Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: A schematic of the mouse Batf2 gene, genotyping results, and the s.c. inoculated mouse tumor model. (A) A schematic of the mouse Batf2 gene, the targeting vector, and the targeted allele. (B) Genotyping PCR of Batf2+/+, Batf2+/−, and Batf2−/− mice. As indicated in A, primers shextra and wild together detect a fragment of the WT genomic locus, while primers shextra and PGKRC2 together detect a fragment of the mutant allele. (C) The relative expression levels of Batf2 mRNA in BMDMs from Batf2−/− or WT littermates following stimulation by LPS (100 ng/mL) and IFN-γ (30 ng/mL) were quantified using qPCR (Left). The Batf2 mRNA levels in BMDMs from Batf2−/− or WT littermates following stimulation with 103 U/mL of recombinant IFN-α were quantified using qPCR (Right). Error bars indicate ±SEM (n = 2). (D) WT and Batf2−/− mice were treated daily with imiquimod cream on shaved back skin for 6 d. The resulting erythema scores on days 4−6 are shown. Error bars indicate ±SEM (n = 6). *P < 0.05. (E) A schematic of the s.c. inoculated mouse tumor model. For this model, 1 × 105 B16-F1 cells were injected s.c. into Batf2−/− mice and WT littermates. The tumor volumes were calculated using the formula (width)2 × length × 0.52. (F–H) Tumor growth kinetics in Batf2−/− (triangle) and WT (square) littermates following an s.c. injection of 1 × 106 Lewis lung carcinoma cells (F), 1 × 106 B16-F10 melanoma cells (G), or 5 × 105 Colon-26 cells (H) per mouse. Average kinetics ± SEM of three (F), five (G), or three (H) mice per group. *P < 0.05.

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Recombinant, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Tumor growth in Batf2−/− mice and WT littermates. (A) BMDMs from WT and Batf2−/− littermates were stimulated with R848 for 8 h, and the total RNA from these cells was subjected to microarray analysis. The log2 ratios of the top 15 most highly expressed genes in WT BMDMs are shown (Left). The log2 ratio for each of the corresponding genes in Batf2−/− BMDMs was subtracted from that of WT, and the results were compared between WT and Batf2−/− (Right). (B) Tumor growth kinetics in Batf2−/− (triangle) and WT (square) littermates following their s.c. injection with 1 × 105 B16-F1 melanoma cells (Left). Average kinetics ± SEM of n = 6–7 mice per group. Tumor size on days 12–14 in Batf2−/− (n = 42) and WT (n = 44) littermates following their s.c. injection with B16-F1 cells (Right). Data are from 10 independent experiments. Bars show means. *P < 0.05; ***P < 0.001. (C) Photographic (Upper) and FDG PET–CT fusion (Lower) images of mice on day 17 after receiving an s.c. injection of B16-F1 cells (arrows). (D) Representative 18F-FDG PET images for tumor detection in Batf2−/− (Lower) and WT (Upper) mice on day 17 after receiving an s.c. injection of B16-F1 cells (arrows). A, anterior; I, inferior; L, left; P, posterior; R, right; S, superior. White cross-hairs indicate the locations of the other two cross-sections. (E) Leukocyte subpopulations in the tumor tissues of WT mice (n = 14) 2 wk post-B16-F1 implantation were sorted, and their relative Batf2 mRNA expression levels were quantified using qPCR. Data are expressed as mean ± SEM of triplicates. CD4T, CD4+ T cells; CD8T, CD8+ T cells; Mono, monocytes; Neut, neutrophils; NK, natural killer cells. (F and G) Histological observation of tumor tissues in a B16-F1 melanoma mouse model: H&E stain (F) and immunofluorescence detection (G). PI (red), CD31 (green), and CD68 (blue) are shown. (H) Immunofluorescence detection of BATF2 (green) and CD68 (blue) in the normal skin from naïve WT mice or in tumor tissues 2 wk after B16-F1 implantation in WT mice. (Scale bar, 100 μm in F–H.) All experiments were performed on male littermates.

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Microarray, Injection, Positron Emission Tomography-Computed Tomography, Expressing, Staining, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Analysis of immune cells within the tumors of WT and Batf2−/− mice. All samples were obtained from the tumor tissues of Batf2−/− and WT littermates 2 wk after B16-F1 implantation. (A) The numbers of tumor-infiltrating CD11b+ F4/80+ cells (Left) and CD45+ cells (Right). Data are from five independent experiments (n = 7–9). Bars show means. (B) Tumor tissue macrophages were examined by fluorescent staining of PI (red) and CD68 (blue). (C) Tumor tissue macrophages were sorted (n = 5–6), and their relative Il12b mRNA expression levels were quantified using qPCR. Data are expressed as mean ± SEM from three independent experiments. (D) The IL-12 p40 expressions of CD45+ CD11b+ F4/80+ macrophages from tumor tissues were analyzed by flow cytometry. Numbers represent the percentages of IL-12 p40+ cells within the CD45+ CD11b+ F4/80+ cell population. Representative plots are shown (Left). Data are expressed as mean ± SEM from three independent experiments (Right, n = 5). (E) Tumor-infiltrating T cells were examined by immunofluorescent staining of CD4 (blue) and CD8 (yellow). (F) Flow cytometric analysis of tumor-infiltrating CD8+ T cells, CD4+ T cells, and CD45+ cells. Numbers indicate the actual numbers of detected cells. Data are expressed as mean ± SEM from two independent experiments (n = 4). (G) Flow cytometric analyses of tumor-infiltrating IFN-γ+ CD4+ T cells and IFN-γ+ CD8+ T cells. Data are expressed as mean ± SEM from four independent experiments (n = 4). (H) Tumor-infiltrating CD4+ T cells were sorted (n = 2–3), and their relative mRNA expression levels of Tbx21 were quantified using qPCR. Data are expressed as mean ± SEM from two independent experiments. (I and J) Flow cytometric analyses of tumor-infiltrating, Ki-67+ CD8+ T cells (I) and Annexin V+ PI+ CD8+ T cells (J). Numbers represent the actual cell numbers and the percentages of these cells within the CD8+ T-cell population. Representative plots are shown (Left). Samples were obtained from one (I) or three pair(s) of mice (J). Data are expressed as mean ± SEM from two independent experiments (Right). All experiments were performed on male littermates. (Scale bar, 100 μm.) In all figures, *P < 0.05; n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Flow cytometric analyses of macrophages, DCs, and T cells. (A) A flow cytometric analysis of CD11b+ F4/80+ splenocytes of tumor-bearing mice. Numbers represent the percentage of cells within the total cell population or the actual cell number. Representative plots are shown (Left). Data are expressed as mean ± SEM from three independent experiments (Right, n = 4). (B–D) Flow cytometric analyses of DCs within the population of splenocytes from tumor-bearing mice (B and C) or within the B16-F1 tumors (D). Representative plots are shown (Left). Data are expressed as mean ± SEM from two independent experiments (Right). Numbers represent either the actual cell number or the percentage of cells within the total cell population (B, n = 3), CD45+ cell population (C, n = 3), or CD45+ CD11c+ cell population (D, n = 4). (E) Mean MFI of MHC class I, class II, CD80, and CD86 in tumor-infiltrating DCs (n = 3). Data are expressed as mean ± SEM from two independent experiments. (F) The Vegfa mRNA expression in BMDMs that had been stimulated with R848 for 4 h was quantified using qPCR. Data are expressed as mean ± SEM from two independent experiments (n = 3). (G) A flow cytometric analysis of CD45− CD31+ endothelial cells in the tumor tissues from Batf2−/− mice and WT littermates. Numbers represent the percentage of cells within the total tumor cell population. Representative plots are shown (Left). Data are expressed as mean ± SEM (Right, n = 2). (H) Flow cytometric analyses of the T-cell populations within the spleen, blood, lymph nodes (LN), and BM