modcs  (PromoCell)

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: The myeloid compartment in B16.F10 melanoma shifts toward suppressive phenotypes as tumors develop (A) Flow cytometry quantification of the percentage of DCs (CD11c+) and phagocytes (CD11b+CD11c-) within CD45 + cells. (B) Flow cytometry quantification of MDSCs populations (moDCs; CD11b + CD11c + Ly6C + , M-MDSCs; CD11b + CD11c − Ly6C + , and G-MDSCs; CD11b + CD11c − Ly6G + ) within CD45 + cells. (C) Flow cytometry quantification of the expression levels of immune-modulatory markers PD-L1, FasL, ARG1, and NOS2 by moDCs, M-MDSCs, and G-MDSCs. (D) Representative CFSE plots for CD8 T cell proliferation after culture alone, co-culture with tumor-derived CD11b + Ly6C − cells, or co-culture with a mix of tumor-derived moDCs and M-MDSCs. Black bar highlights the gated proliferated cells. (E) Flow cytometry quantification of the percentage of proliferating CD8 and CD4 cells cultured alone, co-cultured with tumor-derived CD11b + Ly6C − cells, or a mix of moDCs and M-MDSCs. (F) Representative CFSE plots for CD4 and CD8 T cell proliferation after co-culture with pre-sorted, tumor-derived moDCs or M-MDSCs. Black bar highlights the gated proliferated cells. (G) Quantification of T cell suppression following incubation with pre-sorted, tumor-derived moDCs and M-MDSCs compared to T cells cultured alone. Data are mean ± SEM; ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (A–C) Mixed effect analysis with a Tukey’s post hoc test. (E and G) One-way ANOVA with a Tukey’s post hoc test. (A and B) n = 5 mice for day-5 and day-9 tumors and n = 6 for day-11 tumors, from two independent experiments comparing each cell type at day 5, 9, or 11 time points with the day 0 time point. (C) n = 8 mice for day 5 tumors and n = 6 mice for day 11 tumors from two independent experiments. (E) n = 3 and (G) n = 2 mice performed in duplicate from two different experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Derivative Assay, Cell Culture, Incubation

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: Distribution of CD47 and SIRPα expression across the TME (A) Clustering of stromal populations identified in B16.F10 melanomas and matched draining lymph nodes analyzed from data previously published by Davidson et al. (B) Expression of CD47 and its cognate receptor, SIRPα, distributed across stromal clusters. (C) Violin plots highlighting widespread CD47 but restricted SIRPα expression across stromal subsets. (D) Flow cytometry quantification of CD47 expression at the protein level in T cells, (immunomodulatory) CAF 1, (myofibroblast) CAF 2, myeloid cells, endothelial cells (CD31 + ), and B16.F10 tumor cells. (E) Representative confocal image of a day 11 B16.F10 melanoma showing myeloid populations. Arrows indicate CD11b+Ly6C + SIRPα+ cells. Insets show zoom of selected ROI. Arrowheads depict cells positive for CD11b but negative for Ly6C and SIRPα. DAPI (gray), CD11b (red), Ly6C (green), SIRPα (blue). Scale bar is 50μm. Data are mean ± SEM; ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (D) One-way ANOVA with a Dunnett post hoc test. (D) n = 3 replicates from two independent experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet:

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Purification, Recombinant, Modification, Red Blood Cell Lysis, Cell Isolation, Staining, ATP Assay, Quantitation Assay, Software

Journal: Viruses

Article Title: Detection of Foodborne Viruses in Dates Using ISO 15216 Methodology

doi: 10.3390/v17020174

Figure Lengend Snippet: MNV recovery from whole Medjool dates and RT-qPCR inhibition.

Article Snippet: The viruses were extracted using the ISO 15216-1:2017 standard protocol version for soft fruit (ISO-modA) combined with the eGene-up RNA extraction process (Biomérieux, Montréal, QC, Canada), or the vegetable version combined with either the eGene-up (ISO-modB) or the RNeasy kit (ISO-modC) (Qiagen, Mississauga, ON, Canada) to extract the viral RNA following the manufacturer’s instructions.

