Journal: Oncotarget

Article Title: CD40-activated B cells induce anti-tumor immunity in vivo

doi: 10.18632/oncotarget.7720

Figure Lengend Snippet: A . In the optimal CD40-culture system splenocytes are co-cultured together with CD40L-expressing HeLa cells in the presence of IL-4, ß-mercaptoethanol (ß-ME) and Cyclosporin-A (CsA). Cells are recultivated every 3 days and analyzed or used for experiments after 14 days. B . At every recultivation day the relative increase was assessed by normalizing the relative growth to the percentage of CD3 + and CD19 + cells in the culture as determined by flow cytometry. (n=5). CD40B cells were phenotypically characterized on day 14 by measuring C . the mean fluorescence intensity (MFI) of the activations markers CD80 and CD86 and the MHC molecules I and II D . or the expression of different immunoglobulin subtypes and CD138. MFI values on day 14 were normalized to values on day 0. Significant differences calculated by Student's t-test are indicated by an asterisk: * p ≤ 0.05, ** p ≤ 0.01, ns = not significant. Histograms show representative analyses compared to an unstained control (filled line). Results of five independent experiments are shown.

Article Snippet: For maturation, the medium was supplemented with either 10 ng/ml LPS (Sigma Aldrich) or 1 μg/ml anti-mouse CD40L antibody (clone HM40-3, Acris Antibodies).

Techniques: Cell Culture, Expressing, Flow Cytometry, Fluorescence, Control

Journal: Oncotarget

Article Title: CD40-activated B cells induce anti-tumor immunity in vivo

doi: 10.18632/oncotarget.7720

Figure Lengend Snippet: A-B . For negative controls (Neg. Control) T cells were incubated without stimulating APCs. Dendritic cells were stimulated with LPS (DC LPS) or the CD40L (DC CD40). CD40B cells were used on day 7 (CD40B d7) or day 14 (CD40B d14) of activation. Left column= Typical sequential halving of CFSE fluorescence intensity with each generation was detected by flow cytometry. Right column= CFSE intensity, which was accompanied by upregulation of CD25 expression was detected by flow cytometry. One representative experiment out of 8 is shown. A . CD3 + CD4 + T cells from BALB/C mice were cocultured with the indicated APCs. B . CD3 + CD8 + T cells from BALB/C mice were cocultured with the indicated APCs. C . CD3 + CD4 + T-cell proliferation at various APC-to-T cell ratios in allogenic MLRs with either DC LPS or DC CD40 as APCs. D . CD3 + CD8 + T-cell proliferation at various APC-to-T cell ratios in allogenic MLRs with either DC LPS or DC CD40 as APCs. E . CD3 + CD4 + T-cell proliferation at various APC-to-T cell ratios in allogenic MLRs with either CD40B d7 or CD40B d14 as APCs. F . CD3 + CD8 + T-cell proliferation at various APC-to-T cell ratios in allogenic MLRs with either CD40B d7 or CD40B d14 as APCs. Mean values ± SD of eight independent experiments are shown. Significant differences calculated with Student's t-test are indicated by an asterisk: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For maturation, the medium was supplemented with either 10 ng/ml LPS (Sigma Aldrich) or 1 μg/ml anti-mouse CD40L antibody (clone HM40-3, Acris Antibodies).

Techniques: Control, Incubation, Activation Assay, Fluorescence, Flow Cytometry, Expressing

