Journal: Cancers

Article Title: Circulating Exosomes Inhibit B Cell Proliferation and Activity

doi: 10.3390/cancers12082110

Figure Lengend Snippet: B cells were isolated from citrate blood samples drawn from healthy donors. ( A ) B cells were co-cultured with exosomes for up to 4 days and stimulated with CD40 Ligand (CD40L) and Interleukin 4 (IL-4) on day 1. Each day, B cells were studied by microscopy and counted using trypan-blue stain and an automated cell counter. ( B ) B cell purity as assessed by CD19-positivity of cells was above 90% throughout culture. n = 8 (Day 2), n = 4 (Days 3 & 4). ( C ) Purity of isolated B cells was confirmed by anti-CD19 stain and subsequent FACS analysis. Purity after isolation was always above 98%. ( D ) B cell survival as measured by FACS cell count. B cell counts were reduced after co-culture with both healthy volunteer and HNSCC exosomes. There were no significant differences between HNSCC and NC exosomes. n = 10 (HNSCC exosomes on d2); n = 9 (NC exosomes and PBS on d2); n = 6 (PBS and HNSCC exosomes on d3 & d4); n = 2 (NC exosomes on d3); n = 3 (NC exosomes on d4). ( E ) B cell viability as measured by trypan blue staining and automated counting was significantly lower after co-culture with healthy volunteer exosomes. A similar, but non-significant trend was observed after co-culture with HNSCC exosomes. There were no significant differences between HNSCC and NC exosomes. n ≥ 5 (HNSCC: 5 [d2], 9 [d3], 6 [d4]; NC: 6 [d2], 9 [d3], 5 [d4]; PBS: 5 [d2], 7 [d3], 6 [d4]). **: p < 0.01; *: p < 0.05. HNSCC, exosomes from blood plasma of HNSCC patients. NC = no cancer, exosomes from blood plasma of healthy volunteers.

Article Snippet: After 24 h, B cells were stimulated with 2 μg/mL CD40 Ligand (CD40L, R&D Systems, Minneapolis, MN, USA) and 1500 IU/mL Interleukin 4 (IL-4, CellGenix, Freiburg, Germany).

Techniques: Isolation, Cell Culture, Microscopy, Staining, Cell Counting, Co-Culture Assay, Clinical Proteomics

Journal: Cancers

Article Title: Circulating Exosomes Inhibit B Cell Proliferation and Activity

doi: 10.3390/cancers12082110

Figure Lengend Snippet: B cells were harvested after 2 days of co-culture with either NC or HNSCC exosomes or PBS and stained for FACS analysis. ( A ) Frequency of CD39 + CD73 + regulatory B cells. ( B ) The expression of CD39 on B cells was reduced after co-culture with NC or HNSCC exosomes. ( C ) Expression of CD73 on B cells. ( D ) Expression of CD86 on B cells. ( E ) The expression of CD19 on B cells was increased by stimulation with CD40L and IL-4. **: p < 0.01; *: p < 0.05, n = 8 (HNSCC), n = 6 (NC), n = 5 (Unstim). Unstim = Unstimulated B cells, NC = no cancer (exosomes from blood plasma of healthy volunteers), HNSCC, exosomes from blood plasma of HNSCC patients.

Article Snippet: After 24 h, B cells were stimulated with 2 μg/mL CD40 Ligand (CD40L, R&D Systems, Minneapolis, MN, USA) and 1500 IU/mL Interleukin 4 (IL-4, CellGenix, Freiburg, Germany).

Techniques: Co-Culture Assay, Staining, Expressing, Clinical Proteomics

Journal: Antibodies

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy

doi: 10.3390/antib13020031

Figure Lengend Snippet: Identification of agonistic anti-CD40 antibodies not requiring Fc-effector function. ( A ) Flow diagram of the anti-CD40 antibody discovery campaign. Wildtype rabbits were immunized with recombinant CD40 protein and monoclonal B-cells were FACS-sorted and cultured. Antibodies secreted into the B-cell supernatant medium were screened for the induction of NFκB signaling in a HEK-Blue-CD40L™ cell-based gene reporter assay. Antibody gene sequences were retrieved from B-cells, humanized and cloned as human IgG1-LALA antibodies in HEK-293 FreeStyle™ cells. ( B ) Eighty-eight humanized anti-CD40 hIgG1-LALA antibodies were tested on CD40 expressing HEK 293 cells in the HEK-Blue-CD40L™ cell-based gene reporter assay (grey bars; OD@655 = secreted embryonic alkaline phosphatase activity) and for the induction of IL-12p40 cytokine release by in vitro differentiated dendritic cells (blue bars; measured by ELISA). Asterisks (*) indicate values exceeding the linear range of the ELISA.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L™ (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Recombinant, Cell Culture, Reporter Assay, Clone Assay, Expressing, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Antibodies

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy

doi: 10.3390/antib13020031

Figure Lengend Snippet: Differentiation of novel Fcγ-receptor-independent anti-CD40 antibodies from other clinical antibodies. ( A ) The activity of the four most active CD40 hIgG1-LALA antibodies, MAB 271, 273, 276 and 278, were tested for the activation of in vitro differentiated DCs in direct comparison to different Fc-variants of CP-870,893. IL-12p40, released by DCs, was measured by ELISA. The statistical significance for difference between MAB 273, MAB 271 and the Fc-variants of CP-870,893 was determined by an unpaired t -test. * p < 0.05, ** p < 0.01, ns = not significant. ( B ) CD40 antibodies were incubated with CD40-expressing HEK-Blue-CD40L™ cells, and interference with CD40L binding to CD40 was tested by the addition of increasing concentrations of recombinant CD40L-mIgG2a-Fc-fusion protein. The binding of CD40L was detected by flow cytometry using a fluorescently labeled anti-mouse IgG antibody. ( C ) The competition of anti-CD40 antibodies for binding to human CD40 was tested in a sandwich ELISA. Each antibody was tested both as a coating (vertical label) and as a detection antibody (horizontal label) in combination with any other anti-CD40 antibody. Error bars in ( A , C ) indicate the standard deviation (mean) of triplicates.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L™ (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Activity Assay, Activation Assay, In Vitro, Comparison, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Binding Assay, Recombinant, Flow Cytometry, Labeling, Sandwich ELISA, Standard Deviation

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: Rs1883832 is the fSNP of CD40 . ( A ) Luciferase reporter assay showed that among the three candidate SNPs that scored 5, only rs1883832 had allele-imbalanced luciferase activity. The luciferase reporter with SNP-centered fragment containing either the risk or non-risk allele was transfected into HepG2 cells. Relative luciferase activity was analyzed between the risk (teal) and non-risk (orange) alleles of the three candidate SNPs. ( B ) EMSA showed, among the three candidate SNPs that scored 5, only rs1883832 had allele-imbalanced binding activity of nuclear protein. The 31-bp biotinylated DNA fragments centered on each SNP were established and incubated with nuclear extract from HepG2 cells. The binding activity of nuclear protein in risk allele and non-risk allele was evaluated. rs1883832: T, risk allele; C, non-risk allele; rs6074022: C, risk allele; T, non-risk allele; rs4810485: T, risk allele; G, non-risk allele. Red arrow, allele-specific shift. Lane 1, risk allele probe only; lane 2, non-risk allele probe only; lane 3, risk allele probe with nuclear extract; lane 4, non-risk allele probe with nuclear extract. ( C ) WashU Epigenome Browser showed H3K4me3 (the signals of promoter-associated chromatin modification) enrichment tracks at the rs1883832 locus in HepG2 cells and liver, H3K27ac (the signals of enhancer-specific chromatin modification) enrichment tracks of HepG2, along with DNAse signals (the probability of the existence of enhancer activity) in HepG2, hepatocyte and liver. Experiments were performed in triplicate. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Incubation, Modification

