syngenic agematched cd40 knockout mice ko  (Jackson Laboratory)

Journal: Oncotarget

Article Title: CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer

doi:

Figure Lengend Snippet: MDSC were isolated from the spleens of WT C57BL/6 mice without tumor inoculation (tumor-free; n = 5) or from the spleens and tumors of mice with tumors (diameter = 1 cm; n = 5) grown from subcutaneously injected MFC, RM-1 or LLC cells. CD40 + % MDSC were analyzed using flow cytometry after staining isolated MDSC with PE-conjugated anti-CD40 antibody (CD40 − PE; blue line) or PE-conjugated isotype-matched IgG control antibody (red line). a. Representative flow cytometry images showing minimal CD40 + cell detection in tumor-free mouse spleen, and a dramatic increase in CD40 + % MDSC after tumor formation from all three different types of cancer cells. The highest CD40 + % levels were detected in tumor tissues. b. CD40 + % quantification in different groups of mice. * p < 0.05 and ** p < 0.01, compared to the corresponding tumor tissues.

Article Snippet: Syngenic age-matched CD40 knockout mice (KO) were purchased from the Jackson Laboratory (Maine, USA).

Techniques: Isolation, Injection, Flow Cytometry, Staining, Control

Journal: Oncotarget

Article Title: CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer

doi:

Figure Lengend Snippet: Single cells dissociated from MFC tumors were stained with fluorophore-conjugated anti-CD11b, anti-Gr-1 and anti-CD40 antibodies. The gating strategy for FACS as well as the pre- and post-sorting percentages of CD40 high and CD40 low MDSC are shown.

Article Snippet: Syngenic age-matched CD40 knockout mice (KO) were purchased from the Jackson Laboratory (Maine, USA).

Techniques: Staining

Journal: Oncotarget

Article Title: CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer

doi:

Figure Lengend Snippet: Gene expression profiles of CD40 high MDSC and CD40 low MDSC were examined by microarray analysis. a. Scatter plot of microarray data showing genes that were more than two-fold upregulated (red), less than two fold changed (black) or downregulated by more than two fold (green) in CD40 high compared with CD40 low MDSC. b. Heat map representation of microarray data on genes of interest. Expression levels are indicated on a color scale where red represents higher expression and green lower expression. c. qRT-PCR analysis of eight genes comparing CD40 high and CD40 low MDSC expression levels. The expression level of each target gene relative to an internal control (β-actin) was determined using the 2 −ΔΔCT method. The fold change between the two cell groups is presented.

Article Snippet: Syngenic age-matched CD40 knockout mice (KO) were purchased from the Jackson Laboratory (Maine, USA).

Techniques: Gene Expression, Microarray, Expressing, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer

doi:

Figure Lengend Snippet: Flow cytometry analysis of the percentage of CD11b + Gr-1 + MDSC (MDSC%) within tumor tissues from WT ( n = 6) or KO ( n = 6) mice a and b. , and the CD40 + versus CD40 − MDSC subsets within WT tumor tissues ( c and d. n = 9). a) Representative flow cytometry images demonstrating the gating strategy and a higher MDSC% in tumor tissues from WT mice when compared with KO mice. b) Quantification of the MDSC% determined in a. c) Representative flow cytometry images demonstrating the gating strategy and a significantly higher percentage of CD40 + than CD40 − MDSC in WT tumor tissue. d) Quantification of the flow cytometry data shown in c.

Article Snippet: Syngenic age-matched CD40 knockout mice (KO) were purchased from the Jackson Laboratory (Maine, USA).

Techniques: Flow Cytometry

Journal: Oncotarget

Article Title: CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer

doi:

Figure Lengend Snippet: a. MFC were injected into WT ( n = 6) and KO ( n = 6) mice. Tumor growth was measured and is presented as tumor size at the indicated post-MFC inoculation time points. * p < 0.05, compared with tumors from CD40 KO mice. b. Fifteen days after MFC inoculation, the mice were sacrificed and tumors were excised and photographed. Tumors from WT mice were larger than tumors from KO mice. WT tumors were also irregularly shaped, with rough surfaces and ulcers.

