rneasy kit iso modc  (Qiagen)

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: Depletion of DCs and induction of tolerogenic DC by ADCC suppress T-cell activation. (A) Study design of mixed lymphocyte reaction (MLR) assay after using anti-DCIR antibody to deplete monocyte-derived DCs differentiated and matured in vitro. (B) Gating strategy of flow cytometry analysis on the activation of allogenic T cells. (C–F) T-cell proliferation and activation indicated by the increased T-cell count and CD69 expression on CD4 + (C, D) and CD8 + T cells (E, F). (G–K) Cytokine production from the MLR assay as described in (A). Representative data from 1 of 2 donors are shown, with error bars indicating mean ± SEM.

Article Snippet: For inhibiting the IDO1 activity, indoximod (MCE, Cat#HY-16724) was added together with GM-CSF and IL-4 during MoDC differentiation and MoDC maturation induced by 1 μg/mL CpG ODN2006 (Invivogen, Cat#tlrl-2006) and 500 ng/mL CD40L (PeproTech, Cat#310-02).

Techniques: Activation Assay, Mlr Assay, Derivative Assay, In Vitro, Flow Cytometry, Analysis, Cell Characterization, Expressing, FACS

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: IL-10 signaling is critical for the tolerogenic effect from the NK-programmed DC. (A) Study design of knocking-out transcription factors and evaluating the resultant DC tolerance effects. (B) IL-10, TNF-α, and IL-6 cytokine production was assayed from LPS-stimulated MoDCs after CRISPR-Cas9 editing. CRISPR-KO MoDC effect on T-cell activation in MLR evaluated by flow cytometry showing (C) CD25 expression on CD4 T cells and CD8 T cells ( n = 3 donors) and (D) evaluated MLR secreted cytokines IL-10, TNF-α, and IFN-γ ( n = 3 donors). Means ± SEM are shown, and statistical analysis is determined by one-way ANOVA Fisher least significant difference test compared to the scramble sgRNA control. * P < .05, ** P < .01, *** P < .001, **** P < .0001. (E) Study design of neutralizing IL-10 during the depletion of MoDCs by anti-DCIR–induced ADCC effect. (F) Cell count and (G, H) MFI of DC co-stimulatory/maturation markers (CD40 and CD44) on surviving CD14 + CD11c + MoDCs in the ADCC assay induced by anti-DCIR antibody in combination with or without anti-IL-10 neutralizing antibody ( n = 3 donors). (I) IL-10R expression and (J) cell viability of the MoDCs differentiated by GM-CSF and IL-4 treatment and matured by CpG and CD40L stimulation, along with indoximod, an IDO1 inhibitor ( n = 4 donors). (K) Schematic summary of the tolerogenesis of DCs from the NK engagement induced by DC-targeting antibody (eg anti-DCIR).

Article Snippet: For inhibiting the IDO1 activity, indoximod (MCE, Cat#HY-16724) was added together with GM-CSF and IL-4 during MoDC differentiation and MoDC maturation induced by 1 μg/mL CpG ODN2006 (Invivogen, Cat#tlrl-2006) and 500 ng/mL CD40L (PeproTech, Cat#310-02).

Techniques: CRISPR, Activation Assay, Flow Cytometry, Expressing, Analysis, Control, Cell Characterization, ADCC Assay, Immunopeptidomics, Immune Cell, FACS

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: Surviving monocyte-derived DCs following ADCC exhibit tolerogenic phenotypes. (A) Flow cytometry analysis of house-made anti-DCIR (clone 5E11) antibody binding to the CD14 + monocytes, B cells, T cells, and NK cells from human PBMCs ( n = 4 donors). Means ± SEM are shown, and statistical analysis is determined by one-way ANOVA test with Dunnett correction compared to the isotype staining control. ** P < .01. (B) Flow cytometry analysis of our anti-DCIR (5E11) antibody’s binding to the CD14 + monocytes, monocyte-derived DCs and macrophages ( n = 4 donors). (C) Study design of ADCC assay using anti-DCIR antibody to deplete monocyte-derived DCs differentiated and matured in vitro. (D) Cell count, (E–G) Mean Fluorescence Intensity (MFI) of surface co-stimulatory markers (CD11c, CD40, and CD44), and (H) intracellular IDO1 expression of surviving CD14 + CD11c + monocyte-derived DCs. (I) IL-10 cytokine production, and (J) %Treg (CD25 + CD127 − CD4 + T cells) induction in the ADCC assay as described in (C). Representative data from 1 of 3 donors are shown with error bars indicating mean ± SEM.

