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: Stem Cells

Article Title: Microglia in the spinal cord stem cell niche regulate neural precursor cell proliferation via soluble CD40 in response to myelin basic protein

doi: 10.1093/stmcls/sxae076

Figure Lengend Snippet: Soluble CD40 is the microglia-derived inhibitory factor. (A) Cytokine array analysis reveals 13 factors in the 10-30 kDa molecular weight range that are more highly expressed in MG-spCM with MBP compared to MG - spCM grown in the absence of MBP. The differential intensity was determined by subtracting cytokine intensities of MG-spCM with MBP from MG-spCM minus MBP. ( n = 3 independent experiments) (B) Dose response of mResistin shows inhibition of neurosphere formation at 2000 and 4000 pg/mL ( n = 4 independent experiments, * P < .05, ** P < .01) (C) Dose response of sCD40 shows an inhibitory effect on neurosphere formation at all concentration examined from the spinal cord ( n = 4 independent experiments, **** P < .001). (D) qPCR of CD40 receptor (CD40) and ligand (CD40L) expression in brain and spinal cord derived neurospheres ( n = 3 independent experiments, *** P < .001, * P < .05). (E) Dose response of sCD40 on brain neurosphere formation ( n = 4 independent experiments, **** P < .0001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were purchased from R&D Systems for CD40 (Catalog #MCCD40) and used for the analysis of microglia-derived CM samples as per the manufacturer's instructions for cell culture supernatant samples.

Techniques: Derivative Assay, Molecular Weight, Inhibition, Concentration Assay, Expressing

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: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from blood and spleen tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final dose injection. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (C) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype and function of CNS innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) CD11b + CD45 lo/neg resident microglia and (C) CNS CD11b + CD45 hi infiltrating macrophages and were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. CD11b + cells were isolated from CNS tissue of anti‐CD52‐Ab or vehicle‐treated mice at first day after the final treatment dose. (D) Respiratory burst activity of CD11b + ‐isolated cells. (E) Phagocytosis of FITC‐coated beads by CD11b + isolated cells. Unpaired two‐tailed Student's t test. (F) Cells were left unstimulated or were stimulated with LPS or Poly(I:C)(PIC) for 24 h and cytokine production determined by ELISA. Two‐way ANOVA with Sidak multiple comparisons test, * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery and CNS innate immune cells in EAE mice at three weeks post‐injection. Single‐cell suspensions were obtained from blood, spleen and CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated mice three weeks after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). (C) CNS CD11b + CD45 hi ‐infiltrating macrophages and CD11b + CD45 lo/neg resident microglia were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the function of periphery and CNS innate immune cells in SCID mice at first day post‐injection. SCID mice were treated with either vehicle or 10 mg/kg murine anti‐CD52‐Ab‐Ab for five consecutive days. Single‐cell suspensions were then obtained from blood and spleen tissues first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II, CD40, CD80 and CD86 (all n = 7). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II, CD40, CD80, CD86, and CD206 (all n = 7). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

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