Techniques: Inhibition

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 impairs the function of monocyte-derived Dendritic Cells (A) Alveolar macrophage and dendritic cell populations in bronchoalveolar lavage of patients with lung transplant with and without invasive aspergillosis ( n = 3). (B) Phagocytosis of A. fumigatus conidia (MOI = 1) by dendritic cells untreated, treated with IFNy or FK506 or both for 30 min ( n = 3). (C) Fungal killing of A. fumigatus conidia (MOI = 1) by dendritic cells untreated, treated with IFNy or FK506 or both 6 h post infection, measured by CFUs ( n = 3). (D) Representative ImageStream images of CD83 on moDCs treated with or without IFNy. (E) MFI of CD83 on moDCs untreated, treated with IFNy and/or FK506, infected with A. fumigatus (MOI = 1) or uninfected ( n = 3). (F) Representative ImageStream images of moDCs stained for nucleus (DAPI) and NFATc2 (Cy5), and infected with A. fumigatus (MOI = 1) or uninfected ( n = 3). (G) Timecourse of NFATc2 nuclear translocation of A. fumigatus infected (MOI = 1) moDCs treated with and without FK506 ( n = 3). (H) Time course RCAN-1 expression of Aspergillus infected (MOI = 1) moDCs DCs treated with and without FK506 ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (A) t-test (B, C, E, and H) one-way ANOVA (G) two-way ANOVA. Data represented as mean ± SEM.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Derivative Assay, Infection, Staining, Translocation Assay, Expressing

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 treatment abrogates T cell activation by Dendritic Cells (A) CD4 cell proliferative capacity measured in a mixed leukocyte reaction of T cells and moDC’s, with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment ( n = 3). (B) CXCR3 surface expression on CD4 T cells co-cultured with moDC’s with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment. (C and D) Cytokine secretion by moDCs or MDMs with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment ( n = 3), (C) TNFa, (D) IL-2, (E) IL-10 and (F) IL-12. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (A–F) one-way ANOVA. Data represented as mean ± SEM.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Activation Assay, Infection, Expressing, Cell Culture

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: IFNγ and FK506 treatment alters the transcriptomic response to A. fumigatus in both moDCs and MDMs MoDC’s and MDM’s from healthy volunteers ( n = 3) were pre-treated with FK506 and/or IFNγ, then infected with A. fumigatus (MOI = 1) for 1 h prior to sequencing (A) Gene expression heatmap of top 200 genes (ranked by SD) per unclustered condition. (B) Venn diagram showing overlap of differentially expressed genes in infected moDCs and MDMs.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection, Sequencing, Gene Expression

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: All genes differentially expressed with the FK506 treatment of A. fumigatus infected moDC’s and MDMs

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: Effects of IFNγ on moDC and MDM transcriptional responses during A. fumigatus infection (A) Numbers of differentially expressed genes with IFNγ treatment in moDC’s and MDM’s, with and without A. fumigatus infection. (B and C) Gene ontology of upregulated genes with IFNγ treatment in (B) moDCs and (C) MDMs. (D) Number of differentially expressed genes in infected cells with IFNγ treatment, with and without FK506 treatment.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: All genes differentially expressed by IFNγ treatment during the FK506 treatment of infected moDC’s and MDMs

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 and IFNy treatment alter the epigenetic landscape of A. fumigatus infected DCs (A) Sites of differential H3K27ac binding with the A. fumigatus infection of untreated moDC’s. (B and C) Genome-wide distribution of differential H3K27ac signal around nearest gene transcription start site (TSS, green line) with the A. fumigatus infection of untreated moDCs. (D) Sites of differential H3K27ac binding with the IFN-γ treatment of A. fumigatus infected FK506-treated moDCs. (E and F) Genome-wide distribution of differential H3K27ac signal with respect to nearest gene transcription start site (TSS, green line) with the IFN-γ treatment of A. fumigatus , FK506-treated moDCs.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection, Binding Assay, Genome Wide

