligand loss from APC. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 0, 3 or 6 hours at 37°C. Transendocytosis was established for 3 hours in the absence of anti-CTLA-4 Abs at which point soluble ipilimumab (dotted black line) or tremelimumab (dotted blue line) were spiked into culture. Transendocytosis was continued for a further 3 hours to observe the effect of anti-CTLA4 spike. Soluble anti-CTLA-4 antibodies were also added at timepoint 0h to block transendocytosis from the start of the assay (solid black – ipilimumab or blue – tremelimumab lines). Transendocytosis was also performed in the absence of anti-CTLA-4 treatment (Purple line). (B) % of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI CTV+ CHO cells relative to a negative control where CTLA4 was not expressed (n=3). (C, D) As in A & B except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP cultured at a Jurkat:DG75 ratio of 2:1. Also an additional spike was performed at 6h and the assay was run for a total of 20h. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=2).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Staining, Expressing, Cell Culture, Blocking Assay, Negative Control

Journal: Cellular immunology

Article Title: Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma

doi: 10.1016/j.cellimm.2017.07.002

Figure Lengend Snippet: A–C) Purified CD28-deficient CD4+ T cells were retrovirally transduced with either empty vector (RV), CD28-F51I, CD28-F51V or the CTLA4-CD28 chimera and cocultured with CHO cells expressing either IAd alone (panel A), or coexpressing human CD80 (panel B) or human CD86 (panel C) and stimulated with OVA peptide at the indicated doses. D and E) Purified CD4+ cells as described above were stimulated with plate bound anti-CD3 (.01 μg/ml) alone or in combination with graded doses of plate bound human CD80Ig or CD86Ig and proliferation determined. For all panels, the data shown are the mean ± standard deviation of triplicate wells and are representative of 3–5 independent experiments. Statistical comparisons were made comparing the mutant constructs to the wild type construct, and the wild type to empty vector. **= p<.001 by 2 tailed, multiple comparison, unpaired T testing. Differences were significant at all doses.

Article Snippet: Chinese Hamster Ovary cells (CHO) expressing IA d (gift from Dr K. Murphy, Washington University School of Medicine, St Louis, MO) were stably transfected with plasmids expressing either human CD80 or CD86 (Sino Biologicals, China).

Techniques: Purification, Transduction, Plasmid Preparation, Expressing, Standard Deviation, Mutagenesis, Construct

Journal: Cellular immunology

Article Title: Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma

doi: 10.1016/j.cellimm.2017.07.002

Figure Lengend Snippet: A) Retrovirally transduced CD28-deficient splenocytes were stimulated with OVA (0.03 μM) alone or in the presence of CTLA4Ig at doses ranging from 1–1000 μg/ml and proliferation determined. The percent inhibition at each dose of CTLA4Ig was calculated relative to proliferation in the absence of CTLA4Ig. Results shown are representative results from 3 independent experiments. B) Cells were stimulated as above with the addition of either anti-CD80 or anti-CD86 antibody (10 μg/ml), and proliferation measured. *= p <.05 by 2-tailed t-test adjusted for multiple comparisons, comparing anti-CD86 treatment to either OVA alone or OVA plus anti-CD80. No other comparisons were significantly different. Results shown are representative results from 3 independent experiments.

Article Snippet: Chinese Hamster Ovary cells (CHO) expressing IA d (gift from Dr K. Murphy, Washington University School of Medicine, St Louis, MO) were stably transfected with plasmids expressing either human CD80 or CD86 (Sino Biologicals, China).

Techniques: Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Schematic indicating the results of high-throughput cell-cell screening of B7-1 and p75 NTR against 395 members of the human Ig and TNFR superfamilies. B7-1–expressing cells bound to cells expressing CD28, CTLA-4, and p75 NTR , but not cells expressing PD-L1. p75 NTR –expressing cells bound to cells expressing B7-1 and PTPRF. ( B ) Representative flow plots demonstrating that hB7-1–mCherry cells and PTPRF-mCherry cells bind to cells expressing p75 NTR -GFP cells, but not GFP control cells. ( C ) Binding between B7-1 and p75 NTR was validated by recombinant protein titrations of p75 NTR -mIgG2A and CTLA-4–mIgG2A onto streptavidin beads coated with B7-1–hIgG1 biotin. Binding was detected using an anti-mIgG2A antibody, flow cytometry and binding curves were generated, and B max , EC 50 , and Hill coefficient (coeff.) were calculated using the equation Y = B max × X h /( Kd h + X h ). n = 2–3. Error bars represent SEM. ( D ) Cells expressing B7-1–mCherry from various mammalian species were screened against human p75 NTR -GFP–expressing cells (top left, n = 2–4), mouse p75 NTR -GFP–expressing cells (top right, n = 2–3), rat p75 NTR -GFP–expressing cells (bottom left n = 2–3), or cells expressing only GFP (bottom right, n = 3), indicating that B7-1:p75 NTR interactions are conserved in primates, but not mice. **** P < 0.0001, 1-way ANOVA with multiple comparisons. Each assay represents the indicated number of independent experiments, which each include a single replicate. For mouse B7-1, rat B7-1, and mCherry cells alone, only 2 independent experiments were conducted, each with 2 biological replicates.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: High Throughput Screening Assay, Expressing, Binding Assay, Recombinant, Flow Cytometry, Generated

