Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: IDIOPATHIC NEPHROTIC SYNDROME. Baseline characteristics of patients affected by idiopathic nephrotic syndrome who underwent evaluation of serum levels of sCD40L.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques:

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A-C) Micrographs representative of immunofluorescence staining for nephrin in non-permeabilized GECs: (A) GECs incubated with vehicle alone for 30 min; (B) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (C) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg fusion protein (20 ng/ml), on loss of nephrin induced by hr-sCD40L. Original magnification: ×400 (F-H). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (D) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with various concentrations of hr-sCD40L for 30 min (upper graph), and of the time-course effect of incubation of GECs with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer; dashed bars) or vehicle alone (open bars) on nephrin expression, as detected by immunofluorescence staining (lower graph). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (E-G) Micrographs representative of fluorescein isothiocyanate phalloidin staining of actin microfilaments in permeabilized GECs. (E) GECs incubated with vehicle alone for 30 min; (F) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (G) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg (20 ng/ml), on reorganization of cytoskeleton induced by hr-sCD40L. Original magnification: ×400 (D-I). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (H) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction (dashed bars). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see for details). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (I) Immunoblot of a representative experiment on the effect of hr-sCD40L and PF on nephrin expression as detected by Western blot analysis. GEC lysates were immunoblotted with antibodies anti-nephrin or Beta-actin after incubation with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) or PF (500 ng/ml), in the presence or absence of CD40-muIg fusion protein (20 ng/ml) to inhibit CD40-CD40L interaction. Blots are representative of three independent experiments with similar results.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Immunofluorescence, Staining, Incubation, Expressing, Derivative Assay, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: P alb was determined after the incubation of rat glomeruli for 30 min with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer). A significant increase in glomerular permeability is expressed by values of P alb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to the glomeruli 30 minutes before adding hr-sCD40L (dashed bars) (see for details). At least five animals were studied per each experimental group. * P < 0.05 and ** P < 0.01 versus unstimulated controls; # P < 0.01 versus rh-sCD40L.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Incubation, Permeability

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A-F) Effect of in vivo injection of sCD40L on the glomerular expression of nephrin and podocin. (A-B) Micrographs representative of immunofluorescence staining for nephrin in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L (C) or, as negative control, 200 ng heat-inactivated sCD40L (B) (x63). Bars = 10 μm. (C) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in glomeruli from female C57BL/6 mice injected in vivo with sCD40L or, as negative control, heat-inactivated sCD40L (Ctrl). (D-E) Micrographs representative of immunofluorescence staining for podocin in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L (F) or, as negative control, 200 ng heat-inactivated sCD40L (E) (x63). Bars = 10 μm. (F) Semiquantitative analysis of podocin expression as detected by immunofluorescence staining in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L or, as negative control, 200 ng heat-inactivated sCD40L (Ctrl). Glomerular expression of nephrin and podocin was evaluated by indirect immunofluorescence by confocal microscopy on frozen kidney sections obtained twenty-four hours after injection, as detailed in the Methods section. Six animals were studied per experimental group. (G) Urine protein/creatinine ratio 24 hours after a single injection of 5% bovine serum albumin (BSA–white bars), sCD40L (black bars), or heat-inactivated sCD40L (dashed bars) in female C57BL/6 mice. Each bar is representative of a single experiment.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: In Vivo, Injection, Expressing, Immunofluorescence, Staining, Negative Control, Confocal Microscopy

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: Effect of the partially purified Permeability Factor (PF), prepared from plasmapheresis eluates from patients who presented post-transplant recurrence of FSGS, on nephrin expression in cultured GECs (A) and on Permeability activity of albumin (P alb ) in isolated rat glomeruli (B). (A) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with PF (500 ng/ml) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction. Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see for details). ** P < 0.01 versus unstimulated control and PF + inhibitors of CD40-CD40L interaction. Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (B) Permeability activity of albumin (P alb ) induced by PF on isolated rat glomeruli: P alb was determined after the incubation of rat glomeruli with PF (500 ng/ml), as detailed in the Methods section. A significant increase in glomerular permeability is expressed by values of P alb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to the glomeruli 10 minutes before adding PF. At least five animals were studied per each experimental group.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Purification, Permeability, Expressing, Cell Culture, Activity Assay, Isolation, Immunofluorescence, Staining, Incubation, Derivative Assay