Journal: bioRxiv

Article Title: Single cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells

doi: 10.1101/2024.01.19.576293

Figure Lengend Snippet: Primary human monocyte derived dendritic cells (moDCs) were infected with ZIKV and the ZIKV-inclusive scRNA-seq assay was performed at 24 hpi. (A) Flow cytometry plot of mock and ZIKV-infected cells stained for ZIKV E protein expression using the pan-flavivirus antibody, 4G2. (B) Mock and ZIKV-infected cells were labeled with feature barcoded antibodies, Hash A and Hash B, respectively. They were then mixed 2:1. These barcodes were captured during the reverse transcription reactions alongside host mRNA allowing for mock and ZIKV cells to be distinguished within the same sample computationally. (C) Distribution of normalized viral RNA reads across Hash A (mock) and Hash B (ZIKV) represented as percent of cells in samples with and without ZIKV-specific primer. Cells were labeled as mock (Hash A, ZIKV = 0), bystander (Hash B, ZIKV = 0), and infected (Hash B, ZIKV > 1) and color coded as green, yellow, and red, respectively in the dashed lines on the bar plot (left) and as the dots on the UMAP (right). (D) Percent of cells in ZIKV population (Hash B) that were determined to be infected in samples with and without ZIKV-specific primer. (E) UMAPs of samples with and without ZIKV-specific primer featuring viral RNA. (F) Distribution of normalized viral RNA reads in infected cells across samples with and without ZIKV-specific primer represented as a histogram (left) and violin plot (right). (G) Expression of housekeeping genes across samples with and without ZIKV-specific primer. All graphs comparing samples with and without ZIKV-specific primer are colored red (+ ZIKV-specific primer) and grey (-ZIKV-specific primer).

Article Snippet: CD14+ monocytes were isolated from PMBCs of healthy human blood using the Mojosort TM Human CD14 Selection Kit (BioLegend, Cat# 480026) To generate monocyte derived dendritic cells (moDCs), CD14+ monocytes were stimulated in non-tissue culture treated plates with 100ng/mL GM-CSF (R&D Systems, Cat# 7954-GM-020/CF) and IL-4 (R&D Systems, Cat# 6507-IL-025/CF) in complete RPMI.

Techniques: Derivative Assay, Infection, Flow Cytometry, Staining, Expressing, Labeling, Reverse Transcription

Journal: Frontiers in Immunology

Article Title: Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity

doi: 10.3389/fimmu.2023.1306449

Figure Lengend Snippet: Poly(I:C) improves the adjuvanticity of inactivated spores and YC-NaMA in human monocyte-derived dendritic cells. (A) CD14+ monocytes were magnetically enriched from healthy donor PBMCs After 6 days of differentiation, moDCs were either left unstimulated, or treated with LPS, poly(I:C) or vaccine delivery systems with or without poly (I:C). After 48 hours, cells were surface stained for activation induced markers (MHC-I, MHC-II, CD80, CD86, CD40 and CCR7) for FACS analysis. Culture supernatants were also obtained to quantify secreted inflammatory cytokines (IL-1β, IFN-α2, IFNγ, TNFα, IL-6, IL-18, IL-10, IL-17A, IL-12p70, IL-23, IL-33, CCL2 and CXCL8). (B) Heatmap showing the log10 fold increase in cell-surface and secreted soluble mediators for each treatment group relative to unstimulated group. (C) Heatmap showing P values comparing YC-Nama or Spore with and without Poly(I:C). Data is obtained from the average of two healthy donors. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to compare the groups. Asterisks depicted in A are significant differences from unstimulated. Asterisks in B show significant difference between two groups. P<0.05 = *; P<0.01 = **; P<0.0001 = ****.

Article Snippet: To differentiate CD14+ monocytes to immature moDCs, we used a commercially available dendritic cell generation medium (PromoCell ® , C-28050).

Techniques: Derivative Assay, Staining, Activation Assay