Article Snippet: Syngenic age-matched CD40 knockout mice (KO) were purchased from the Jackson Laboratory (Maine, USA).

Techniques: Injection

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: Expression of costimulatory molecules and MHC II by MDSC upon IFN-γ stimulation. Bone marrow Percoll fraction 2 cells, which contain MDSC, were cultured in the presence or absence of IFN-γ (100 ng/mL). Twenty-four hours later, cells were stained with fluorochrome-conjugated anti-Gr-1, anti-CD115, anti-CD40, anti-CD80, anti-CD86, and anti-I-Ab or isotype control. A, induction of CD40 on MDSC by IFN-γ. Flow cytometric data obtained from one representative experiment are presented as dot plots. B, significant induction of CD40 by IFN-γ. The results obtained from six tumor-bearing mice are presented. C, constitutive expression of CD80 and CD86 on MDSC.

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: Expressing, Cell Culture, Staining, Control

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: Requirement of CD40 for MDSC-mediated T-cell suppression and Treg expansion in vitro. A, Treg induction by WT versus CD40 KO MDSCs. Purified CD4+ T cells were cocultured with WT or CD40 KO MDSCs at 4:1 ratio for 5 d followed by flow cytometry to assess the presence of CD4+CD25+Foxp3+ Tregs. Data are gated on CD4+ populations (n =6). B, expansion of CD4+CD25+Foxp3+ Tregs by WT, but not CD40 KO, MDSCs. CFSE-labeled, purified CD4+CD25– T cells and CD4+CD25+ Tregs from naïve mice were cocultured with WT or CD40 KO MDSCs at 4:1 ratio in the presence of anti-CD3/anti-CD28 for 3 d. Proliferation was assessed by flow cytometry. Histograms of CD4+CD25– population were gated on CD4+ cells, whereas those of CD4+CD25+ population were gated on Foxp3+ cells. Data from one representative of three reproducible experiments. C, CFSE-labeled, purified CD4+CD25+ Tregs from naïve OT-II mice were cocultured with WT or CD40 KO MDSCs at 4:1 (T cell to MDSC) ratio in the presence of irradiated OVA-EL4 cell (3,000 rad) at 10:1 ratio in the presence of IL-2 for 4 d. In the transwell experiment, MDSCs and Treg were added in the upper and lower chambers, respectively. Proliferation (CFSE dilution) was assessed by flow cytometry. Data from one representative of three reproducible experiments. Mean ± SD of each group has presented in results (n = 6).

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: In Vitro, Purification, Flow Cytometry, Labeling, Irradiation

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: Reversal of MDSC mediated immune suppression by anti-CD40. HA-TCR tumor (HA)–specific Thy-1.2 T cells were sorted from recipient HA-MCA26 tumor-bearing mice. Three mice per group were used in each of three reproducible and independent experiments. A, proliferative response of sorted Thy-1.2 T cells. Data are expressed as stimulation index (SI) relative to the cpm of T-cell proliferation in the absence of peptide (rat immunoglobulin versus anti-CD40; P < 0.001). B, IL-10 and TGF-β secretion by Thy-1.2 T cells. The concentrations of IL-10 and TGF-β in the culture supernatants were measured by ELISA (**, P < 0.001). C, prevention of Treg development by anti-CD40 in vivo. Foxp3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was assessed by RT-PCR of total RNA derived from sorted T cells. D, intracellular staining of Foxp3 in recovered tumor (HA)–specific T cells.

