Journal:

Article Title: A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

doi: 10.1046/j.1365-2249.2001.01473.x

Figure Lengend Snippet: Surface phenotype of bone marrow derived DC. The DC consisting of 90–95% CD11c+ cells were analysed by flow cytometry for the expression of I-A, β2-microglobulin, CD80, CD86 and CD40 (▪) or by staining with secondary antibody alone (□). Two I-A specific mAbs, Ox-6 (shown here) or 10·2.16 were used with identical result. The results are representative of three different experiments carried out three weeks apart.

Article Snippet: The antibodies used in the study were the MHC class II specific rat antimouse I-A k clone MRC Ox-6 (Serotec, Oxford, UK) and anti-I-A g7 clone 10·2.16 (kindly provided by EP Reich, Anergen Inc), rat antimouse CD80 (B7·1) clone IG10 (Pharmingen, San Diego, USA), rat antimouse CD86 (B7·2) clone GL1 (Cambridge Bioscience, UK); rat antimouse CD40 clone 3/23 (Serotec, UK), hamster antimouse CD11c clone N-418 (produced by ICRF, London, UK); FITC-conjugated rabbit antirat Ig (Dako, Ely, UK); FITC-conjugated rabbit anti‐mouse Ig (Dako, Denmark); and mouse anti‐mouse β2-microglobulin clone S19·8 (kindly provided by M Millrain, CSC, UK).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Staining

Journal: bioRxiv

Article Title: Failed down-regulation of PI3K signaling makes autoreactive B cells receptive to bystander T cell help

doi: 10.1101/2023.01.23.525206

Figure Lengend Snippet: A) Schematic representation of the experimental protocol. One group of OTII x TCRα KO mice was treated with agonistic anti-CD40 antibody on days 6 and 9 post initiation of tamoxifen treatement. B) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ SHP-1 deficient B cells in WT C57BL/6 mice and OTII x TCRα KO mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=4-5/group, representative cytograms shown). C) Distribution of the YFP+ E4+ SHP-1 deficient Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. D) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). E) Quantification of the number of CD45.1+ E4+ cells/spleen in the indicated transfers 14 days after tamoxifen treatment. F) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ WT Ars/A1 B cells in WT C57BL/6 mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=5/group, representative cytograms shown). G) Distribution of the YFP+ E4+ WT Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. H) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). Data shown are representative of at least two replicate experiments. Error bars represent mean ± SEM. Two -tailed unpaired Student’s t test was used. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: In VivoMab anti-mouse CD40, (Clone:FGK4.5/FGK45; Bio X Cell) was injected i.v. or i.p. (200 μl at 0.25mg/ml in sterile PBS) for stimulation.

Techniques: Enzyme-linked Immunospot, Two Tailed Test

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Model of SPD-gp100-CD40L. Amino acids 25 to 596 (sequence KVPRNQD to EAGLGQV) of human gp100 were inserted between amino acids 105 and 106 of murine SPD within the SPD-CD40L fusion construct. (b) Cartoon of expected SPD-gp100-CD40L 4-trimer structure showing trimers of CD40L (gray circles) and trimers of gp100 (black circles) extending from the SPD collagen-like domain. (c) Western blot analysis. 293T cells were transfected with DNA plasmid encoding gp100 or SPD-gp100-CD40L fusion protein. After 48-hour culture, supernatant was collected and run on an SDS-PAGE gel in the presence of reducing agent. Western blot was performed using a polyclonal antibody to gp100.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Sequencing, Construct, Western Blot, Transfection, Plasmid Preparation, SDS Page

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Representative SEC elution profile of SPD-gp100-CD40L as monitored by the scattering intensity, expressed in terms of the excess Rayleigh ratio (R(θ)), at three scattering angles of 42° (tall gray line), 90° (black line) and 138° (short gray line) as a function of elution volume (V). Note that SPD-gp100-CD40L apparently elutes as a major species (megamer) and as a minor species (polymer, evident as a shoulder of the major elution peak). (b) Representative partial Zimm plot obtained from analytical SLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at three scattering angles of 42°, 90° and 138°—represents a linear fit. (c) Representative autocorrelation function plot obtained from analytical DLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at a scattering angle of 90°—represents non-linear least squares fit.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Polymer, Protein Concentration

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) In vitro activity of SPD-CD40L and SPD-gp100-CD40L was determined using a cell-based CD40 NF-κB enzymatic reporter system. An equivalent amount of 293T supernatant from pcDNA3.1, pSPD-CD40L or pSPD-gp100-CD40L transfected cells was incubated with 293-CD40-SEAP NF-κB reporter cells at various dilutions. (b) In vitro activity of supernatant from SPD-gp100-CD40L transfected 293T cells was evaluated on mouse bone marrow derived mouse DC and compared to supernatant from 293T cells transfected with empty vector pcDNA3.1. Error bars represent mean + SEM (n=3). * p<0.05, ** p<0.01 by Student’s t test compared to pcDNA3.1 supernatant.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: In Vitro, Activity Assay, Transfection, Incubation, Derivative Assay, Plasmid Preparation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA/GVAX therapeutic vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of mice on day 0. Mice were then immunized by i.m. injection of PBS or pSPD-gp100-CD40L on day 3, 10, and 17. GVAX, B16-F10 tumor cells expressing GM-CSF, were irradiated at 5,000 rad and 1 × 106 cells injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume per group (n=5). (c) Survival analysis was based on the date of death or when tumor size reached >1500 cm2.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA or GVAX vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of the mice on day 0. Mice were immunized i.m. with PBS, pSPD-gp100-CD40L + pIL-12, pSPD-gp100-CD40L + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. For GVAX therapy, B16-F10 tumor cells expressing GM-CSF (GVAX), were irradiated at 5,000 rad and 1 × 106 cells were injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice. (d) Tumor growth kinetics of individual mice from each treatment arm.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected into the left flank of the mice on day 0. Mice were then immunized i.m. with PBS, pgp100, pgp100 + pIL-12, pgp100 + pGM-CSF, pgp100 + pIL-12 + pGM-CSF, pgp100-IRES-SPD-CD40L + pIL-12 + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection