Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Monoallelic expression of FL reduces FL production. (A) Semi-quantitative RT-PCR of FL transcripts in BM cells from C57BL/6 (B6), FL+/+ x RAG1-GFP/+, FL+/− x RAG1-GFP/+, or FL−/− x RAG1-GFP/+ mice. The cDNA was serially diluted 1:1, 1:3, and 1:9 for semi-quantitative analysis. Beta-actin was used as a loading control. Data are representative of two BM samples for each genotype. (B) Quantification of FL transcripts. Intensity data are the average of two BM samples for each genotype. (C) Concentration of FL (pg/mL) in the serum of C57Bl/6 (B6), FL+/−, and FL−/− mice as determined by ELISA. Data represents the mean ± S.D. (*p ≤ 0.0001) FL concentration in serum from ≥ 5 mice/genotype and 2 independent experiments. P-values were determined using the Students T-test.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: FL haploinsufficiency reduces LHP and RAG1 locus activation. (A) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP x FL+/+, RAG1-GFP x FL+/−, and RAG1-GFP x FL−/− mice (top panels). Differential expression of Flt3 and VCAM-1 in LSK+ BM cells to discriminate HSC/MPP, GMLP, and LMPP (bottom panels). Boxed regions indicate Flt3hi GMLP. (B) Histogram of boxed region from (A) bottom panels, indicative of Flt3 expression in Flt3hi GMLP in different FL genotypes. (C) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP+ x FL mice to distinguish HSC, MPP, and LHP. A littermate GFP- control was analyzed in each experiment to determine GFP+ gates (data not shown). (D) Flow cytometric analysis of Lin- BM cells to distinguish CLP: Lin- c-kitlo IL-7R+ (Top panels). CLP were further discriminated by Flt3 and Sca-1 expression (middle panels). GFP expression within Lin- c-kitlo IL-7R+ Flt3+ Sca-1+ CLP (bottom panels). GFP+ gates were determined by analysis of Lin- c-kitlo IL-7R- Flt3+ cells which do not express GFP (data not shown). (E) Flow cytometric analysis of BCP. Pre-Pro-B/Pro-B cells are B220+ CD43+, Pre-B cells are B220+ CD43-, and naïve/mature B cells are B220hi CD43-. Data are representative of ≥ 5 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Activation Assay, Quantitative Proteomics, Expressing, Control

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Id1 does not rescue the lymphoid defect in FL−/− mice. (A) Flt3, tcfe2a, rag1, and ebf1 and (B) Scl and id1 were analyzed from FACS sorted GMLP and LMPP from C57Bl/6 (B6) and FL+/− mice by quantitative PCR. (A+B) Data are representative of 2 independent experiments with BM pooled from ≥ 5 mice/genotype. Error bars represent the mean ± SEM. (C) Flow cytometric analysis of BM from C57Bl/6 (B6), Id1−/−, FL−/−, and FL−/− Id1−/− mice stained with antibodies for lineage markers, c-kit, and Sca-1 to determine LSK+ cells (top panels). LSK+ cells stained with antibodies to Flt3 and VCAM-1 to distinguish HSC/MPP, GMLP, and LMPP (bottom panels). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Flt3 regulates the survival, but not the proliferation of LHP. (A-B) Flow cytometric analysis of bone marrow to examine BrdU incorporation 12 hours after 2 mg were injected i.p. into FL+/+ and FL−/− mice in c-kit+ Sca-1+ (SK+) (A) and c-kit+ Sca-1- (B). (A) Total BrdU incorporation in SK+ cells (top panels). BrdU incorporation in SK+ cells using Flt3 as additional criteria (bottom panels). (B) Total BrdU incorporation in c-kit+ Sca-1- cells (top panels). BrdU incorporation in c-kit+ Sca-1- cells using Flt3 as additional criteria (bottom panels). BrdU quadrants are set on the IgG isotype control for each genotype (data not shown). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments. (C-D) Annexin V staining and intracellular Mcl-1 expression in LSK+ cells (C) and CLP (D). FL+/+ represented by filled histogram, FL−/− indicated by solid line, and thin line depicts isotype control. (C) LSK+ cells were fractionated into Flt3lo and Flt3+hi subsets (top panels). Overlaid histograms depicting Annexin V staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (bottom panels). (D) CLP fractionated into three subsets: Flt3neg, Flt3lo, and Flt3hi (top panels). Overlaid histograms depicting Annexin V staining in CLP subsets (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in CLP subsets (bottom panels). Data are representative of 3 independent experiments (C+D).

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: BrdU Incorporation Assay, Injection, Control, Staining, Expressing

Journal: Immunity

Article Title: Activation of p53 in immature myeloid precursor cells controls differentiation into Ly6c + CD103 + monocytic antigen-presenting cells in tumors

doi: 10.1016/j.immuni.2017.12.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Tumors were disaggregated by treating for 1 hr with collagenase, DNAse and hyaluronidase in RPMI 1640 medium, as described ( Sharma et al., 2015 ). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-Flt3L neutralizing antibody (goat polyclonal) R&D Systems Cat#AF427 anti-IL-12p40 neutralizing antibody (clone C17.8) BioXcell Cat#BE0051 anti-CTLA4 blocking antibody (clone 9D9) BioXcell anti-PD-1 blocking antibody (clone J43) Laboratory of Dr. Hideo Yagita, or from BioXcell ( Agata et al., 1996 ), Cat#BP0033-2 anti-PDL2 blocking antibody (clone TY25) Laboratory of Dr. Hideo Yagita, or from BioXcell ( Yamazaki et al., 2002 ), Cat#BE0112 anti-PD-L1 blocking antibody (clone MIH7) Laboratory of Dr. Miyuki Azuma ( Tsushima et al., 2003 ) CD4 (mouse) (clone RM4-5) BD-Bioscience Cat#553051 CD8 (mouse) (clone 53-6.7) BD-Bioscience Cat# 561092 CD86 (mouse) (clone GL1) BD-Bioscience Cat#553692 CD11c (clone HL3) BD-Bioscience Cat#561119 Ly6c (mouse) (clone AL-21) BD-Bioscience Cat#560592 IFNγ (mouse) (clone XMG1.2) BD-Bioscience Cat#554412 CD24 (clone M1/69) BD-Bioscience Cat#561079 CCR7 (mouse) (clone 4B12) BD-Bioscience Cat#560682 Foxp3 (mouse) (clone FJK-16s) eBioscience Cat#11-5773 granzyme B (mouse) (clone NGZB) eBioscience Cat#12-8898 PD1 (mouse) (Clone: J43) eBioscience Cat#12-9985 PDL1 (mouse) (clone MIH5) eBioscience Cat#12-5982 CD103 (mouse) (M290) BD-Bioscience Cat#557495 Ly6c (mouse) (clone HK1.4) eBioscience Cat#45-5932 CD69 (mouse) (clone H1.2F3) eBioscience Cat#11-0691 NOS2 (mouse) (C-11) Santa Cruz Biotech.

Techniques: Blocking Assay, Virus, Recombinant, Adjuvant, Isolation, Software

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 1. Flt3L-Ag85B is expressed by DNA vaccines in a functional form. (A) Schematic representation of pCDNA3 vectors expressing Flt3L, M. tuberculosis Ag85B (p85B), and the Flt3L-Ag85B fusion protein (pFlt-85). (B) Secretion of Flt3L by DNA-transfected HEK 293 cells. Flt3L in the culture medium of cells 3 days after transfection was detected by ELISA. (C) Generation of DCs from bone marrow progenitors using culture medium from DNA-transfected HEK 293 cells. The generation of CD11c MHC-II cells on day 6 after addition of HEK 293 supernatants to bone marrow cells was determined by flow cytometry. (D) Total number of CD11c MHC-II cells in culture.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Vaccines, Functional Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cytometry