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: Western blot analysis of sCD40L in PF preparations obtained from plasmapheresis eluates of different FSGS patients with elevated P alb (lanes A-F), and from a normal patient with negative P alb (lane G). In the former case, PF was prepared utilizing plasma from FSGS patients undergoing plasmapheresis and separated on Protein A at different conditions; in the second case, normal plasma without P alb activity was obtained as unbound to Protein A material. PF preparations (5μg) were immunoblotted with anti-sCD40L antibody, which identified two bands, at 17 and 34 kDa, corresponding to the monomeric and dimeric forms of the molecule, respectively. hr-sCD40L (500 ng) was used as positive control. Each lane corresponds to an individual patient.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Western Blot, Activity Assay, Positive Control

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A) Serum sCD40L levels in healthy controls and in patients with idiopathic nephrotic syndrome (NS), subdivided according to clinical features ( i . e . age at onset and response to steroids). Baseline clinical characteristic of the patients with idiopathic nephrotic syndrome are reported in . Children who presented proteinuria under the 1 st year of age underwent biopsy and had a diagnostic molecular approach for several genes implicated in NS; they were classified as congenital NS (cNS). (B) Serum sCD40L levels in healthy controls and in patients with steroid-resistant idiopathic nephrotic syndrome according to the levels of proteinuria (< or > 0.5 g/day). (C) Serum sCD40L levels in patients with nephrotic syndrome and a biopsy-proven diagnosis of FSGS or of iMN. Patients with FSGS were further subdivided according to their age in < or > 18 years. Patients with iMN were always older than 40 years.All patients had eGFR >60 ml/min at the time of serum sampling for sCD40L measurement.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Diagnostic Assay, Sampling

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Proliferative response of human B cells (A) and cytokine secretion of human MDDCs (B and C) to a dose range of various anti-hCD40 IgG4 Abs incubated with and without a constant low dose of human sCD40L. Data represent averages from seven (11B6, 12B4, CP, and IgG4) or three (12E12 and 24A3) independent experiments on two different donors normalized for maximum proliferation (80 ± 22%) versus baseline replication without Ab or sCD40L (range 6 ± 4%) or without Ab but with sCD40L (11 ± 7%). In the MDDC assay, the maximum response for each cytokine within the experiment was set at 100%. Data represent duplicate tests within two independent experiments using different donors normalized relative to the maximum secretion of each cytokine tested. All the in-house mAbs had the parental mouse mAb V region grafted to human H and L chain in C regions. Error bars are SD of the mean.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Incubation

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Inhibition of CD40L binding by anti-CD40 Abs. A titration series of anti-hCD40 human IgG4 (upper panel) or mouse (lower panel) mAbs was added to a constant amount of human CD40 ectodomain human Fc (bottom panel) or mouse Fc (upper panel) fusion protein, incubated on ice for 1 h, and then tested for binding to human CD40L stably transfected L cells. The experiment was replicated using 100 ng/ml of the CD40 Fc reagents with identical conclusions.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Inhibition, Binding Assay, Titration, Incubation, Stable Transfection, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Proliferative response of human B cells (A) and cytokine secretion responses (B and C) and activation marker mean fluorescence intensity responses (MFI, D–F) of human MDDCs to a dose range of anti-hCD40 hIgG4 mAbs with or without directly fused CD40L or incubated with a constant low dose of soluble CD40L. Data in (A) represent two independent experiments from three donors normalized for maximum proliferation versus baseline replication without Ab. The anti-hCD40 IgG4, 24A3, 12E12, and 12B4 mAbs used had the parental mouse mAb V regions grafted to human H and L chain C regions, whereas the anti-hCD40 11B6 had either the mouse (for the Ab alone) or the human (for the fusion protein Ab–CD40L) parental mAb V regions grafted to H chain IgG4 and L chain C regions. In (B) and (C), note that the data for Ab and Ab + sCD40L is identical to that represented in (Fig. 1B and (​(1C,1C, except that the scale is expanded ∼5-fold to accommodate the correspondingly higher maximal responses by the anti-hCD40–CD40L Abs. Data represent a single experiment. (D–F) represent an independent experiment with a different normal donor. The MFI data in (D)–(F) support cytokine data shown in Fig. 1. Similar data were obtained in additional experiments, including with other donors (Supplemental Fig. 2).

