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: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Design of the coiled-coil peptide delivery platform. (A) Anti-CD40 antibody constructs of IgG1 or IgG2 isotypes were connected with E domains of various lengths (EIAALEK)3-6 via a peptide linker (4GS)x2to the C-terminal of the light chain, heavy chain or both light and heavy chains. (B). Combination of antibodies connected with E domains with K domains fused with OVA peptides leads to the formation of complexes. Created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: K4-OVA peptides strongly bind E4 domains connected to anti-CD40 antibody constructs and aid formation of stable complexes capable of cell binding and CD40 activation. Binding interactions between E and K coiled coil peptides as determined by ELISA and bio-layer interferometry (BLI). ELISA plates were coated with human CD40 antigen followed by addition of antibody constructs of either IgG1 (A) or IgG2 (B) isotype connected with E4 domains. Biotinylated K4 domains fused with OVA peptides were added followed by detection using streptavidin HRP. Representative BLI affinity measurements to determine binding kinetics of (C) mAbLCE4-IgG1 or (D) mAbLCE4-IgG2 antibody constructs interacting with K4 domains fused with OVA peptides. Antibody constructs connected with E domains were captured on anti-fab 2nd generation (FAB2G) sensors and assayed against serially diluted K4-OVA peptides in solution. Antibody-peptide complex formation and binding of the complexes to B16-F10 CD40 expressing and B16-F10 wt cells was assessed by flow cytometry. IgG1 anti-CD40 antibodies connected with E4 were mixed with equimolar amounts of biotynylated K4-OVA to enable complex formation followed by staining using PE streptavidin and flow cytometry analysis. (E) Cells incubated with 5 µg mAbLCE4 + 0.5 µg biotinylated K4-OVA peptides, (F) cells incubated with 5 µg mAbLCE4, (G) cells incubated with 0.5 µg biotinylated K4-OVA peptides, (H) cells without any compounds. Data shown is representative of two independent experiments. (I) a reporter assay was performed to assess the ability of IgG1 anti-CD40 antibody constructs connected with E domains to induce CD40 activation. CD40 reporter cells (promega) were incubated with FcγRI (CD64) CHO expressing cells or wildtype CHO cells in the presence of mAbLCE3 complexed with K4-OVA peptides, mAbLCE3 and mAb control. CD40 activation was measured according to the manufacturer’s instructions.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining, Incubation, Reporter Assay, Control

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Stability characterization of anti-CD40 antibody constructs connected with E domains. (A) Ability of the constructs to withstand shear stress was determined by measuring A280 before and after rapid agitation. (B) Constructs were incubated in human serum or BSA for seven days after which a Mono ELISA was performed. (C) Stability of the constructs in un optimized buffer (PBS) was analyzed by incubation at room temperature (RT) or 40 °C for 2 weeks followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies. (D) Constructs were subjected to three rounds of freeze (-80 °C)-thawing (37 °C) at 24, 48, and 72 hours followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Shear, Incubation, Enzyme-linked Immunosorbent Assay, High Molecular Weight

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Combining the coiled coil platform with potential antigen carrying nanocarriers. (A) An illustration of formation of antibody-peptide-bead complexes from interactions between antibody-peptide conjugate constructs, biotinylated K6-OVA peptides and streptavidin labeled beads (utilized to mimic antigen loaded nanocarriers). Antibody-peptide-bead complexes were prepared first before addition to cells. (B) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes and stained with both PE anti-streptavidin and anti-human lgG Fc-PE. (C) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only PE anti-streptavidin. (D) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only anti-human lgG Fc-PE. (E) Unstained B16-F10 CD40 and B16-F10 wt cells without any compounds. All stainings were followed by FACS analysis. The data depicted here is representative of two independent experiments. was created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Labeling, Incubation, Staining

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Coiled-coil technology enables efficient OVA peptide cross-presentation and activation of OVA-specific CD8+ T cells. (A) Mice treatment plan. On day 0 and day 7, non-tumor bearing male hCD40tg mice were subcutaneously treated with different compound formulations. Seven days after the second treatment (day 14), mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN) were removed for FACS analysis of the percentage of OVA peptide-specific CD8+ T cells. (B) Mice were treated with vehicle (PBS), or molar equivalent amounts of the peptides alone or in combination with mAbLCE4 IgG1 construct. (C) a similar treatment set up as that in (B) was used and additional molecules were included (mAbLCE4 -IgG2 + K4-OVA, mAbLCE4 -IgG2+ OVA peptides, mAb wt + OVA peptides). Four to five mice were used per group in each experiment. The graphs show the mean ± SD of data from each experiment. Statisctical analysis was performed using a Mann-Whitney test, * p < 0.05; ** p < 0.01. (D) a postulated mode of action of the technology. After internalization of the antigen-peptide complexes, DCs process and cross-present antigenic peptides to T-cells that are activated to kill tumor cells. (E) An example of a potential application of the technology could be in personalized neoantigen cancer vaccines. From patient tumors, neoantigens are identified and characterized. Neoantigens fused with K domains are manufactured and combined with off-the-shelf anti-CD40 antibodies connected with E domains. Finally, patients are vaccinated with the mixture. were created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Activation Assay, Construct, MANN-WHITNEY, Vaccines, Immunopeptidomics