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 2. Immunogenicity and protective efficacy of DNA encoding murine Flt3L and M. tuberculosis Ag85B. (A) Splenocytes from immunized mice were cultured with 3 g/ml Ag85B, and the level of IFN- released was determined by ELISA. (B) For determination of protective efficacy, 4 weeks following the final vaccination mice were infected by the aerosol route with 100 CFU of M. tuberculosis H37Rv. Four weeks after challenge, the bacterial load, expressed as the log10 CFU (mean standard error of the mean), was analyzed in the lung. The significance of differences between groups was determined by ANOVA. The error bars indicate standard errors of the means, and the data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Immunopeptidomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Aerosol

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 3. Construction and in vivo immunogenicity of BCG:Flt3L. BCG Pasteur was transformed with the control pMV261 plasmid (BCG:Ct) or plasmid pJEX73 (BCG:Flt3L), and the presence of Flt3L in cell lysates was determined by immunoblotting with the anti-c-myc MAb 9E10 (A). The presence of Flt3L in the culture supernatants of rBCG strains was determined by murine Flt3L-specific ELISA (B). To assess the immunogenicity, mice were not vaccinated (striped bar) or were vaccinated with 5 105 CFU of BCG:Ct (open bars) or BCG:Flt3L (solid bars), and at the indicated time points splenocytes (C) or DLN cells (D) were stimulated with 1 g/ml BCG lysate. The number of IFN--secreting cells was determined by an enzyme-linked immunospot assay. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct- vaccinated animals (asterisk, P 0.05) was determined by ANOVA. The data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: In Vivo, Immunopeptidomics, Transformation Assay, Control, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 4. Influence of BCG:Flt3L on DC numbers in vaccinated mice. Mice were vaccinated subcutaneously with 5 105 CFU of rBCG, and at the indicated time points the numbers of CD11c MHC-II cells in the spleen (A, C, and E) and DLNs (B, D, and F) were determined by flow cytometry. The cells were further defined as cells that were B220 (C and D) or B220 (E and F). Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated animals; solid bars, BCG:Flt3L-vaccinated animals. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct-vaccinated animals (asterisk, P 0.05) was determined by ANOVA. The data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Cytometry

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 5. Protective efficacy of BCG secreting Flt3L. Mice were immunized subcutaneously with 5 105 CFU of BCG:Flt3L or BCG:Ct, and 12 weeks after immunization they were challenged by the aerosol route with M. tuberculosis H37Rv. Four weeks postchallenge the bacterial loads, expressed as log10 CFU (means standard errors of the means), were analyzed in the (A) lungs and (B) spleens of mice. The statistical significance between naı¨ve and rBCG-vaccinated groups (asterisk, P 0.01) was analyzed by ANOVA. The data are representative of three individual experiments. Unv, unvaccinated mice. (C and D) For assessment of rBCG growth in vivo, mice were infected i.v. with 1 106 CFU of BCG:Flt3L (F) or BCG:Ct (E). At 1, 14, 28, and 56 days postinfection the bacterial loads were assessed in the lungs (C) and spleens (D). The statistical significance between groups (asterisk, P 0.05) was analyzed by ANOVA.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Aerosol, In Vivo, Infection

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 6. Safety of BCG:Flt3L in immunodeficient mice. (A) RAG- 1/ mice were infected i.v. with 1 106 CFU of BCG:Flt3L (F) or BCG:Ct (E), and survival was monitored over time. The significance of differences in survival was determined by the Mantel-Cox log rank test (asterisk, P 0.001). (B and C) RAG-1/ mice were vaccinated subcutaneously with 5 105 CFU of rBCG strains, and the numbers of CD11c MHC-II cells in the DLNs (B) and spleens (C) of the vaccinated mice were determined by flow cytometry. Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated mice; solid bars, BCG:Flt3L-vaccinated mice. There were no statistically significant dif- ferences between groups as determined by ANOVA. The data are representative of one of two individual experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Infection, Cytometry