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Activation Assay, Marker, Fluorescence, Incubation

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Fusion of CD40L to anti-hCD40 11B6 increases both efficacy and potency of cell killing directed via engagement of CD40 ectodomain fused to FAS transmembrane and intracellular domains. CHO cells stably transfected with a construct expressing human CD40 ectodomain fused to FAS transmembrane and intracellular residues were incubated for 4 d with a dilution series of anti-hCD40 IgG4 Abs. Cells were then incubated with MTT for colorimetric detection of mitochondrial reduction activity as a surrogate for viability. Nontransfected CHO cells are not affected by any of these tested agents (not shown). Some of these data are repeated in (Fig. 6 along with additional controls and constructs.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Stable Transfection, Transfection, Construct, Expressing, Incubation, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Superagonist activity of CD40L fused to anti-hCD40 11B6 is independent of CD40L positioning. In (A), SPR analysis of soluble cohesin–human CD40 ectodomain protein (200 nM injected for 6 min plus 5-min dissociation time at 25 μl/min) binding to solid-phase anti-hCD40 Abs with or without directly linked human CD40L linked to either H or L chain C termini. CD40LL indicates fusion to the L chain, CD40LH indicates fusion to the H chain, 11B61 has mouse V regions on human IgG4 H chain and human κ L chain, and 11B62 is a variant of humanized 11B6 with an alternate VL sequence. Cartoons show the CD40L configuration in black, and F indicates constructs that use CD40L attachment via a Flex V1 linker. (B) shows cytokine secretion responses of human MDDCs to a dose range of anti-hCD40 11B6 IgG4 Abs with directly fused human CD40L fused to H or L chain C termini with and without a flexible linker region. Data represent normalized and averaged responses for TNF-α, IL-15, and IL-12p40 with two different donors. Error bars are the SEM. The cartoons shown below each titration series indicate the L chain or H chain CD40L fusion isoforms, and F indicates the presence of the Flex V1 linker; CD40L is the black domain; megaCD40L is shown as the three black domains joined; and the mAb without CD40L is CP. (C) shows the efficacy and potency of cell killing directed by engagement of human CD40 ectodomain fused to FAS transmembrane and intracellular domains on CHO cells. In the graph, h indicated humanized 11B6 mAb, and m indicates the original mouse V region. The error bars are the SD based on internal replicates of the experiment. Similar data were obtained in an independent experiment (data not shown).

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Activity Assay, Injection, Binding Assay, Variant Assay, Sequencing, Construct, Titration

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: SPR analysis of soluble CD40 ectodomain binding to solid-phase anti-hCD40 Abs with or without directly linked human CD40L. The curves shown are actual RU values normalized compared with the buffer control channels. The residual SD of the fitted curves from the actual data curves for the four panels were 3.5 (2.6% of residual SD relative to the maximum response of the curve set), 6.3 (2.7%), 2.4 (0.7%), and 20.4 (5.9%). In similar analyses for the anti-hCD40 11B6-CD40L isoforms (described in (Fig. 6), all the CD40L fusion variants retained >25% of the bound CD40 after a washout period of 250 s, indicating highly stabilized off-rates compared with the parental anti-hCD40 11B6 mAb. The mAb anti-hCD40 11B6-CD40L had the parental mouse mAb V regions grafted to human H and L chain C regions.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Binding Assay

