Journal: PLoS Medicine

Article Title: Inhibition of Endothelial Activation: A New Way to Treat Cerebral Malaria?

doi: 10.1371/journal.pmed.0020245

Figure Lengend Snippet: HBEC-5i were left unstimulated or were activated with LMP-420 alone, TNF alone, TNF with LMP-420, LT alone, or LT with LMP-420. After an overnight incubation, cells were detached and analyzed by flow cytometry after immunolabeling for ICAM-1, VCAM-1, and CD40. Results are expressed as dotplots of mean fluorescence intensity of surface expression of the antigens measured in six experiments (Dunn's test, ** corresponds to p < 0.01).

Article Snippet: HBEC-5i were then harvested and labeled by indirect labeling using mouse anti-human CD54 (ICAM-1 [84H10]) and CD106 (VCAM-1 [1G1]) antibodies (Beckman-Coulter Immunotech, Marseille, France), CD40 monoclonal antibody (mAb) (B-B20, Diaclone, Besançon, France), and CD36 mAb (FA6–152, gift from L. Edelman, Institut Pasteur Paris) as the first step.

Techniques: Incubation, Flow Cytometry, Immunolabeling, Fluorescence, Expressing

Journal: Cancer Immunology Research

Article Title: Using Antigen-Specific B Cells to Combine Antibody and T Cell–Based Cancer Immunotherapy

doi: 10.1158/2326-6066.cir-16-0236

Figure Lengend Snippet: Figure 2. Antigen-specific CD40B cells induce a specific T-cell response. A, Purified OVA-B cells were stimulated with the CD40 ligand and analyzed for the expression of different activation markers. The MFI was normalized to values of the same population before stimulation. Histograms show representative analyses compared with anisotype control. B, In antigen-presentation assays,T cells from OT-II or OT-I mice were cocultured with different unpulsed or protein-pulsed APCs in a ratio 1:1, i.e., polyclonal (Poly) CD40B cells, OVA-CD40B cells, and CD40L-matured dendritic cells (CD40 DC). T-cell proliferation was determined by a decrease of CFSE intensity as shown in representative CFSE plots. C–E, Mice were immunized with protein-pulsed APCs, i.e., CD19þ-enriched polyclonal CD40B cells (Poly), CD19þ-enriched OVA-CD40B cells (OVA), or DCs. PBSþ IFA served as negative control (Neg). C, The specific lysis of peptide-pulsed target cells was determined by a decrease of the CFSE signaling in flow cytometry analyses. D, CD3þ CD8þ T cells from vaccinated mice were stained for the presence of OVA peptide-specific T cells. E, Specific lysis after injection of different types of protein-pulsed APCs at different concentrations. Bar charts show mean values þ SD of four independent experiments. Significant differences calculated with (A þ E) a two-tailed t test (B–D) or ordinary one-way ANOVA with multiple comparisons are marked by an asterisk. ns, not significant; , P 0.02; , P 0.0035; , P 0.0006; , P 0.0001.

Article Snippet: Generation of antibody-secreting plasma cells For in vitro generation of antibody-secreting plasma cells, B cells from mice were resuspended at a concentration of 1 106 cells/ mL DMEM medium (Life Technologies) supplemented with different stimuli, including IL4 (1 U/mL, Immunotools), IL21 (50 ng/mL, Immunotools), antibody to mouse CD40 (1 mg/mL, Acris Antibodies), and OVA-biotin tetramers (0.2 mg/mL OVABiotin þ 0.5 mg/mL streptavidin-PE, BioLegend).

Techniques: Expressing, Activation Assay, Control, Immunopeptidomics, Negative Control, Lysis, Cytometry, Staining, Injection, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: Using Antigen-Specific B Cells to Combine Antibody and T Cell–Based Cancer Immunotherapy