of naïve or tumor-bearing mice. Numbers represent the percentage of cells within the CD3+ T-cell population. Representative plots are shown (Left). Data are expressed as mean ± SEM (Right, n = 3). All experiments were performed on male littermates. In all figure parts, *P < 0.05; **P < 0.01; n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Tumor growth in BM chimeric mice. (A) Lethally irradiated WT mice received BM transplants from Batf2−/− or WT littermates. Eight weeks after BM transplant, the chimeric mice were each injected s.c. with 1 × 105 B16-F1 cells, and tumor growth was monitored (Left). Tumor sizes on day 18 are shown (Right). Error bars indicate ±SEM. (B) A flow cytometric analysis was performed on the tumor tissues of BM chimeric mice 2 wk after they received an implantation of B16-F1 cells. The populations of CD45+ CD11b+ F4/80+ macrophages from tumor tissues and their IL-12 p40 expressions were analyzed by flow cytometry. Numbers represent the percentages of F4/80+ cells within the CD45+ CD11b+ cell population (Upper) and of IL-12 p40+ cells within the CD45+ CD11b+ F4/80+ cell population (Lower). (C and D) An analysis of T cells within the tumor tissues of BM chimeric mice. Numbers represent the percentage of cells within the tumor-infiltrating CD45+ CD3+ cell (C) or CD8+ T-cell (D) populations. Representative plots from two independent experiments are shown. (E) BM chimeric mice were each injected s.c. with 1 × 106 Lewis lung carcinoma cells, and tumor growth was monitored (Left). Tumor sizes on day 12 are shown (Right). Error bars indicate ± SEM. (F) A photographic image of BM chimeric mice taken 15 d after the mice received an s.c. injection of Lewis lung carcinoma cells (E). Bars show means. In all figure parts, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Irradiation, Injection, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Analysis of the T-cell activities in Batf2−/− mice and WT littermates. (A) Splenic CD8+ T cells were isolated from Batf2−/− mice and WT littermates without tumors (naïve) and then stimulated with PMA and ionomycin (ION) for 4 h in the presence of monensin for the last 2 h. The IFN-γ expressions in these CD8+ T cells were analyzed by flow cytometry. Data are from two independent experiments (n = 4). (B) Naïve splenic CD8+ T cells isolated from Batf2−/− mice or WT littermates were transferred i.v. into Rag2−/− recipients. After 24 h, the recipient mice were injected s.c. with B16-F1 tumor cells (schematic, Left). Tumor growth was monitored (Right) in the mice with Batf2−/− CD8+ T cells (triangle) or WT CD8+ T cells (square). Average kinetics ± SEM of n = 3 mice per group. (C) In vivo migration assay. Naïve CD8+ T cells isolated from WT splenocytes (n = 14) were labeled with CFSE, and 2 × 107 of these CD8+ T cells per mouse were injected i.v. into Batf2−/− or WT littermates. After 20 h, lymphocytes were obtained from the spleen, lymph nodes, and tumor tissues and analyzed by flow cytometry (schematic, Left). Data are representative of three independent experiments (Right). Numbers represent the percentage of cells within the CD8+ T-cell population. (D) Th1 differentiation assay. Naïve CD4+ T cells from Batf2−/− mice or WT littermates were purified by cell sorting. The cells were cultured in an anti-CD3 antibody-coated plate in the presence of mIL-12, anti–IL-4, mIL-2, and anti-CD28. ELISAs were performed to assess the changes in the culture medium IFN-γ levels over time (Left). After 7 d, the cells were analyzed by flow cytometry (Right). Data are representative of two independent experiments. (E) MHC II-restricted antigen-presentation experiments. CFSE-labeled OT-II transgenic CD4+ T cells were incubated with the splenic CD11c+ DCs from Batf2−/− mice or WT littermates in the continuous presence of 1 mg/mL or 0.1 mg/mL chicken ovalbumin or with 1 μM Ova peptide (323-339) together with 1 µg/mL CpG1826. The percentages of CD4+ CFSElow cells were assessed by flow cytometry after 72 h. A