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: ANXA2 is the regulatory protein which preferentially binds to the risk allele T of rs1883832. ( A ) The schematic of FREP was diagrammed. Briefly, the FREP constructed with BamHI (cyan) and EcoRI (green) restriction sites flanking a 31-bp sequence (light blue) centered on the fSNP of interest (red), and two 20-bp sequences (orange) for PCR were attached to magnetic dynabeads. After incubation of nuclear extract, EcoRI digestion deleted the 3′ DNA binding proteins followed by removal of the 5′ non-specific DNA complex by BamHI digestion. With magnetic separation and wash, the remaining fraction was analyzed by mass spectrometry (MS). ( B ) MS discovered that several proteins may bind with the rs1883832 region. The silver stain (left) showed proteins pulled down from lane 1 (non-specific sequence) and lane 2 (test sequence with rs1883832). The red arrow indicated the specific bands sent for MS analysis. The peptide spectrum count was listed (right). ( C ) ANXA2 regulated CD40 expression. Western blot was performed to assess the expression of CD40 after siRNA-mediated silencing of ANXA2. ( D ) ChIP-qPCR (left) and ChIP-PCR (right) were conducted to investigate endogenous binding of ANXA2 to the rs1883832 region. The agarose gel electrophoresis of PCR products for Input and ChIP, respectively. Light blue arrows indicated the marker bands. The enrichment with ANXA2-specific antibody compared with IgG antibody, as well as between negative control (siCtrl) and ANXA2-si3 HepG2 cells, was evaluated. ( E and F ) ChIP experiment combined with TA cloning demonstrated that ANXA2 preferentially bound to the risk allele T of rs1883832. (E) A sequencing trace was performed to explore the genotypes on rs1883832 in hepatocarcinoma cell lines. Rs1883832 DNA fragments from both input and ChIP samples were cloned into TA vectors. (F) The number of clones containing allele T or C in each group was counted. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Construct, Sequencing, Incubation, DNA Binding Assay, Mass Spectrometry, Silver Staining, Expressing, Western Blot, ChIP-qPCR, Binding Assay, Agarose Gel Electrophoresis, Marker, Negative Control, TA Cloning, Clone Assay

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 exerts anti-HBV effect in HBV-producing cells, HBV-transfected and HBV-infected cells. ( A – C ) Inhibition of CD40 or CD40L using antagonistic antibodies, or knockdown of CD40 via siRNA reversed suppression of HBV replication and transcription. HepG2.2.15 cells transfected with CD40 siRNA or siCtrl were stimulated with CD40L (1 μg/ml) by preincubation with anti-CD40 antibody (5 μg/ml) or anti-CD40L antibody (0.1 μg/ml) for 1 h. The level of extracellular HBV DNA load was determined using the Hepatitis B Viral DNA Quantitative Fluorescence Diagnostic Kit (A). The expression of intracellular HBsAg and HBcAg was analyzed by western blot (B). HepG2.2.15 cells were transfected with CD40 siRNA or siCtrl for 72 h and the expression of intracellular HBV total RNA and HBV 3.5-kb RNA was measured by qPCR (C). ( D – E ) Decreased expression of CD40 facilitates HBV transcription. HBV-transfected HepG2 cells were transfected with CD40 siRNA or siCtrl for 72 h. HepG2-NTCP cells were infected with 1000 genome equivalents/cell of HBV for 24 h and then transfected with CD40 siRNA or siCtrl for 96 h. Expression of intracellular HBV total RNA, 3.5-kb RNA, HBsAg and HBcAg was analyzed as indicated (D and E). ( F – H ) Overexpression of CD40 resulted in the suppression of HBV replication and transcription. HepAD38 cells were transfected with CD40 plasmid or vector for 72 h and HBV DNA load in cell supernatant was determined as indicated (F). HBV-transfected HepG2 cells were transfected with CD40 plasmid or vector for 72 h. HepG2-NTCP cells were infected with 1000 genome equivalents/cell of HBV for 24 h and then transfected with CD40 plasmid or vector for 96 h. Expression of intracellular HBV total RNA, 3.5-kb RNA, HBsAg and HBcAg was analyzed as indicated (G and H). Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Transfection, Infection, Inhibition, Knockdown, Fluorescence, Diagnostic Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 activates JAK–STAT signaling pathway. ( A – B ) Total RNA from HepG2.2.15 transfected with CD40 siRNA or siCtrl was extracted and used for mRNA sequencing analysis. GO enrichment analysis (A) and KEGG enrichment analysis (B) were performed by hypergeometric test. P value was adjusted by the false discovery rate (FDR) method. ( C ) The expression of STAT1, p-STAT1, STAT3, p-STAT3 and IRF9 was detected in CD40-silenced cells, and CD40-overexpressed cells with or without preincubation of STATs inhibitor (nifuroxazide 20 μM) for 24 h. ( D ) The level of IRF9 in nucleus and cytoplasm was examined in HBV-transfected HepG2 and Huh7 cells. ( E ) Immunofluorescence assay was utilized to observe the subcellular location of IRF9. Red for IRF9, blue for DAPI. ( F ) Luciferase activity was analyzed after ISRE reporter plasmids co-transfection into cells with CD40 siRNA/siCtrl or CD40 plasmid/vector for 48 h. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Transfection, Sequencing, Expressing, Immunofluorescence, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: CD40 induces BST2 expression. ( A – B ) BST2 was selected as downstream target gene of JAK–STAT pathway. Several JAK–STAT pathway related genes in HepG2.2.15 (A) and HepAD38 (B) transfected with CD40 siRNA were detected by qPCR. ( C – E ) CD40 modulated BST2 expression via JAK–STAT pathway. In HBV-transfected and HBV-infected model, the level of BST2 was detected in CD40-silenced cells, and CD40-overexpressed cells with or without preincubation of STATs inhibitor (nifuroxazide 20 μM) for 24 h (C). HBV-transfected HepG2 and Huh7 cells transfected with CD40 siRNA or siCtrl were seeded in 24-well plate (2 × 10 4 cells/well) and cultured for 48 h, and then the expression of BST2 was analyzed by immunofluorescence assay (D and E). Red for BST2, blue for DAPI. ( F – G ) CD40 is positively associated with BST2. Correlation of CD40 and BST2 in liver tissues was plotted by the GTEx database (F). Correlation of CD40 and BST2 in HBV infected liver tissues from published data (GSE84044) was plotted (G). Experiments were performed in triplicate.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Expressing, Transfection, Infection, Cell Culture, Immunofluorescence

Journal: Human Molecular Genetics

Article Title: A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis

doi: 10.1093/hmg/ddac284

Figure Lengend Snippet: Suppression of JAK/STAT/BST2 axis abrogates CD40-induced inhibitory effect of HBV. ( A – C ) Overexpression of BST2 restrained HBV replication and transcription. HBV DNA load in HepAD38 cells supernatant was determined. Intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg in HBV-transfected HepG2, or HBV-infected HepG2-NTCP cells transfected with BST2 plasmid or vector were analyzed as indicated. ( D – E ) Knockdown of BST2 partially abrogated overexpressed CD40-induced suppression of HBV replication and transcription. CD40 plasmid and BST2 siRNA were sequentially transfected into HBV-transfected Huh7 and HBV-infected HepG2-NTCP cells, and then intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg were detected. ( F – G ) Inhibition of the JAK/STAT pathway with nifuroxazide decreased BST2 expression, and restored HBV replication and transcription which were suppressed by CD40. HBV-infected HepG2-NTCP cells and PHHs were transfected with CD40 plasmid for four days and then treated with nifuroxazide for one more day. Expression of intracellular HBV total RNA, HBV 3.5-kb RNA, HBsAg and HBcAg was analyzed. Data represent the mean ± SD of three independent experiments. * P < 0.05, * * P < 0.01, * * * P < 0.001.

Article Snippet: HepG2.2.15 or HepAD38 cells transfected with CD40 siRNA/siCtrl were stimulated with CD40 ligand (CD40L;1 μg/ml; Cell Signaling Technology, Danvers, MA, USA), neutralizing by preincubation with monoclonal antagonistic antibody against CD40 (anti-CD40; 5 μg/ml; R&D Systems, Minneapolis, MN, USA) or antagonistic antibody against CD40L (anti-CD40L; 0.1 μg/ml; Ancell, Bayport, MN, USA) for 1 h. HepG2.2.15 or HepAD38 cells were seeded in a 6-well plate (5 x 10 5 cells/well) overnight and then transfected with CD40 plasmid/vector.

Techniques: Over Expression, Transfection, Infection, Plasmid Preparation, Knockdown, Inhibition, Expressing