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Anti-hCD40 11B6-CD40L enhances CD40 cluster formation. (A) MDDC were incubated in culture media for 6 h with 100 nM anti-hCD40 12E12 (top panels) or with 100 nM anti-hCD40 11B6-CD40L (bottom panels) at 37°C on cover slips. Both the mAbs used to induce CD40 cluster formation on DCs had the parental mouse mAb V regions grafted to human H and L chain C regions. (B) Human CD40-mCherrry–tagged CHO cells were incubated in culture media for 1 h alone (top panels), with 10 nM anti-hCD40 12E12 (middle panels), or with 10 nM anti-hCD40 11B6-CD40L (bottom panels) at 37°C on cover slips. (C) In the left panel, MDDC were incubated for 6 h in culture media at 37°C on cover slips with 100 nM anti-hCD40 11B6-CD40L or anti-hCD40 12E12 fused at the H chain C terminus to a flex V1 Doc Var1 module (21, 22) in stable noncovalent association with a Cohesin-eGFP fusion protein. In the right panel, human CD40-mCherry CHO cells were treated for 1 h with 10 nM of the indicated anti-hCD40 mAbs and analyzed by spatial image correlation spectroscopy. The scale difference between left and right panels is due to differences in the fluorescence intensity between the cell types because of the nature of the fluorescent probes. The statistical significance between the 11B6-CD40L and 12E12 for both panels was p < 0.0001. ****p < 0.0001, ***0.0001 < p < 0.001.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Incubation, Spectroscopy, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Anti-hCD40 11B6-CD40L enhances CD40-mediated internalization defined by resistance to acid stripping. Human CD40-CHO cells were incubated in culture medium for various times with 100 nM mCherry-labeled anti-hCD40 12E12 (A), anti-hCD40 11B6-CD40L (B), or anti-hCD40 11B6 (C) at either 37 or 0°C. Cells were then either washed with PBS or treated with isotonic acid stripping buffer (pH 2.5) for 1 min (labeled H+), neutralized, and then washed with PBS. Total or acid-resistant (i.e., presumed internalized) label was then measured every 30 min by fluorescence. Signal for total binding from the 30–270 min time points was averaged and set to 100% to normalize the data between three independent replicate experiments. Background fluorescence values with cells alone or 100 nM mCherry not conjugated to anti-hCD40 mAbs were 2 ± 1%. All the mAbs had the parental mouse mAb V regions grafted to human H and L chain C regions.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Stripping Membranes, Incubation, Labeling, Fluorescence, Binding Assay

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Fractionation of anti-hCD40 11B6-CD40 isoforms by SEC (A and B) and DC cytokine production assays (C and D) and analysis of surface activation marker mean fluorescence intensity responses (MFI) (E and F). Shown in gray are fractionation profiles of two independent anti-hCD40 11B6-CD40L forms (i) and (iv) superimposed on indicated m.w. marker protein profiles. Supplemental Fig. 3 shows a closer image of the relevant fractionation profiles, as well as SEC analysis on the same column of m.w. markers and anti-hCD40 11B6 without fused CD40L. The MDDC activation assays of dialyzed and concentrated fractions corresponding to SEC profiles (A and B) are shown in panels (C–F) as a titration series from left to right of 10, 1, 0.1, and 0.01 nM.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Fractionation, Activation Assay, Marker, Fluorescence, Titration

Journal: The Journal of Immunology Author Choice

Article Title: Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties

doi: 10.4049/jimmunol.2000704

Figure Lengend Snippet: Effects on immune cells and serum cytokines of anti-hCD40 11B6, anti-hCD40 11B6-CD40L, and anti-hCD40 CP 4 h after i.p. injection to human CD40 transgenic mice. Control mice were left untreated. (A) Immune cells within PBMCs and splenocytes were stained with cell type and cell activation markers and then analyzed by flow cytometry. (B) Cytokine production from blood was analyzed. Data are pooled from three independent experiments using identical reagents, procedures, and personnel as limited by animal handling constraints. All test mAbs were formatted on human IgG4 H chain and human κ L chain C regions. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, p ≡ 0.05.

Article Snippet: Abbreviations used in this article anti-hCD40 anti-human CD40 BD Becton Dickinson CD40L CD40 ligand CHO-S Chinese hamster ovary subline S CP CP-870, 893 DC dendritic cell hIgG1 human IgG1 MDDC human myeloid-derived DC ref reference RT room temperature RU resonance unit sCD40L monomeric soluble CD40L SEC size-exclusion chromatography SPR surface plasmon resonance

Techniques: Injection, Transgenic Assay, Staining, Activation Assay, Flow Cytometry

Journal:

Article Title: CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

doi: 10.1046/j.1365-2249.1999.00764.x

Figure Lengend Snippet: The effect of CD40 engagement on IL-6 production by human gingival fibroblasts (HGF). HGF (5 × 104/well) were stimulated for 72 h with 30 Gy-irradiated CD40L+ Jurkat J39.8/50 cells or CD40L− Jurkat SPV.WT cells (1 × 104 cells/well) (a) or sCD40L (1 μg/ml) (d) in the absence or presence of IFN-γ (1000 U/ml). Where indicated, blocking CD40 MoAb (500 pg/ml) (b) or CD40L MoAb (500 pg/ml) (c) were added to the cultures stimulated with CD40L+ Jurkat cells in the presence of IFN-γ. Results in (a) are representative of nine and results in (b,c,d) are representative of two independent experiments. All results are expressed as the mean value of triplicate cultures. Error bars indicate the s.d. *P < 0.001.

