Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were inoculated with mock or VZV infected ARPE-19 epithelial cells for 2 days then analysed for infection by flow cytometry. (A) Representative flow cytometry plots of mock or VZV-S infection, examining surface VZV gE:gI expression on live T cells (CD3 + CD56 – ), CD3 + CD56 + lymphocytes, and NK cells (CD3 – CD56 + ). (B) Frequencies of live gE:gI + lymphocytes in total (shaded), compared to specific populations: T cells, CD3 + CD56 + lymphocytes, and NK cells (n = 19). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean. Statistical analysis was performed between specific lymphocyte populations. **p < 0.01, ****p < 0.0001 (RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test). (C) Representative flow cytometry plots of vOka infection, examining surface gE:gI expression on live T cells (CD3 + CD56 – ), CD3 + CD56 + lymphocytes, and NK cells (CD3 – CD56 + ) (n = 3).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: (A) Healthy human donor PBMCs were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface VZV gE:gI expression from one representative donor and graphs show frequency of live gE:gI + NK cells (CD3 – CD56 + ) (top panels), CD3 + CD56 + lymphocytes (middle panels), and T cells (CD3 + CD56 – ) (bottom panels). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean (n = 8). ***p < 0.001, ****p < 0.0001 (two-tailed paired t test). (B & C) Healthy human donor CD56 + -selected lymphocytes were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface gE:gI expression from one representative donor and graphs show frequency of live gE:gI + NK cells (B) or CD3 + CD56 + lymphocytes (C). Symbols represent individual donors, consistent across (B & C) (n = 7). *p < 0.05 (two-tailed Wilcoxon matched-pairs signed rank test).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: NK cells (CD3 – CD56 + ) were FACS sorted from healthy human donor CD56 + -selected lymphocytes following mock or VZV infection for 1 day. (A & B) Staining by IFA of sorted VZV cultured (left panels) or mock cultured (right panels) NK cells for IE63 (A), pORF29 (B) or respective isotype control, with DAPI (n = 3). (C) Sorted VZV cultured NK cells were added to ARPE-19 epithelial cell monolayers. Four days later monolayers were fixed and infectious centres detected with IFA by staining for IE63 and gE:gI, with DAPI. One representative experiment of five is shown.

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Staining, Cell Culture, Control

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: (A) Healthy human donor PBMCs were infected with VZV for 2 days then analysed for infection by flow cytometry. Plots show gating strategy for CD56 bright and CD56 dim NK cells (CD3 – CD56 + ) (left panel), with respective surface VZV gE:gI expression (right panels) from one representative donor (n = >7). (B & C) CD3 – CD56 bright (B) and CD3 – CD56 dim (C) NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently infected with VZV for 2 days before analysis by flow cytometry. Plots show surface gE:gI expression from one representative donor (n = 2).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Isolation

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (A) Diagram describes gating strategy and tSNE analysis workflow for samples shown in (B & C). (B & C) tSNE plots show marker expression levels for single parameters on individual cells in the tSNE map for mock and VZV cultured NK cells after 2 days, either untreated (B) or in the presence of IL-2 (C). Arrowheads indicate the CD56 bright NK cell subset, and the outlined population indicates the localisation of VZV + NK cells. One representative experiment of three is shown. (D & E) Plots show CD57 expression between mock and VZV cultured NK cells (D) and between bystander and VZV + NK cells (E), from one representative donor. Graphs show respective frequencies of CD57 + NK cells when untreated or with IL-2 (shaded) for four donors. Bars indicate mean. (F) Histograms show CD16 expression for mock, bystander and VZV + NK cells from one representative donor. Graph shows frequency of CD16 + NK cells when untreated or with IL-2 (shaded) for six donors. Bars indicate mean. *p < 0.05, **p < 0.01, ***p < 0.001 (Friedman test with Dunn’s multiple comparisons test).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Marker, Expressing, Cell Culture

