Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: PBMCs of subjects were recovered from liquid nitrogen and stimulated with CD40L at concentrations ranging from 0 to1000ng/mL and phosphorylation of P65, P38, ERK and JNK were monitored at different time points by instant fixation, permeabilization and staining with antibody master mixes containing cell surface markers and phospho-epitope antibodies. Single cells were then analyzed by flow cytometry. A) Gating strategy to identify cell subtypes within PBMCs. Cells were first gated on FSC-A and SSC-A to exclude cell debris. After singlet gating, cells were gated on CD3 versus CD4 to identify CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD4−) and monocytes (CD3dimCD4dim with higher SSC-A than lymphocytes). B cells were then identified in the CD3−CD4− population based on the presence of CD20 expression. Memory B cells (CD20+CD27+) and naïve B cells (CD20+CD27−) were further identified in total B cell pools based on the presence or absence of CD27 expression. B–C) Phosphorylation of P65 was analyzed in indicated cell subsets. The median fluorescent intensity (MFI) was based on 2000–40,000 single-cell measurements depending on cell type. B) Overlaid histograms show the time course of P65 phosphorylation in memory and naïve B cells of a healthy donor after stimulation with 1µg/mLCD40L. C) Overlaid histograms show the dose-response of P65 phosphorylation in memory and naïve B cells at 15min after stimulation with indicated concentrations of CD40L. Black histograms show the unstimulated samples corresponding to basal phosphorylation levels. Data are representative of 9 healthy individuals tested.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Phospho-proteomics, Staining, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A) PBMCs from treatment-naïve RRMS patients (RRMS_TN) and healthy donors (HD) were analyzed by multi-parameter phosflow at different time points post-stimulation with 2ng/mL CD40L, gating on memory and naïve B cell populations, as indicated. Scatterplots of the MFI for p-P65, p-P38, p-ERK, and p-JNK in memory and naïve B cells are shown. A) Data shown are phosflow of PBMCs isolated from leukopheresis pack of treatment-naïve RRMS patients (n=12, closed circles) and healthy donors (n=9, open circles). B) Western blot analysis of CD40-induced pP65 and pP38 in purified CD19+ B cells from healthy donors and RRMS patients. CD19+ B cells were magnetically isolated from PBMCs from treatment-naive RRMS patients (RRMS-TN, n=6) and healthy donors (HD, n=6) using CD19 microbeads. Isolated CD19+ B cells were left unstimulated (labeled as "-") or stimulated with 2ng/mL CD40L for 15 minutes (labeled as "+"). After stimulation, the lysates were harvested and analyzed by Western blot with antibodies recognizing the phosphorylated form of P65 and P38 Antibodies. Level of β-actin was shown as a loading control. C) Mean Pixel Values (band density) of Western blot data in B were acquired using Image Studio software. The levels of pP65 and pP38 were then normalized to β-actin level. Data shown were fold induction of pP65 and pP38 after CD40L stimulation compared to unstimulated condition.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation, Western Blot, Purification, Labeling, Control, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: CD40 stimulation and phosflow were performed as described in Figure 2 on PBMCs isolated from blood of treatment-naïve SPMS patients (n=12, closed squares), treatment-naïve NMO patients (n=5, inverted triangles) and their controls isolated from blood of healthy donors (n=9, open circles). Data shown is representative of two repeated experiments. Significant differences in MFI values were calculated by Student’s t test (P<0.05).