time course of changes in the percentages of the proliferating CD4+ T cells is shown. Data are expressed as mean ± SEM of duplicates. All experiments were performed on male littermates. Bars show means. In all figures, n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Isolation, Flow Cytometry, Injection, In Vivo, Migration, Labeling, Differentiation Assay, Purification, FACS, Cell Culture, Transgenic Assay, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: In vitro protein expression and mRNA levels in BMDMs from WT or Batf2−/− mice. (A) Levels of IL-12 p40, TNF-α, and IL-6 secreted by BMDMs following stimulation by serially diluted concentrations of R848 for 24 h were measured by ELISA. Data are from five independent experiments (n = 10). (B) Relative expression levels of Il12b, Batf2, Tnf, Il6, Ifnb1, and Nos2 in BMDMs stimulated by R848 (100 ng/mL) for 4 h were quantified using qPCR. Data are expressed as mean ± SEM from two independent experiments (n = 3). (C) The mRNA levels of Batf2 and Il12b in BMDMs that had been treated with DOTAP for 4 h were quantified using qPCR. BMDMs were treated with either DOTAP alone, DOTAP formulations of RNA, or RNA alone. RNA was isolated from B16-F1 cells. Data are from three independent experiments (n = 3) and expressed as mean ± SEM. All experiments were performed on male littermates. Bars show means. In all figures, *P < 0.05; **P < 0.01; n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Analysis of the role of Batf2 in CD8+ T-cell proliferation and antitumor responses. (A) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+ T cells from OT-I transgenic mice and BMDMs from Batf2−/− or WT littermates pulsed with Ova 257–264 peptide (SIINFEKL) were cocultured at decreasing dilutions (OT-I:BMDM = 1:1–1:1/8) together with R848. Proliferation of OT-I cells was assessed after 60 h by flow cytometry. (B) CFSE-labeled CD8+ T cells and BMDMs from Batf2−/− or WT littermates with SIINFEKL peptide were cocultured with recombinant IL-12 at decreasing dilutions for 60 h (OT-I:BMDM = 1:1/2). (C) BMDMs (1 × 106) from Batf2−/− or WT littermate donor mice were transferred into Batf2−/− recipient mice on days 0, 4, and 8. Control mice were injected with PBS in the same protocols. Tumor growth was monitored in mice that had received Batf2−/− BMDMs (triangle), WT BMDMs (square), or PBS (circle). (D) Batf2−/− (open triangle) and WT (open square) littermates were injected intraperitoneally with recombinant IL-12 on days 4, 3, and 1 before tumor implantation as well as 24 h after tumor inoculation. Control Batf2−/− (closed triangle) and WT (closed square) mice were injected with PBS in the same protocols. (E) Batf2−/− (open triangle) and WT (open square) littermates were injected intraperitoneally with R848 on days 4, 3, and 1 before tumor implantation and then twice per week after tumor inoculation. Control Batf2−/− (closed triangle) and WT (closed square) mice were injected with PBS in the same protocols. (F) Batf2−/− (open triangle) and WT (open square) littermates were injected intraperitoneally with anti–IL-12 p40 neutralizing antibody at 1 mg/mouse, with follow-up doses of 0.5 mg every 5 d. Control Batf2−/− (closed triangle) and WT (closed square) mice were injected with control IgG in the same protocols. After 1 × 105 B16-F1 cells per mouse were injected, the tumor growth was monitored (C–F). Average kinetics ± SEM of 5 (C), 3–4 (D), 3–4 (E), or 6 (F) mice per group. All experiments were performed on male littermates. In all figures, *P < 0.05; **P < 0.01; n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Labeling, Transgenic Assay, Flow Cytometry, Recombinant, Injection, Tumor Implantation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Analysis of the mechanism responsible for the effect of BATF2 on IL-12 p40 expression. (A) A schematic of the luciferase reporter construct with the mouse IL-12 p40 promoter and its sequence are shown. Underlined sequences indicate the locations of oligonucleotide