doi: 10.1158/2326-6066.cir-16-0236

Figure Lengend Snippet: Figure 4. Stimulation of OVA-B cells with IL21 leads to PC differentiation. OVA-B cells (OVA) of immunized mice or na€ve B cells of nonimmunized mice (Polyclonal) were stimulated with the indicated stimuli for three days. A, Cells were analyzed by flow cytometry for expression of CD138. B, The amount of anti-OVA IgG1 was determined by ELISA. Bar charts show mean values þ SD in three independent experiments. Significant differences calculated with two-way ANOVA are marked by an asterisk. , P 0.02; , P 0.01. C, Anti-OVA IgG2b, IgG3 and IgM titers in supernatants of cultures were determined by ELISA. Polyclonal B cells were treated with LPS and served as positive control. Mean values of three independent experiments of absorption at 450 nm at different 10-fold dilutions are shown. D, The affinity of anti-OVA IgG1 antibodies in supernatants of cultures that were stimulated with OVA þ IL4 þ IL21 þ anti-CD40 was determined by calculating the LogIC50 from competition ELISAs with decreasing concentrations of soluble OVA (10-fold dilution). Shown are mean values of 10 independent experiments. E and F, Supernatants of OVA-B-cell cultures that were stimulated with OVA þ IL4 þ IL21 þ anti-CD40 were incubated with splenocytes from C57BL/6 mice and OVA-expressing EG.7 cells. EG.7 cells þ splenocytes were used as negative controls (Neg. Control). E, The percentages of CD69þ activated NK1.1þ CD3 cells were determined by flow cytometry after 3 hours. Values were normalized to internal control without EG.7 target cells. Shown are mean values þ SD of five independent experiments. Significant differences calculated with two-tailed t test are marked by an asterisk. , P 0.0001. F, The percentage of 7AADþ EG.7 cells was determined by flow cytometry after 24 hours. 2% DMSO were used as positive control; an irrelevant IgG1 antibody was used as additional negative control. Shown are mean values þ SD of five independent experiments. Significant differences calculated with one-way ANOVA are marked by an asterisk. , P 0.0001.

Article Snippet: Generation of antibody-secreting plasma cells For in vitro generation of antibody-secreting plasma cells, B cells from mice were resuspended at a concentration of 1 106 cells/ mL DMEM medium (Life Technologies) supplemented with different stimuli, including IL4 (1 U/mL, Immunotools), IL21 (50 ng/mL, Immunotools), antibody to mouse CD40 (1 mg/mL, Acris Antibodies), and OVA-biotin tetramers (0.2 mg/mL OVABiotin þ 0.5 mg/mL streptavidin-PE, BioLegend).

Techniques: Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Positive Control, Incubation, Control, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: Divergent hIgG agonism and specific FcγR-binding requirement. a Diagram showing the OVA-specific CD8 + T-cell response model. In brief, FcγR-humanized (hFCGR Tg ) or -deficient (FcγRα −/− ) mice were adoptively transferred with OT-I cells on day-1, immunized intraperitoneally (i.p.) with 2 μg of DEC-OVA in the presence of control or anti-CD40 antibodies on day 0. Splenocytes were harvested to quantify OVA-specific CD8 + T cells on day 6. b , c Representative FACS profile ( b ) and quantification ( c ) showing the percentage of OT-I cells (CD45.1 + TCRVα2 + ) among CD8 + T cells in mice treated and analyzed as in ( a ) together with 30 μg of indicated control or anti-mCD40 antibodies. Numbers of mice: b , c three hFCGR Tg mice for Ctrl IgG, five hFCGR Tg , and three FcγRα −/− mice for other groups. d – f Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells ( d , e ) or cell count ( f ) in FcγR-humanized mice treated and analyzed as in ( a ) together with 10 μg ( d , e ) or 30 μg ( f ) of indicated control or anti-mCD40 antibodies (the N297A mutation abrogates Fc–FcγR binding) and with/without FcγRIIB-blocking antibody 2B6 (150 μg per mouse) ( f ). Numbers of mice: d four mice per group; e five mice per group; f two mice for Ctrl IgG, five mice for αmCD40:G2, six mice for αmCD40:G2 + 2B6. g Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells in mice of indicated genotypes (FcγR-deficient (FcgRα −/− , five mice per group), FcγRIIB-deficient (Fcgr2b −/− , five mice per group) or humanized (Fcgr2b −/− hFCGR2B Tg , four mice per group)) treated and analyzed as in ( a ) together with 10 μg of indicated control or IgG2 anti-mCD40 antibodies. Each symbol represents an individual mouse. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired two-tailed t test ( c , g ), one-way ANOVA with Holm–Sidak’s post hoc ( d – f ). Source data ( c – g ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Binding Assay, Control, Cell Counting, Mutagenesis, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: CH1-hinge basis of agonistically inactive IgG3 and superior IgG2. a – c Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells ( a ) or cell count ( b , c ) in FcγR-humanized mice treated and analyzed as in Fig. together with indicated control or anti-mCD40 antibodies ( a 3.16 μg per mouse; b , c 10 μg per mouse). Numbers of mice: a six mice for Ctrl IgG, six to seven mice per group for others; b , c three mice for Ctrl IgG, four to five mice per group for others. d – f Representative FACS profile ( d ) and quantification ( e , f ) showing the percentage of OT-I cells (CD45.1 + TCRVα2 + ) among CD8 + T cells in human CD40/FcγR-transgenic mice treated and analyzed as in Fig. together with control or anti-human CD40 antibodies of indicated clones (Clones 21.4.1 and 3.1.1 have been described in Patent No.:US 7,338,660; Clone 21.4.1 in the IgG2 form is also known as CP-870,893) and constant domains (30 μg per mouse). Numbers of mice: d – f five to six mice per group. Each symbol represents an individual mouse. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; one-way ANOVA with Holm–Sidak’s post hoc. Source data ( a – c , e , f ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Cell Counting, Control, Transgenic Assay, Clone Assay