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) hB7-1 residues that when mutated showed more than 25% losses in binding to CD28 (blue) or both CD28 and CTLA-4 (green) mapped onto crystal structure of hB7-1 (PDB: 1I8L) ( B ) hB7-1 residues that when mutated showed more than 25% losses in binding to p75 NTR (red) mapped onto crystal structure of hB7-1 (PDB: 1I8L). ( C ) Same residues as in B , except with monomer of CTLA-4 shown. ( D ) CD28-hIgG1 competes for binding to B7-1 with p75 NTR . n = 6. ( E ) CTLA-4–mIgG2A competes for binding to B7-1 with p75 NTR . n = 3. Each assay represents the indicated number of independent experiments, which each include a single replicate. Error bars represent SEM.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) p75 NTR residues identified as important for hB7-1 binding mapped onto crystal structure of rat p75 NTR bound to NT3 (PDB: 3BUK) affect binding to B7-1 (red), make contacts with NT3 and also affect binding to B7-1 (brown), and make H bond contacts with NT3 (blue). ( B ) The hB7-1–binding site modeled onto the dimeric structure of p75 NTR bound to an NT3 dimer (PDB: 3BUK). ( C ) Ligand-competition experiments showing that NGF and hB7-1 compete for binding to p75 NTR at high concentrations. n = 3. ( D ) Cells expressing p75 NTR -F136K and F136H demonstrate binding to B7-1N82E–expressing cells as well as WT B7-1–expressing cells. **** P < 0.0001, 1-way ANOVA with multiple comparisons. n = 4. ( E ) Crystal structures of hB7-1 and p75 NTR indicating proposed interaction between p75 NTR -F136 and B7-1–N82. Each assay represents the indicated number of independent experiments, which each include a single replicate. Error bars represent SEM.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Binding Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Representative IF images of WT hippocampal neurons treated for 2 hours with proNGF (10 nM), hB7-1–Fc (750 nM), or hB7-2–Fc (750 nM) and stained for actin, PSD95, and MAP2. ( B ) Quantification of PSD95 puncta density with indicated treatments. Recombinant hB7-1–Fc but not hB7-2–Fc induced a decrease in PSD95 density in cultured neurons, similarly to proNGF treatment. ( C ) Representative IF images of p75 –/– hippocampal neurons treated with proNGF (10 nM), hB7-1–Fc (750 nM), or hB7-2–Fc (750 nM) and stained for actin, PSD95, and MAP2. ( D ) Quantification of PSD95 puncta density with indicated treatment. Recombinant hB7-1–Fc failed to induce a decrease in PSD95 density in cultured neurons. ( E ) Representative IF images of WT hippocampal neurons treated with hB7-1–Fc (750 nM) or hB7-2–Fc (750 nM) and stained for actin, GluR1, bassoon, and MAP2. ( F ) Quantification of bassoon puncta density with indicated treatment. ( G ) Quantification of GluR1 puncta density with indicated treatment. ** P < 0.01, 1-way ANOVA with Dunnett’s multiple-comparisons test. n = 3 independent experiments. Experiments under each condition were performed in triplicate, and 13 to 15 dendritic segments were analyzed per condition. Scale bar: 20 μm. Error bars represent SEM. See also .