Article Snippet: For inhibiting the IDO1 activity, indoximod (MCE, Cat#HY-16724) was added together with GM-CSF and IL-4 during MoDC differentiation and MoDC maturation induced by 1 μg/mL CpG ODN2006 (Invivogen, Cat#tlrl-2006) and 500 ng/mL CD40L (PeproTech, Cat#310-02).

Techniques: Derivative Assay, Flow Cytometry, Analysis, Binding Assay, Staining, Control, ADCC Assay, In Vitro, Cell Characterization, Fluorescence, Expressing, FACS

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: Epigenetic remodeling of chromatin accessibility induces the transcriptional regulation of tolerogenic phenotypes in NK-programmed DCs. (A) Study design of bulk RNA-seq and ATAC-seq analysis on the surviving MoDCs from the NK–DC co-culture (weak NK engager) and MoDC cultured alone collected before and after LPS stimulation. (B) Principal component analysis (PCA) plot of the RNA-seq results using edgeR showing the 4 groups as defined in (A). (C) Gene expression changes identified by DGE analysis comparing the effects of LPS stimulation in DCs cultured alone or co-cultured with NK cells (weak NK engager). (D) Gene expression changes identified by DGE analysis comparing DC (weak NK-engager) vs DC alone. (E) Correlation of changes in chromatin accessibility determined by ATAC-seq and changes in gene expression determined by bulk RNA-seq in DCs alone compared to DCs co-cultured with NK cells (weak NK-engager). (F) Genome browser plots showing chromatin accessibility indicated by ATAC-seq peaks in proinflammatory cytokines genes (eg IL-1B, IL-6) and DC co-stimulatory marker genes (eg CD40 and CD11c) loci. Highlighted peaks indicate promoters that have lower accessibility in DC (weak NK-engager) as compared to DCs alone (DEseq, FDR <0.05). (G) Predicted top 20 most enriched transcription factors motifs based on the differentially chromatin accessible peaks identified by the ATAC-seq results.

Article Snippet: For inhibiting the IDO1 activity, indoximod (MCE, Cat#HY-16724) was added together with GM-CSF and IL-4 during MoDC differentiation and MoDC maturation induced by 1 μg/mL CpG ODN2006 (Invivogen, Cat#tlrl-2006) and 500 ng/mL CD40L (PeproTech, Cat#310-02).

Techniques: RNA Sequencing, Analysis, Co-culture Assay, Cell Culture, Gene Expression, Marker, Co-Culture, Significance Assay

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: IL-10 signaling is critical for the tolerogenic effect from the NK-programmed DC. (A) Study design of knocking-out transcription factors and evaluating the resultant DC tolerance effects. (B) IL-10, TNF-α, and IL-6 cytokine production was assayed from LPS-stimulated MoDCs after CRISPR-Cas9 editing. CRISPR-KO MoDC effect on T-cell activation in MLR evaluated by flow cytometry showing (C) CD25 expression on CD4 T cells and CD8 T cells ( n = 3 donors) and (D) evaluated MLR secreted cytokines IL-10, TNF-α, and IFN-γ ( n = 3 donors). Means ± SEM are shown, and statistical analysis is determined by one-way ANOVA Fisher least significant difference test compared to the scramble sgRNA control. * P < .05, ** P < .01, *** P < .001, **** P < .0001. (E) Study design of neutralizing IL-10 during the depletion of MoDCs by anti-DCIR–induced ADCC effect. (F) Cell count and (G, H) MFI of DC co-stimulatory/maturation markers (CD40 and CD44) on surviving CD14 + CD11c + MoDCs in the ADCC assay induced by anti-DCIR antibody in combination with or without anti-IL-10 neutralizing antibody ( n = 3 donors). (I) IL-10R expression and (J) cell viability of the MoDCs differentiated by GM-CSF and IL-4 treatment and matured by CpG and CD40L stimulation, along with indoximod, an IDO1 inhibitor ( n = 4 donors). (K) Schematic summary of the tolerogenesis of DCs from the NK engagement induced by DC-targeting antibody (eg anti-DCIR).

Article Snippet: For the MLR experiment in , PBMCs were isolated from fresh human whole blood and treated with 40 ng/mL human IL-4 and 100 ng/mL human GM-CSF for 3 days to induce MoDC differentiation, followed by CpG ODN2006 (Invivogen, Cat# tlrl-2006) and CD40L (PeproTech, Cat#310-02) to promote MoDC maturation in addition to anti-DCIR antibody to induce antibody-dependent cellular cytotoxicity (ADCC) for 2 to 3 days.

Techniques: CRISPR, Activation Assay, Flow Cytometry, Expressing, Control, Cell Characterization, ADCC Assay

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