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: CD3 – CD56 + CD57 – NK cells and CD3 – CD56 + CD57 bright NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently mock or VZV infected with or without IL-2 (200 U/ml) for 2 days before analysis by flow cytometry. (A) Diagram describes experimental design of isolating NK cells on CD57 expression, then infecting, and subsequently analysing for infection and phenotype changes. (B) Plots show surface VZV gE:gI expression between subsets from one representative donor. Graph shows frequency of VZV + NK cell subsets when untreated or with IL-2 (shaded) for three donors. Bars indicate mean. (C) Plots show subsequent CD57 expression between mock, bystander and VZV + CD57 – NK cells (left panels) and CD57 versus gE:gI expression for VZV cultured CD57 – NK cells (middle panels), from one representative donor. Graphs show frequency of CD57 expression on mock, bystander and VZV + CD57 – NK cells for three donors. Bars indicate mean. *p < 0.05 (two-tailed paired t test). (D) Histograms show CD16 expression for mock, bystander and VZV + CD57 – NK cells (left panel) and CD57 bright NK cells (right panel) for one representative donor (n = 3).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Isolation, Infection, Flow Cytometry, Expressing, Cell Culture, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (Left panels) Representative plots show CCR4 (A) or CLA (B) expression against CD56 expression for mock, bystander and VZV + NK cells. (Right panels) Representative plots show CCR4 (A) or CLA (B) expression versus VZV gE:gI expression for VZV cultured NK cells. Data are representative of five donors.

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Cell Culture

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: miR-130a & miR-145, and TβRII mRNA in Gr-1 + CD11b + cells. a Left panels: qRT-PCR for fold changes of TβRII transcript in Gr-1 + CD11b + cells isolated from the spleen of 4T1 tumor-bearing mice (Tumor) compared to that from healthy control mice (Healthy) ( n = 3). Right panel: Immunofluorescence images of TβRII (green) and DAPI (blue) in Gr-1 + CD11b + cells. scale bar=10 μm. b Stability of TβRII and GAPDH transcripts in Gr-1 + CD11b + cells derived from the spleen of 4T1 tumor-bearing mice (T) compared to those from healthy control mice (N). Total RNA was isolated at the indicated times after Actinomycin D treatment. 18sRNA was used to normalize TβRII and GAPDH expression. The experiment was performed three times with three biological replicates in each group. c Strategies to identify miRNAs targeting TβRII. d Heat map of differentially expressed miRNAs in Gr-1 + CD11b + cells from 4T1 tumor-bearing mice (Tumor) compared to the healthy control (Healthy) ( n = 3). Yellow and blue colors indicate increased and decreased expression, respectively. Below: Schematic diagram showing the location of miR-130a and miR-145 consensus binding sites in 3′-UTR of TβRII transcript. e Expression of the indicated miRNAs by qRT-PCR. qRT-PCR of miR-130a and miR-145 in Gr-1 + CD11b + myeloid cells sorted from the spleen of healthy control or tumor-bearing mice, with miR-19a and miR-93 as controls. f Relative luciferase activity of HeLa cells transfected with pGL3-TβRII-3′-UTR vector and miR-16, miR-130a, or miR-145 mimics by electroporation. The miR-16 was used as a negative control. g Time course experiments for the expression of miR-130a (left), miR-145 (middle) and TβRII (right) in sorted Gr-1 + CD11b + myeloid cells as measured by qRT-PCR. Y -axis: fold changes; X -axis: days after tumor injection in the MFP ( n = 3/each time point). Right panel: Spearman analysis of the correlations of miR-130a or miR-145 level with TβRII level. r : Pearson’s correlation coefficient. h qRT-PCR for expression of miR-130a (left), miR-145 (middle) and TβRII (right) in myeloid subsets CD11b + Ly6C + and CD11b + Ly6G + sorted from spleens of 4T1 tumor-bearing (Tumor) or healthy control mice (Healthy). The data are represented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Isolation, Control, Immunofluorescence, Derivative Assay, Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Electroporation, Negative Control, Injection

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: Ectopic expression of miR-130a & miR-145 reduced tumor metastases. a Top panel: the construct of miR-130a or miR-145 expressions under control of a CD11b promoter, with GFP and WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element) in 3′-UTR. * indicates transcription starting site (TSS); numbers indicate CD11b promoter sequence relative to TSS. Lower panels: microscopy (left), flow cytometry analysis of CD11b + myeloid cells differentiated from HS/PCs with the lentiviral expression vector in the culture with 4T1 tumor supernatant (right). b flow cytometry of GFP expression in CD11b + myeloid cells (left), and CD11b + Ly6C + monocytic and CD11b + Ly6G + granulocytic subsets in GFP + myeloid cells (middle) with quantitative data (right). c Fold changes of miR-130a (left), and miR-145 (middle) as well as TβRII (right) in sorted GFP + and GFP – myeloid cells by qRT-PCR, with HS/PCs as a negative control. Data in ( b - c ) are from ex vivo culture as illustrated in ( a ). d TβRII fold changes by qRT-PCR from sorted peripheral blood Gr-1 + CD11b + myeloid cells of tumor-bearing mice that received miR-130a, miR-145, and TβRII shRNA-engineered bone marrow transplantation. e Lung metastasis of 4T1 cells in mice that received miR-engineered bone marrow transplantation as noted above. 4T1 cells were injected in MFP number 2 and metastatic nodules in the lung were assessed 28 days later ( n = 5–13 mice). The data are represented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Expressing, Construct, Control, Virus, Sequencing, Microscopy, Flow Cytometry, Plasmid Preparation, Quantitative RT-PCR, Negative Control, Ex Vivo, shRNA, Transplantation Assay, Injection