probes used in DNA-binding assays and EMSAs. Potential transcription factor binding consensus sequences mentioned in the text are boxed and labeled. (B) The mouse IL-12 p40 promoter luciferase reporter construct was transfected into RAW 264.7 cells along with a Batf2 construct. Cells were activated with LPS or R848 at 24 h posttransfection, and luciferase activity was measured 24 h after stimulation (Left). The mouse IL-12 p40 promoter luciferase reporter construct was cotransfected with increasing amounts of Batf2 construct, and cells were activated with LPS (Middle) or R848 (Right). Luciferase activities relative to empty vector control are shown as a representative mean ± SEM. (C) Deletion-mutant (Upper) and substitution-mutant (Lower) analyses of the IL-12 p40 promoter. Mutants were transfected into RAW 264.7 cells with a Batf2 or empty construct. Twenty-four hours after transfection, cells were activated with LPS for 24 h and compared with nonactivated control cells. The result shown for each mutant represents the mean ± SEM of data from three independent experiments. (D) DNA-binding activities of p65 in RAW 264.7 cells transfected with Batf2 or empty constructs were measured with a TransAM Flexi NF-κB Transcription Factor Assay Kit. (E) ChIP analysis was performed using RAW 264.7 cells overexpressing Myc-tagged Batf2. Immunoprecipitation was performed using the following antibodies: normal rabbit IgG (control IgG) and anti–c-Myc or anti–NF-κB p65. Sample DNA was analyzed by PCR using primers that detect sequences containing the Il12b promoter. (F) Data from coimmunoprecipitation experiments assessing BATF2 binding to p50 and p65. For the p50 analysis, cell extracts from RAW 264.7 cells overexpressing both Nfkb1 (p50) and Myc-tagged Batf2 were prepared. After immunoprecipitation with anti-p50 antibody or normal IgG (Control), samples were analyzed with anti–c-Myc antibody by Western blotting. The interactions between Myc-tagged BATF2 and p65 were analyzed similarly. Data are representative of at least two independent experiments. In all figures, *P < 0.05; n.s., not significant (P > 0.05).

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Expressing, Luciferase, Construct, Sequencing, Binding Assay, Labeling, Transfection, Activity Assay, Plasmid Preparation, Mutagenesis, Transcription Factor Assay, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: Analysis of the mechanism responsible for the effect of BATF2 on IL-12 p40 expression. (A) A luciferase reporter construct of the mouse IL-12 p40 (Left) or IL-6 (Right) promoter was transfected into RAW 264.7 cells along with a Batf2 construct. The cells were activated with R848 at 24 h posttransfection, and the luciferase activity was measured 24 h after stimulation. Data are shown as a mean ± SEM, *P < 0.05. (B) EMSAs were performed with nuclear extracts from RAW 264.7 cells transfected with Batf2 or empty constructs. After cells were activated with LPS for 1 h, nuclear extracts (NE) were obtained. The radiolabeled probes contained positions −110 to −69 of the gene sequence, to detect the C/EBP binding site (Left), or positions −132 to −110, which included the NF-κB half-site (Right). Similar results were obtained from two independent experiments. (C) The p50 and p65 expressions in nuclear and cytosolic extracts of RAW 264.7 cells transfected with Batf2 or empty control vector and activated with LPS or R848 for 1 h were analyzed by Western blotting. Data are representative of at least two independent experiments.

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: Expressing, Luciferase, Construct, Transfection, Activity Assay, Sequencing, Binding Assay, Plasmid Preparation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

doi: 10.1073/pnas.1708598114

Figure Lengend Snippet: A schematic model of the antitumor effect of Batf2 through IL-12 p40 up-regulation in TAMs.

Article Snippet: Myc-tagged Batf2 was purchased from OriGene technologies (MR203728).

Techniques: