Journal: EMBO Molecular Medicine

Article Title: Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models

doi: 10.15252/emmm.201607296

Figure Lengend Snippet: A VACV‐neutralizing antibodies were measured in virus‐treated rats 15 and 35 days after implantation ( n = 4–5 rats per group). B Protection from subcutaneous tumor challenge after virus‐induced tumor clearance. AY‐27 cells were implanted in the flanks of cured rats ( n = 6) and naïve age‐matched control rats ( n = 4). C–E T‐cell proliferation after co‐culturing with bone marrow‐derived dendritic cells (BMDCs). CD4 + and CD8 + cells were co‐cultured with BMDCs and proliferation assayed with CellTrace Violet. The representative plots show CD4 + and CD8 + T‐cell proliferation after co‐culture with either mock‐pulsed (C) or with tumor‐lysate‐pulsed BMDCs (D). Panel (E) shows the percentage of CD4 + and CD8 + T cells that proliferated in response to BMDC stimulation ( n = 3). F, G Ex vivo upregulation of CD107a by CD8 + T cells from challenged rats. (F) CD3 + cells were incubated +/− BMDCs for 1 h in the presence of anti‐CD107a antibody, incubated for 5 h with monensin and brefeldin A, and then stained with anti‐CD4 and anti‐CD8 antibodies. Events were gated for viable CD8 + T cells. Panel (G) shows the percentage of CD107a + CD8 + T cells +/− BMDC stimulation ( n = 3). H IFN‐γ released after 24‐h co‐culture of CD3 + cells with BMDCs ( n = 3–5). I T cells activated ex vivo by tumor‐lysate‐pulsed DCs are cytotoxic. After 6 days of co‐culture with BMDC, CD3 + cells were incubated for 18 h with 10,000 target cells and at different effector‐to‐target ratios. Lysis was determined by LDH assay. RK3E are normal rat kidney cells ( n = 2–3 performed in duplicate). Data information: Mean ± SEM is shown. Two‐way ANOVA followed by Tukey's multiple comparison test was used in (A), (B), and (H). For (A), significance was determined against the ∆ J2R group. Two‐tailed Student's t‐ test was used in (E) and (G).

Article Snippet: CD3 + cells were isolated from this preparation using a MagCellectTM Rat CD3 + T‐cell isolation kit following the manufacturer's protocol (R&D Systems).

Techniques: Virus, Control, Derivative Assay, Cell Culture, Co-Culture Assay, Ex Vivo, Incubation, Staining, Lysis, Lactate Dehydrogenase Assay, Comparison, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: 4PD Functionalized Dendrimers: A Flexible Tool for In Vivo Gene Silencing of Tumor-Educated Myeloid Cells.

doi: 10.4049/jimmunol.1600833

Figure Lengend Snippet: FIGURE 6. 4PD-mediated in vivo silencing of STAT3 and C/EBPb restores the efficacy of antitumor vaccines. (A) CT26 tumor–bearing (25 mm2) mice (n = 3) were injected once with 4PD loaded with BrUTP and STAT3 and/or C/EBPb shRNA. At 2, 24, 72, and 120 h postinjection, single-cell suspensions from the tumors were labeled with Abs against CD11b, Ly6G, Ly6C, F4/80, CD206 (to identify gMDSCs, mMDSCs, TAMs, and M1/M2 TAMs), and anti BrU Ab to identify the in vivo–transfected cells (n = 3 mice per group per time point). Data were derived from two independent experiments. The table shows the ANOVA p values comparing the effect of treatment on each population at each time point. (B) CT26 tumor–bearing mice (n = 5 per group) were treated i.v. with 4PD loaded with STAT3- and/or C/EBPb-specific shRNAs. Twenty-four hours after the last injection, T cells were magnetically sorted, CFSE labeled, and tested in MLTCs against CT26. (C, D and E) Starting 9 d after challenge, CT26 tumor–bearing mice were treated i.v. three times a week with PBS or with 4PD loaded with STAT3-specific shRNA, C/EBPb-specific shRNA, or with both shRNAs (20 mg per mouse). At 10 and 17 d after challenge, mice were vaccinated via electroporation with pcDNA3 (D) or with gp70 encoding pcDNA3 (E). Tumor growth was monitored. *p , 0.05 versus control.

Article Snippet: The following Abs were used for flow cytometry analysis: allophycocyaninor Brilliant Violet (BV)711–conjugated rat anti-mouse CD11b (clone M1/70; BD), PerCp-Cy5.5–conjugated rat anti-mouse Ly6G and Ly6C (clone RB68C5; BioLegend), allophycocyanin-Cy7–conjugated rat anti-mouse Ly6G (clone 1-A8), V450-conjugated rat anti-mouse Ly6C (clone AL-21), PEconjugated rat anti-mouse CD124 (clone mIL4R-M1), PE-Cy7–conjugated rat anti-mouse F4/80 (clone BM8; all from BD), FITC-conjugated rat antimouse F4/80 (clone A3-1; AbD Serotec), BV650-conjugated rat anti-mouse CD206 (clone C068C2; BioLegend), PE-Cy7–conjugated hamster antimouse CD11c (clone HL3; BD), eFluor 450–conjugated rat anti-mouse by guest on A pril 13, 2017 http://w w w .jim m unol.org/ D ow nloaded from CD49b (clone DX5; eBioscience), PE-conjugated mouse anti-mouse I-A[d] (clone AMS-32.1), allophycocyanin-conjugated hamster anti-mouse CD80 (clone 16-10A1), FITC-conjugated rat anti-mouse CD86 (clone GL1), allophycocyanin-Cy7–conjugated rat anti-mouse CD4 (clone GK1.5), PEconjugated rat anti-mouse CD25 (clone 3C7), PerCP-conjugated hamster anti-mouse CD3 (clone 145-2C11; all from BD), PerCP–eFluor 710– conjugated rat anti-mouse CD3 (clone 17A2; eBioscience), allophycocyaninCy7–conjugated rat anti-mouse CD19 (clone ID3; BD), allophycocyanin rat anti-mouse Foxp3 (clone FJK-16s; eBioscience), Pacific Blue– or BV711conjugated rat anti-mouse CD8 (clone 53-6.7; BD), allophycocyaninconjugated mouse anti BrdU (clone Bu20a; eBioscience), FITC-conjugated mouse anti-human CD33 (clone HIM3-4), allophycocyanin-H7–conjugated mouse anti-human CD14 (clone MfP9), Pacific Blue–conjugated mouse anti-human CD11b (clone ICRF44; all from BD), allophycocyaninconjugated mouse anti-human IL-4Ra (clone 25463; R&D Systems), and BV711-conjugated mouse anti-human HLA-DR (clone L243; BioLegend). p-STAT6 AF647 Ab (clone J71-773.58.11; BD) was used with Phosflow Perm Buffer IV, as per the manufacturer’s instructions.

Techniques: In Vivo, Vaccines, Injection, shRNA, Labeling, Transfection, Derivative Assay, Electroporation, Control