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: miR-130a transgenic mice showed a decreased metastasis. a GEM strains crossbred to produce a myeloid-specific Tet-inducible miR-130a transgenic mouse. b Fold change of miR-130a in Gr-1 + CD11b + myeloid cells from miR-130a transgenic mice treated with doxycycline (DOX), which was quantified and normalized to that of untreated mice. c miR-130a expression in CD3 + T cells, CD19 + B cells, and Gr-1 + CD11b + myeloid cells from the splenocytes of the miR-130a transgenic mice treated with DOX ( n = 3). d Left panel: qRT-PCR of TβRII in Gr-1 + CD11b + myeloid cells from the spleen of E0771 tumor-bearing miR-130a transgenic mice with DOX treatment ( n = 3); right panel: Western blot of TβRII. e Number of E0771 metastatic nodules in miR-130a transgenic mice treated with DOX compared with control mice (Left panel: spontaneous metastasis, n = 12; Right panel: experimental metastasis, n = 7–9). f Metastasis nodule counts of Lewis lung carcinoma in miR-130a transgenic mice treated with DOX compared with control mice after tail vein injection. Lung nodules were counted after 14 days ( n = 9). Representative lungs stained by Indian ink on the right panels. The data was represented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Transgenic Assay, Expressing, Quantitative RT-PCR, Western Blot, Control, Injection, Staining

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: miR-130a & miR-145 reprogram Gr-1 + CD11b + cells and enhance host immunity. a TβRII Western blot (left) and qRT-PCR (right) of Gr-1 + CD11b + cells from 4T1 tumor-bearing mice 24 h after miR control, miR-130a and miR-145 electroporation. b qRT-PCR of TβRII after electroporation of miR-130a or miR-145 with or without miRNA antisense inhibitors. c M1/M2 cytokine ratio post treatment with miRNA electroporation with or without miR-130a or miR-145 antisense inhibitors ( n = 5). The ratio of M1/M2 cytokines was calculated by dividing each M1 cytokine (TNFα, IL-12, GM-CSF) to M2 cytokine (IL-10, TGFβ1, IL-4) as described in Material and Methods. d Left panels: flow cytometry of IFNγ + CD8 + T cells from splenocytes of TCR-HA transgenic mice co-cultured with Gr-1 + CD11b + myeloid cells with or without miRNA, which were pulsed with HA peptide. Right panels: quantitative data. The Gr-1 + CD11b + cells (Mye) to splenocytes (Spl) ratio is indicated for each panel. e IFNγELISPOT of splenocytes from TCR-HA transgenic mice co-cultured with Gr-1 + CD11b + cells following miRNA electroporation. f CTL assays: flow cytometry of B16 melanoma tumor cells (Sytox blue positive are dead) expressing hgp100 co-cultured with splenocytes from Pmel-1 transgenic mice and Gr-1 + CD11b + cells from spleens of miR-130a transgenic, quantitative data on right. The percentage of targeted dead tumor cells = total dead cells + subtraction of dead cells from a single culture of tumor cells, myeloid cells, and splenocytes. All data are presented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Western Blot, Quantitative RT-PCR, Control, Electroporation, Flow Cytometry, Transgenic Assay, Cell Culture, Expressing

