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