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Evaluating the potential of the technology to evoke anti-tumor responses in vivo . (A) Human CD40 transgenic mice were subcutaneously (s.c) administered with (0.2x10 6 ) MB49-EpCam-OVA tumor cells on day 0 followed by s.c. treatment on the right outer flank with either vehicle (PBS), 1.5 µM free OVA-peptides, 0.4 µM of mAbLCE4, 1.5 µM K4-OVA peptides, 0.4 µM of mAbLCE4 in combination with 1.5 µM K4-OVA peptides or 0.4 µg of mAbLCE4 in combination with 1.5 µM OVA peptides on day 10 and 17. (B) Tumor growth and (C) survival over time were monitored. The graphs show the mean (+SEM) of 7–10 mice per group in one experiment. Statistical analysis of tumor volumes was performed on days 21–34 using a Mann-Whitney test. Survival analysis was conducted using Kaplan-Meier log-rank test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns- not statistically significant.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: In Vivo, Transgenic Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP-IC, and mainly with MP, induce the activation of autologous B cells from patients with RA. (A) From left to right: representative light microscopy pictures of MDM unstim alone; MDM unstim co-cultured with B cells; MDM differentiated in the presence of RMP or RMP-IC from patients with RA and co-cultured with B cells. (B) Representative histograms of CD80 expression on B cells from HC (top) and patients with RA (below) cultured alone (light blue) and with anti-BCR plus CD40L (yellow) or co-cultured with MDM differentiated without (Unstim, black) or with RMP (gray) and RMP-IC (green). Blue histograms represent the FMO control. (C) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with RA ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (D) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with RA ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with RMP and RMP-IC. (E) BAFF and APRIL (Top panel) levels in supernatants of MDM from patients with RA ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with RMP and RMP-IC. IgG and IgM (below panel) levels in supernatants from co-cultures of MDM differentiated with or without RMP and RMP-IC with autologous B cells from HC ( n = 5) and RA ( n = 5) patients. Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Light Microscopy, Cell Culture, Expressing, Control, Positive Control

Journal: Frontiers in Immunology

Article Title: Proinflammatory Differentiation of Macrophages Through Microparticles That Form Immune Complexes Leads to T- and B-Cell Activation in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02058

Figure Lengend Snippet: MDM differentiated with MP and MP-IC induce the activation and plasmablast differentiation of autologous LB from patients with SLE. (A) The frequency of CD80, CD86, CD69, and CD95 in B cells from patients with SLE ( n = 7) and HC ( n = 6) co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (B) The frequency of dead B cells (positive for LIVE-DEAD probe) from patients with SLE ( n = 7) and HC ( n = 6) cultured alone (Unstim, in complete medium) and with anti-BCR plus CD40L (positive control) or co-cultured with MDM differentiated without (Unstim) or with LMP and LMP-IC. (C) BAFF and APRIL levels in the supernatants of MDM from patients with SLE ( n = 5) and HC ( n = 5) differentiated without (Unstim) or with LMP and LMP-IC. (D) Representative gating strategy to determine the frequency of plasmablasts after the co-culture of B cells with MDM differentiated without (Unstim) or with LMP and LMP-IC. (E) The frequency of plasmablasts from B cells cultured alone (Unstim, in complete medium), with anti-BCR plus CD40L (positive control), or co-cultured with autologous MDM from patients with SLE ( n = 7) and HC ( n = 6) differentiated without (Unstim) or with LMP and LMP-IC. (F) IgG and IgM levels in supernatants from the co-cultures of MDM differentiated with or without LMP and LMP-IC with autologous B cells from HC ( n = 5) and patients with SLE ( n = 5). Comparisons among the groups were performed using ANOVA II and the Bonferroni post-hoc test.

Article Snippet: Recombinant human (rh) CD40 Ligand (CD40L), rhIFN-γ, and rhIL-4 were purchased from R&D Systems; rhIL-2 from Biolegend (San Diego, CA, USA) and the affinity-purified F(ab') 2 fragment anti-human IgM (anti-BCR) and F(ab') 2 anti-IgG fragment Alexa Fluor 488 conjugated from Jackson ImmunoResearch (New Baltimore, PA, USA).

Techniques: Activation Assay, Cell Culture, Positive Control, Co-Culture Assay