Journal: Oncotarget

Article Title: CD40-activated B cells induce anti-tumor immunity in vivo

doi: 10.18632/oncotarget.7720

Figure Lengend Snippet: A-C . Left column = Specific lysis was determined by injecting 1 × 10 7 target cells labeled with different amount of CFSE ip. into vaccinated B6 mice 4 days after the last immunization. The ratio of OVA-pulsed target cells versus unpulsed target cells was determined by flow cytometry one day later and the specific lysis was calculated. Right column = Production of IFN-γ was determined by flow cytometry after restimulating purified lymphocytes of vaccinated OT-I mice with peptide. Mean values of frequencies of restimulated cells were normalized to corresponding values of unstimulted cells. Mice were immunized with OVA-peptide pulsed CD40B cells (CD40B +) or unpulsed CD40B cells (CD40 -). Negative controls (Neg. Control) were immunized with PBS+IFA alone, positive controls (Pos. Control) were immunized with Peptide + CpG + IFA. Bar charts show mean values ± SD of four independent experiments with three mice per group. Significant differences calculated with Student's t-test are indicated by an asterisk: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. A . Three different injection routes were tested for vaccination with peptide-pulsed CD40B cells; i.e. intravenous (iv), intraperitoneal (ip) or subcutaneous (sc). B . Different cell numbers were tested for vaccination with peptide-pulsed CD40B cells; i.e. 1 × 10 5 , 1 × 10 6 and 1 × 10 7 . C . Different vaccination intervals were tested for vaccination with peptide-pulsed CD40B cells; i.e. injection on day 0 and 7 (2 inject.) or on day 0, 7 and 14 (3 inject.). Peptide-pulsed DCs stimulated with LPS or CD40L served as additional control. D . CD3 + CD8 + T-cells from spleens of vaccinated B6 mice were stained for the presence of OVA peptide-specific T-cells. Bar charts show mean values ± SD of four independent experiments with three mice per group. Significant differences calculated with Student's t-test are indicated by an asterisk: * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: For maturation, the medium was supplemented with either 10 ng/ml LPS (Sigma Aldrich) or 1 μg/ml anti-mouse CD40L antibody (clone HM40-3, Acris Antibodies).

Techniques: Lysis, Labeling, Flow Cytometry, Purification, Control, Injection, Staining

Journal: Oncotarget

Article Title: CD40-activated B cells induce anti-tumor immunity in vivo

doi: 10.18632/oncotarget.7720

Figure Lengend Snippet: A . Expression of the homing molecules CXCR4, CXCR5, CCR7 and CD62L in CD40B cells from luciferase + mice on day 14 was compared to the expression before stimulation. Histograms show representative analyses of four independent experiments compared to an unstained control (filled line). B . CD40B cells from luciferase + mice were injected intraperitoneally into E.G7 tumor-bearing B6 mice. Homing behavior was measured in the IVIS system on day 1 or day 6 after injection. Representative pictures of five independent experiments with three mice per group are shown. C . B6 mice were treated with 1 × 10 6 or 1 × 10 7 TRP2-peptide pulsed CD40B cells (CD40Bs +) or unpulsed CD40B cells (CD40Bs -) three times in an interval of seven days before challenging them with B16.F10 melanomas 7 days after the last vaccination. Alternatively, mice were vaccinated with 1 × 10 6 LPS or CD40L stimulated DCs. Injection of PBS alone served as negative control (Neg. Control). Tumor growth was assessed daily. One representative experiment out of two independent experiments with three mice per group is shown. Significant differences calculated with paired Student's t-test are indicated by an asterisk: ** p ≤ 0.01, *** p ≤ 0.001. D . B6 mice were treated with 1 × 10 6 OVA-protein pulsed CD40B cells (CD40Bs +) three times in an interval of seven days before challenging them with E.G7 lymphomas 7 days after the last vaccination. As positive control, mice were vaccinated with 1 × 10 6 LPS stimulated DCs. Injection of PBS alone served as negative control (Neg. Control). Tumor growth was assessed daily. One representative experiment of four independent experiments with three mice per group is shown. Significant differences calculated with paired Student's t-test are indicated by an asterisk: ns = not significant, *** p ≤ 0.001.

Article Snippet: For maturation, the medium was supplemented with either 10 ng/ml LPS (Sigma Aldrich) or 1 μg/ml anti-mouse CD40L antibody (clone HM40-3, Acris Antibodies).

Techniques: Expressing, Luciferase, Control, Injection, Negative Control, Positive Control