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Staining, Recombinant, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Representative IF images of hippocampal neurons cocultured with HEK293–hB7-1, HEK293–hB7-2, or HEK293–hB7-1N82E. Differences in ( B ) PSD95 density and ( C ) MAP2 morphology in dendrites in direct contact with HEK-hB7 cell lines were quantified. ( D ) Representative IF images of hippocampal neurons cocultured with HEK293–hB7-1, HEK293–hB7-2, or HEK293–hB7-1N82E stained for bassoon, mCherry, MAP2, and actin. ( E ) Quantification of bassoon puncta density with indicated treatment. *** P < 0.001; **** P < 0.0001, 1-way ANOVA, Tukey’s multiple-comparisons test. n = 3 independent experiments. Experiments under each condition were performed in triplicate, and 13 to 15 dendritic segments were analyzed per condition. Error bars represent SEM. Scale bar: 20 μm.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Representative IF images of WT hippocampal neurons cocultured with HEK293–hB7-1, HEK293–hB7-2, or HEK293–hB7–1N82E treated with or without 375 nM abatacept. Differences in ( B ) MAP2 morphology in dendrites in direct contact with HEK-hB7 cell lines were quantified. Treatment with 375 nM abatacept could prevent the MAP2-positive process degeneration induced by the HEK293–B7-1 cell line. *** P < 0.001, 2-way ANOVA. n = 3 independent experiments in which 12 to 14 individual neurons were analyzed per treatment per experiment. Scale bar: 20 μm. ( C ) Representative IF images of p75 –/– hippocampal neurons cocultured with HEK293–B7-1, HEK293–B7-2, or HEK293–B7-1N82E treated with or without 375 nM abatacept. ( D ) No differences in MAP2 morphology in dendrites in direct contact with HEK-B7 cell lines (HEK293–B7-1, HEK293–B7-2, or HEK293–B7-1N82E treated with or without 375 nM abatacept) were found. P = 0.1860, 2-way ANOVA. n = 3 independent experiments. P < 0.01, 2-way ANOVA with multiple comparisons (WT coculture). Scale bar: 20 μm. See also . n = 3 independent experiments in which 12 to 14 individual neurons were analyzed per treatment per experiment. Error bars represent SEM.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Cells were harvested at 24 hours after stimulation and stained for hB7-1 using an Alexa Fluor 488–conjugated anti-human B7-1 antibody. Antibody-stained THP-1 cells were analyzed by flow cytometry. Representative FACS plot of PMA-treated THP-1 cells (gray), THP-1 cells treated with both PMA and LPS (red), and HEK293 cells stably overexpressing hB7-1 (blue). ( B ) Quantification of n = 2 flow cytometry experiments. ( C ) Representative IF images of hippocampal neurons cocultured with Thp1 cells treated with PMA with or without LPS (1 μg/ml) with or without abatacept (375 nM). Original magnification, 43.7 × 16.09. ( D ) Differences in MAP2 morphology in dendrites in direct contact with THP-1 were quantified. ** P < 0.001, 1-way ANOVA, Tukey’s multiple-comparisons test. n = 3 independent experiments. Each treatment was performed in triplicate, and 10 to 15 dendrites were analyzed for each condition. Error bars represent SEM.

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Staining, Flow Cytometry, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor

doi: 10.1172/JCI157002

Figure Lengend Snippet: ( A ) Golgi-stained WT pyramidal neurons 3 hours after in vivo injection of hB7-1–Fc (100 ng), hB7-2–Fc (100 ng), or saline into the dSubiculum area of the hippocampal formation at P75. ( B ) There was a significant reduction in total spine density after 3 hours of hB7-1, compared with saline and the hB7-2 group. n = 4 mice/condition. ( C ) Golgi-stained p75 –/– pyramidal neurons 3 hours after in vivo injection of hB7-1–Fc (100 ng), hB7-2–Fc (100 ng), or saline into the dSubiculum area of the hippocampal formation at P75. n = 4 mice/condition ( D ) There were no significant changes in total spine density after 3 hours for the hB7-1 or hB7-2 group compared with the saline group. n = 4 mice/condition. ( E ) Golgi-stained WT pyramidal neurons 3 hours after in vivo injection of hB7-1–Fc (200 ng), hB7-2–Fc (200 ng), or saline into the dSubiculum area of the hippocampal formation at P75. ( F ) There was a significant reduction in total spine density after 3 hours of hB7-1, compared with the saline and hB7-2 groups. n = 4 mice/condition. ( G ) Golgi-stained WT pyramidal neurons 24 hours after in vivo injection of hB7-1–Fc (200 ng), hB7-2–Fc (200 ng), or saline into the dSubiculum area of the hippocampal formation at P75. ( H ) There was a significant reduction in total spine density after 24 hours of hB7-1, compared with the saline and hB7-2 groups. n = 4 mice per condition. The apical dendrite segment 50 to 150 μm away from the cell soma was chosen for quantification. * P < 0.05, 1-way ANOVA, post hoc Tukey’s test. Scale bar: 20 μm. 15–20 neurons/brain. Data are represented as mean ± SEM. Error bars represent SEM. See also .

Article Snippet: C57BL/6 mice (2.5 months old) were used for stereotactic microinjection of hB7-1–Fc or hB7-2–Fc (100 ng; Sino Biological; 10698-H03H and 10699-H03H).

Techniques: Staining, In Vivo, Injection