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: Enzyme-linked Immunosorbent Assay, In Vivo, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Staining

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: CD40 expression by MDSC is required for MDSC mediated tumor specific T cells immune suppression in vivo. After 9 d of adoptive transfer, tumor (HA)–specific CD4 Thy-1.2+ T cells were sorted from recipient HA-MCA26 tumor-bearing mice (Thy1.1+). One of three reproducible experiments was presented. Three mice per group were used in each of three independent experiments. A, proliferative response of sorted tumor-specific T cells. Data are expressed as stimulation index (SI) relative to the cpm of T-cell proliferation in the absence of peptide (W/MDSC versus W/O MDSC; P < 0.001, and no significant difference in other group). B, reduction in Foxp3 expression by tumor (HA)–specific T cells recovered from mice that also received CD40-deficient MDSCs. Foxp3 gene expression was assessed by RT-PCR on total RNA prepared from sorted T cells (WT MDSC versus WT MDSC + anti-CD40; P < 0.001).

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: Expressing, In Vivo, Adoptive Transfer Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: CD40 is essential for Treg expansion, immune suppression, and tumor promotion mediated by MDSCs. OVA-B16–bearing MaFIA mice (CD45.2) were left untreated or depleted of CD115+ cells followed by adoptive transfer of CD45.1 OT-II T cells and reconstitution of WT or CD40 KO MDSC. A, tumor (OVA)–specific CD4+CD25+Foxp3+ Tregs in the tumor. Tumor infiltrated leukocytes were isolated and stained with antibodies against CD45.1, CD4, CD25, and Foxp3 or isotype controls followed by flow cytometry. Contour plots gated on CD45.1+CD4+ population are presented. One of three reproducible experiments (n = 3–4 per group) was presented. B, the number of tumor (OVA)–specific Tregs in the tumor. The numbers of CD45.1+ OT-II Tregs in the tumor were calculated (second versus first column, P < 0.001; fourth versus third column, P = 0.004). C, proliferative response of tumor (OT-II)–specific T cells reisolated from the recipient MaFIA mice. CD45.1+ OT-II T cells were reisolated from the spleen of recipient mice and stimulated with OVA peptide (1 μg/mL) in the presence of irradiated naïve splenocytes for 3 d. [3H]Thymidine was added in the last 8 h of culture (second versus first column, P = 0.002; fourth versus third column, P = 0.009). D, tumor weight in various treatment groups. Tumors were resected from tumor-bearing mice and weighed (second versus first column, P = 0.011; fourth versus third column, P = 0.006). Data were combined from three reproducible experiments (n = 9).

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: Adoptive Transfer Assay, Isolation, Staining, Flow Cytometry, Irradiation

Journal:

Article Title: Immune Stimulatory Receptor CD40 Is Required for T-Cell Suppression and T Regulatory Cell Activation Mediated by Myeloid-Derived Suppressor Cells in Cancer

doi: 10.1158/0008-5472.CAN-09-1882

Figure Lengend Snippet: Effect of the combination of anti-CD40 and Adv/mIL-12 plus anti-4-1BB mAb on antitumor immunity. Mice bearing large MCA26 tumors were randomly assigned to four treatment groups. The long-term survival rate of mice treated with Adv/mIL-12 + anti-4-1BB + anti-CD40 (n = 12) is significantly higher than that of mice treated with Adv/mIL-12 + anti-4-1BB + rat immunoglobulin (n = 20; **, P < 0.01, log-rank test) and that of mice treated with DL312 + anti-4-1BB + anti CD40 (n = 12) or Adv/mIL-12 + anti-CD40 + rat immunoglobulin, respectively (n = 12; **, P < 0.01, log-rank test). All of the mice in the DL312 + rat immunoglobulin treatment group (n = 10) died on or before day 30 after tumor implantation. The combined results from two separate and reproducible experiments are presented.

Article Snippet: Wild-type (WT) BALB/c, CD40-deficient BALB/c, ovalbumin (OVA)–specific MHC class II–restricted TCR-transgenic (OT-II) CD45.1 C57BL6, and CD40 knockout (KO) CD45.2 C57BL6 mice were purchased from National Cancer Institute and The Jackson Laboratory.

Techniques: Tumor Implantation