Article Snippet: The following stimuli were used: 30 Gy-irradiated CD40L + Jurkat J39.8/50 cells or CD40L − Jurkat SPV.WT cells (1 × 10 4 cells/well) or soluble trimeric human rCD40L (sCD40L) (1 μg/ml; kindly provided by Immunex Research and Development Corp.).

Techniques: Irradiation, Blocking Assay

Journal:

Article Title: CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

doi: 10.1046/j.1365-2249.1999.00764.x

Figure Lengend Snippet: The effect of CD40 engagement on IL-8 production by human gingival fibroblasts (HGF). HGF (5 × 104/well) were co-cultured for 72 h with 30 Gy-irradiated CD40L+ Jurkat J39.8/50 cells or CD40L− Jurkat SPV.WT cells (1 × 104 cells/well) (a) or sCD40L (1 μg/ml) (d) in the absence or presence of IFN-γ (1000 U/ml). Where indicated, blocking CD40 MoAb (500 pg/ml) (b) or CD40L MoAb (500 pg/ml) (d) were added to the cultures stimulated with CD40L+ Jurkatt cells in the presence of IFN-γ. Results in (a) are representative of nine and results in (b,c,d) are representative of two independent experiments. All results are expressed as the mean value of triplicate cultures. Error bars indicate the s.d. *P < 0.001.

Article Snippet: The following stimuli were used: 30 Gy-irradiated CD40L + Jurkat J39.8/50 cells or CD40L − Jurkat SPV.WT cells (1 × 10 4 cells/well) or soluble trimeric human rCD40L (sCD40L) (1 μg/ml; kindly provided by Immunex Research and Development Corp.).

Techniques: Cell Culture, Irradiation, Blocking Assay

Journal:

Article Title: CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

doi: 10.1046/j.1365-2249.1999.00764.x

Figure Lengend Snippet: The effect of CD40 engagement on matrix metalloproteinase (MMP) production by human gingival fibroblasts (HGF) in the presence of IFN-γ. HGF (5 × 105/well) were stimulated for 72 h with or without sCD40L (1 μg/ml) in the absence or presence of IFN-γ (1000 U/ml). Cell-free supernatants were analysed by Westen blotting for MMP-1, MMP-2, MMP-3 and TIMP-1 protein expression. Data shown are representative of four independent experiments in which HGF maintained in medium alone (no cytokine) showed a detectable level of MMP-3 expression in Western blot. Isotype-matched control antibodies did not detect any immunoreactive bands (data not shown).

Article Snippet: The following stimuli were used: 30 Gy-irradiated CD40L + Jurkat J39.8/50 cells or CD40L − Jurkat SPV.WT cells (1 × 10 4 cells/well) or soluble trimeric human rCD40L (sCD40L) (1 μg/ml; kindly provided by Immunex Research and Development Corp.).

Techniques: Expressing, Western Blot, Control

Journal:

Article Title: CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

doi: 10.1046/j.1365-2249.1999.00764.x

Figure Lengend Snippet: The effect of CD40 engagement on matrix metalloproteinase (MMP) production by human gingival fibroblasts (HGF) in the presence of IL-1β or tumour necrosis factor-alpha (TNF-α). HGF (5 × 105/well) were stimulated for 72 h with or without sCD40L (1 μg/ml) in the absence or presence of IL-1β (100 U/ml) or TNF-α (10 ng/ml). Cell-free supernatants were analysed by Westen blotting for MMP-1, MMP-2, MMP-3 and TIMP-1 protein expression. Data shown are representative of four independent experiments in which HGF maintained in medium alone (no cytokine) showed an variable detectable level of MMP-3 expression in Western blot. Isotype-matched control antibodies did not detect any immunoreactive bands (data not shown).

Article Snippet: The following stimuli were used: 30 Gy-irradiated CD40L + Jurkat J39.8/50 cells or CD40L − Jurkat SPV.WT cells (1 × 10 4 cells/well) or soluble trimeric human rCD40L (sCD40L) (1 μg/ml; kindly provided by Immunex Research and Development Corp.).

Techniques: Expressing, Western Blot, Control