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: Impact of both IgG CH1-hinge and Fc on anti-CD40 antibody antitumour activities. MC38 ( a , b ) and MO4 ( c ) tumour volumes in FcγR-humanized mice following treatment with control or anti-mCD40 antibodies of indicated constant domains. After tumour cells were subcutaneously inoculated and established in FcγR-humanized mice, mice were treated i.p. twice on day 0 (the day when mice with palpable tumours receive their first treatment) and day 3 with 31.6 μg/mouse of control or anti-CD40 antibodies of indicated constant domains (the N297A mutation abrogates Fc–FcγR binding), and monitored for tumour growth. For mice inoculated with MO4 tumour cells in ( c ), each treatment also included 2 μg/mouse of DEC-OVA. Shown are tumour growth curves of individual mice ( a ) or mouse groups ( b , c ). Numbers of mice: a , b seven to eight mice per group; c seven mice per group except six mice for αmCD40:G2(N297A). Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, chi-square test ( a ), and two-way ANOVA with Holm–Sidak’s post hoc ( b , c ) were used for group comparison. Source data ( a – c ) are provided as a Source Data file. A representative of two independent experiments is shown ( a , b )

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Control, Mutagenesis, Binding Assay, Comparison

Journal: Nature Communications

Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility

doi: 10.1038/s41467-019-12097-6

Figure Lengend Snippet: The superior rigidity of IgG2 CH1-hinge. a A diagram showing anti-mCD40 antibody TR-FRET. Anti-mCD40 antibody molecules mixed with CD40-Tb and CD40-D2 (mCD40 labeled with Tb donor and D2 acceptor fluorochromes, respectively) can simultaneously bind CD40-Tb and CD40-D2, and emit TR-FRET signal quantified as the relative ratio of detected 665 nm fluorescence to 620 nm fluorescence (Em665/Em620) upon stimulation. b A diagram of the model showing that hinge flexibility of hIgG anti-mCD40 antibodies correlates with TR-FRET signal levels. Left, anti-mCD40 antibodies with little hinge flexibility do not trigger TR-FRET signal due to the large distance between CD40-Tb and CD40-D2; middle, hinge flexibility can bring CD40-Tb and CD40-D2 close enough to trigger TR-FRET signal; right, anti-mCD40 antibodies with large hinge flexibility give rise to stronger TR-FRET signal due to more molecules with closer CD40 binding sites. c – e TR-FRET signal levels of anti-mCD40 antibodies of indicated constant domains. Shown are relative TR-FRET signal levels (Em665/Em620) plotted against the concentration of control IgG or the anti-mCD40 antibodies of indicated constant domains. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, two-way ANOVA with Holm–Sidak’s post hoc. Source data ( c – e ) are provided as a Source Data file. A representative of two independent experiments is shown

Article Snippet: Serially diluted control IgG or anti-CD40 antibodies (3.16–0.00316 μg ml −1 ) were then added and incubated for 1 h. After washing for three times, horseradish peroxidase-conjugated detection antibody (anti-human IgG Fc-HRP, Bethyl Laboratories, 100 ng ml −1 ) was added and incubated for 1 h. The plate was then washed and developed with TMB peroxidase substrate (KPL) in the dark for 20–40 min.

Techniques: Labeling, Fluorescence, Binding Assay, Concentration Assay, Control

Journal: Bioengineering & Translational Medicine

Article Title: Viral vector‐based gene therapies in the clinic

doi: 10.1002/btm2.10258

Figure Lengend Snippet: Examples of current clinical trials for adenovirus‐based in vivo gene therapies, grouped by indication

Article Snippet: Ad , EnAd , Anti‐CD40 antibody , Metastatic cancer, epithelial tumor , NG‐350A (PsiOxus Therapeutics Ltd) , Intratumoral , NCT03852511 (Phase 1).

Techniques: In Vivo, Plasmid Preparation, Sequencing, Infection

Journal: Bioengineering & Translational Medicine

Article Title: Viral vector‐based gene therapies in the clinic

doi: 10.1002/btm2.10258

Figure Lengend Snippet: Examples of current clinical trials for herpes simplex virus (HSV)‐based in vivo gene therapies, grouped by indication

Article Snippet: Ad , EnAd , Anti‐CD40 antibody , Metastatic cancer, epithelial tumor , NG‐350A (PsiOxus Therapeutics Ltd) , Intratumoral , NCT03852511 (Phase 1).

Techniques: In Vivo, Plasmid Preparation