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: Gene networks targeted by miR-130a & miR-145. a Identification of miRNA targeted genes from TargetScan mouse database, which was intersected with mRNA expression microarray data comparing tumor Gr-1 + CD11b + cells with those from healthy control mice. Seven targets were common for miR-130a and -145. b IPA analysis of gene networks targeted by miR-130a (purple), miR-145 (blue), or both (orange) involving TGFβ and IGF pathways. c Validation of the major pathway mediators comparing tumor-associated myeloid cells with those from healthy controls, qRT-PCR (left) and Western blot (right). d qRT-PCR (left) and Western blot (right) from Gr-1 + CD11b + cells ex vivo treated with miR-130a or -145 or control mimics. e Immunofluorescence images of TβRII (Green), IGF1R (red), and DAPI (blue) in Gr-1 + CD11b + cells from the spleen of 4T1 tumor-bearing mice. scale bar: 10 μm. f M1/M2 cytokine ratio post restorations of TβRII, IGF1R, and IRS1 in Gr-1 + CD11b + cells that overexpress miR-130a or miR-145. The ratio of M1/M2 cytokines was calculated by dividing each M1 cytokine (TNFα, IL-12, GM-CSF) to M2 cytokine (IL-10, IL-4) as described in Material and Methods. g–l metastasis reduction by IGF1R inhibitor NT157: g The number of metastatic nodules of 4T1 tumor-bearing Tgfbr2 MyeKO and WT mice treated with NT157 ( n = 7–10). h The number of metastatic nodules in 4T1 tumor-bearing mice that received IGF1R inhibitor NT157. i qRT-PCR of ARG1, TGFβ1, and IL-10 in Gr-1 + CD11b + cells from 4T1 tumor-bearing mice treated with NT157 ( n = 5). j The number of metastatic nodules from mice bearing E0771 tumor treated with NT157 ( n = 5–6). k qRT-PCR of ARG1, TGFβ1, and IL-10 in Gr-1 + CD11b + cells from E0771 tumor-bearing mice treated with NT157 ( n = 4). l The number of metastatic nodules of 4T1 tumor-bearing mice treated with NT157 with CD8α neutralizing antibody or IgG as control ( n = 10). Mice with similar tumor weight were used for metastatic nodule counts (Supplementary Fig. ). Data are presented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Expressing, Microarray, Control, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Ex Vivo, Immunofluorescence

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: Reduced metastasis and enhanced anti-tumor immunity by miRNA mimics. a qRT-PCR of miR-130a and −145 in the plasma and Gr-1 + CD11b + cells (left), as well as TβRII in Gr-1 + CD11b + cells (right) from 4T1 tumor-bearing mice ( n = 6/each time point) at indicated times after systemic delivery of miRNA mimics through TVI. b qRT-PCR of miR-130a (left) or miR-145 (right) in Gr-1 + CD11b + myeloid cells, CD3 + lymphocytes, and CD19 + B cells sorted from peripheral blood of mice ( n = 3) 24 h after injection of miRNA mimics. c Metastasis nodule counts from mice that received miR control, miR-130a, and miR-145 mimics as a single agent or in combination with Paclitaxel (PAC) ( n = 13). d Flow cytometry of IFNγ + CD8 + cells from spleens of TCR-HA transgenic mice co-cultured (ex vivo) with Gr-1 + CD11b + cells from 4T1 tumor-bearing mice that were treated with miR-130a or miR-145 mimics. e Upper panels: miRNA in situ hybridization: miR-145 mimic distribution (green) in metastatic lungs of 4T1 tumor-bearing mice 12 h after miR mimic injection, with DAPI (blue), CD11b (red), and E-cadherin (Magenta). Scale bar: 20 μm. Lower panel: the quantitative data of miRNA mean fluorescent intensity in myeloid cells or tumor cells in metastatic lung of mice with miRNA mimic injections ( n = 23–30). f Flow cytometry of IFNγ + CD8 + T cells at metastasized lung from mice treated with miR-130a or miR-145 mimics ( n = 3). g CD8 T cell proliferation assay: flow cytometry of CSFE-labeled CD8 + T cells from splenocytes of TCR-HA transgenic mice co-cultured with sorted Gr-1 + CD11b + myeloid cells from the spleen of mice treated with miR-130a or miR-145 mimic injection (left), with quantitative data (right). h CTL assays: flow cytometry of 4T1.2 tumor cells (Sytox blue positive are dead) expressing HA co-cultured with splenocytes from TCR-HA transgenic mice and Gr-1 + CD11b + cells from the spleen of mice treated with miR-130a or miR-145, quantitative data (right). The percentage of targeted dead tumor cells = total dead cells minus dead cells from single culture of tumor cells, myeloid cells, and splenocytes. i Number of metastasis in 4T1 tumor-bearing mice treated with miR mimics with CD4 or CD8α neutralizing antibody or IgG control. The data are presented as mean ± SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For sorting, CD3 + CD4 + or CD3 + CD8 + T cells, CD3 – CD19 + B cells, and Gr-1 + CD11b + , CD11b + Ly6C + or CD11b + Ly6G + myeloid cells, or GFP + or GFP – Gr-1 + CD11b + myeloid cells were sorted by the FACSAria flow cytometer (BD Biosciences) or magnetically activated cell sorting (MACS) with CD11b and Gr-1 microbeads per the manufacturer’s protocol (Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Clinical Proteomics, Injection, Control, Flow Cytometry, Transgenic Assay, Cell Culture, Ex Vivo, In Situ Hybridization, Proliferation Assay, Labeling, Expressing

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