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A–B) PBMCs from RRMS patients (n=12 with 1 data point from 12 RRMS patients, closed circles) and healthy donors (n=12 with 2 data points from 6 healthy individuals, open circles) were analyzed by multi-parameter phosflow at different time points post-CD40L (2ng/mL) stimulation, gating on memory (A) and naïve (B) B cell populations, as indicated. Scatterplots of the MFI for p-IKKα/β are shown. Data shown is representative of two repeated experiments. Significant differences in MFI values were calculated by Student’s t test (P<0.05). (C–F) PBMCs from RRMS patients were stimulated with 2ng/mL CD40L in the presence or absence of TCPA-1 inhibitor at indicated concentrations. The cells were instantly fixed 15 minutes after the stimulation and analyzed by phosflow to detect p-P65 and p-P38. Cells stimulated with CD40L in the presence of DMSO served as a vehicle control. Data shown is from one RRMS patient as a representative of experiments from 3 individual RRMS patients.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A–D) Following CD40 stimulation for 15 minutes, the level of p-P65 and p-P38 in memory B (A and C) and naïve B cells (B and D) from healthy donors (n=9, open circles), RRMS patients (n=8) before (RRMS_TN (T0), closed circles) and after (RRMS_A+C (T1), closed triangles) the therapy were included. The levels of p-P65 and p-P38 were detected at 15 minutes after stimulation with CD40L at 2ng/mL. P values were calculated by Student’s t test and were shown in the figure. E–F) EDSS scores were calculated before (T0) and after (T1) the combination treatment with Avonex and Cellcept. Changes in EDSS score (ΔEDSS) were assessed by subtracting the EDSS at the time of sampling (EDSST1) with the baseline EDSS before any treatments (EDSST0). The peak MFI values for p-P65 (15 minutes after CD40 stimulation) in memory (A) and naïve B cells (B), were plotted relative to ΔEDSS as indicated.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Sampling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: PBMCs isolated from blood of treatment-naïve RRMS patients (n=12, closed circles) and GA-treated RRMS patients (n=8, closed triangle) were analyzed by multi-parameter phosflow at 15 minutes post-CD40L (2ng/mL) stimulation, gating on memory and naïve B cell. A and C) P65 phosphorylation of memory B (upper panel) and naïve B cells (lower panel). B and D) P38 phosphorylation of memory B (upper panel) and naïve B cells (lower panel). P values were calculated by Student’s t test and were shown in the figure.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: CD40-CD40L interaction recruits TRAFs (TRAF2, 3, 5 and 6) which bind to the CD40 cytoplasmic domain and mediates the activation of multiple signaling pathways including canonical and non-canonical NFκB pathways, PI3K pathway, as well as phosphorylation of MAP kinases including P38, ERK and JNK. For canonical NFκB activation, TRAF2 or TRAF6 mediates signaling activation and phosphorylates the subunits (IKKα, IKKβ and IKKγ) in the IKK complex, which further induces the phosphorylation of IκB. The phosphorylation of IκB induces its degradation and this process leads to phosphorylation and nuclear translocation of NFκB, where it acts as a transcription activator. In normal B cells (left), the CD40 signaling plays an essential role in B cell proliferation, survival and cytokine production. In B cells of RRMS patients (right), we found enhanced CD40-mediated canonical NFκB signaling while the MAPK pathway remains unaffected. A prediction of the model is that dysregulation of CD40-mediated NFκB signaling leads to the hyper-proliferation of MS B cells upon CD40 engagement as we described previously. In addition, our data points to the potential of therapeutic interventions including Avonex/Cellcept combination therapy and GA therapy to correct this signaling dysregulation.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Translocation Assay

Journal: Nature Communications

Article Title: Nanomechanical binding mechanism of ligands drives agonistic activity

doi: 10.1038/s41467-025-61929-1

Figure Lengend Snippet: a Schematic illustration of SMFS experiments, in which the unbinding forces between hIgG, coupled to the AFM tip via a PEG crosslinker, and the transmembrane hCD40 receptor were measured. b Exemplary force-distance curves from the SMFS experiments with distinct rupture events due to single (red) hIgG:hCD40 bond dissociation, and simultaneous (blue) or sequential (black) dissociation of two hIgG:hCD40 bonds. c Experimental pdfs (black curves) of unbinding forces for different contact times. Pdfs peaks were fitted with multi-Gaussians, representing unbinding forces of one (red) and two (blue) hIgG:CD40 bonds. The frequency of two bonds increased with longer contact times. d , e Increase of probability of hIgG binding P(t) and probability of second bond formation P 2 (t) over contact time, fitted using pseudo-first-order kinetics of a bimolecular reaction. f Unbinding force versus loading rate plot of hIgG2A. Data points (grey) were divided into loading rate segments. Mean and standard deviation of the first Gaussian (red dots) obtained from unbinding force pdfs of each segment were fitted using the Bell-Evans model (red fit). Higher forces were in good agreement with a Markov binding model (blue fit), predicting unbinding forces for parallel breakage of two identical bonds. Data presented are from three independent experiments.

Article Snippet: The cantilevers were then covered with a pre-mixed solution containing 100 μl of 1.5 μM His 6 -tagged human CD40 ligand (hCD40L; AcroBiosystems, CDL-H5248) and 4 μl of 5 mM NiCl 2 and stored overnight at 4 °C.

Techniques: Binding Assay, Standard Deviation

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Overview of the treatment scheme. i.t: intratumorally; i.v: intravenously; ICOSL: inducible co-stimulator ligand.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques:

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Viral DNA extraction from CsCl-purified virus particles using proteinase K. The AdV-D24-ICOSL-CD40L sequencing library was made using a Nextera XT kit (Illumina). The size of the library was assessed by high-resolution agarose gel electrophoresis. The presence of the 24-bp deletion in the E1A CR2 domain, the CMV-ICOSL-IRES-CD40L expression cassette inserted in place of the E3 region and the hybrid Ad5/3 fiber were confirmed. Analysis of sequence coverage did not detect the presence of a virus sub-population with a genome characterized by a major rearrangement such as a large insertion or deletion. The figure was generated by SnapGene ® .

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: DNA Extraction, Purification, Sequencing, Agarose Gel Electrophoresis, Expressing, Generated

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of ICOSL and CD40L expression in tested melanoma cells. ( A ) ICOSL concentration was detected on supernatants collected 24–48 h after treatment using ELISA kit. ( B ) CD40L was detected from supernatants collected 24–48 h after treatment using an ELISA kit according to the manufacturer’s instructions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (ns = not significant, p > 0.05). ( C ) Transgene expression. The expression of ICOSL and CD40L from AdV-D24-ICOSL-CD40L was assessed by infecting A549 cells with the virus, harvesting proteins 48 h after the infection and detecting ICOSL and CD40L by Western blot. 1–4: protein extracts from A549 cells infected with AdV-D24-ICOSL-CD40L clones #1–4. N: protein extract from non-infected A549 cells. M: PageRuler Prestained Protein Ladder (ThermoScientific # 26616). ICOSL MW: 70 kDa (due to heavy protein glycosylation); CD40L MW: 30 kDa.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Infection, Western Blot, Clone Assay

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of cell viability by MTS assay (cell cytotoxicity assay). ( A ) Cell viability was evaluated 72 h post-infection with AdV-24-ICOSL-CD40L and AdV-24 at the concentration of 100 VP/cell and combination with anti-PD1 (pembrolizumab). Data are expressed as the percentage of viable cells according to MTS cell viability assay protocol (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega). Statistical analysis was carried out with a Mann–Whitney test to compare two groups ( p > 0.05). ( B , C ) Microscopic photographs visualizing the morphology and cytopathic effect (CPE) of the cells representing the investigated groups and assessed by MTS assay at 48 and 72 h post-infection (magnification 10×) (** = p ≤ 0.001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: MTS Assay, Cytotoxicity Assay, Infection, Concentration Assay, Viability Assay, Proliferation Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Immunogenic cell death assessment. ( A ) Evaluation of calreticulin (CRT) exposure by melanoma cell lines after treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured 48 h post-treatments with anti-calreticulin antibody staining AlexaFluor ® 488 and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( B ) Assessment of ATP release after the treatment. ATP concentration in a supernatant was evaluated 72 h after infection with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. ( C ) Evaluation of high-mobility group box 1 (HMGB-1) release after treatment with oncolytic adenoviruses and the combination with anti PD-1. HMGB-1 level was measured from the supernatant collected 72 h after infection with ELISA kit (MBL International, Woburn, MA, USA), according to manufacturer’s dispositions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (* = p ≤ 0.05; ** = p ≤ 0.001, *** = p ≤ 0.0001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Infection, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Antitumor efficacy of oncolytic vectors and the combination therapy with anti PD-1 antibody in murine melanoma B16V (1 × 10 6 cells/flank, one mouse had 2 tumors/6 tumors per group) xenograft immunocompetent C57BL/6 model. ( A ) Tumor volume (mm 3 ) measured through the study. The treatment was performed once per day on days 1–6. The mice were treated according to the scheme with viruses (i.t.) and anti PD-1 antibody (i.v.) ( B ) At the end of the study, mice were sacrificed and tumors harvested for weight assessment. ( C , D ) Tumor volume measurement on days 6 and 20. ( E ) Body weight measurements throughout the study. ( F ) Survival profile was calculated by Kaplan–Meier test. ( H ) Evaluation of CRT exposure after the treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured in the end of the study (after mice sacrifice) with anti-calreticulin antibody staining and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( G ) Assessment of ATP release after the treatment. ATP concentration from the tumors was evaluated in the end of the study (after mice sacrifice) with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. Error bars, mean ± SEM, * = p ≤ 0.05, *** = p ≤ 0.0001.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Cell Viability Assay

Journal: Frontiers in immunology

Article Title: Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

doi: 10.3389/fimmu.2022.919854

Figure Lengend Snippet: FIGURE 9 | IL-21 rescued CD40L and anti-IgM induced cell death in human primary B cells. (A) Representative graphs of CFSE labelling cells showed no differentiation was induced by any stimulatory factors in 3 days. (B) Representative graphs showed that IL-21 could suppress cell death induced by CD40L and anti- IgM in terms of increased number of DAPI- cells. (C) Representative graphs showed that IL-21 was able to induce CD38+ B cell differentiation both in the absence and presence of CD40L.

Article Snippet: Cells were then seeded into 96-well round bottom plate at the density of 2E5 cells/100μl, and then challenged with 50 ng/ml IL-21 and 1 μg/ml CD40L (#6420-CL/CF, R&D Systems, Minneapolis, USA) or 2 μg/ml anti-IgM.

Techniques: Cell Differentiation