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

Article Title: Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.

doi: 10.4049/jimmunol.179.11.7767

Figure Lengend Snippet: FIGURE 2. HCMV activates PDCs. A, HCMV alters PDC morphology. PDCs were incubated overnight with medium or TB40/E at an MOI of 5 and examined by light microscopy and Evans blue staining of cytospin preparations. At 24 h, mock-infected PDCs showed plasmacytoid morphology, whereas TB40E-infected PDCs formed clusters, developed dendrites, and had larger cytoplasm. B and C, HCMV induces partial maturation of PDCs. PDCs were mock-infected (mock inf), stimulated with CpG-A (3 g/ml), or infected with TB40/E or VR1814 (MOI of 5) overnight. The stimuli were removed, cells were washed, and fresh medium was added. After 24 h, expression of CD83, HLA-DR, BDCA-2, CD80, and CD86 was measured by FACS. B, Values are means SEM of seven donors in independent experiments. , p 0.05. Y-axis values indicate MFI. C, Open histograms depict cells stained with isotype-matched control Ab. Representative data from one of seven experiments are shown.

Article Snippet: For cell surface staining, we used Abs against CD1a, CD3, CD4, CD8, CD14, CD19, CD20, CD38, CD80, CD86, HLA-DR, and mouse isotype controls (BD Pharmingen); CD83 (Immunotech); and BDCA-2 and BDCA-4 (Miltenyi Biotec).

Techniques: Incubation, Light Microscopy, Staining, Infection, Expressing, Control

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

Article Title: Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.

doi: 10.4049/jimmunol.179.11.7767

Figure Lengend Snippet: FIGURE 4. Soluble factors secreted by HCMV-infected DCs contribute to B cell activation. A and B, PDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered through 0.1-m filters to eliminate viral particles. B cells (2.5 105 cells/well) were cultured in 200 l of supernatant from PDC cultures in 96-well round-bottom plates. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 g/ml). A, After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means SD. , p 0.05 (n 4 donors in independent experiments). B, For proliferation assay, B cells were labeled with 1 M CFSE, incubated with PDC supernatant for 5 days, harvested, and analyzed by FACS. Data from one representative of three donors in independent experiments are shown. C, MDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered. B cells (2.5 105 cells/well) were cultured in 200 l of supernatant from MDC cultures. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 g/ml). After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means SD. , p 0.05 (n 4 donors in independent experiments).

Article Snippet: For cell surface staining, we used Abs against CD1a, CD3, CD4, CD8, CD14, CD19, CD20, CD38, CD80, CD86, HLA-DR, and mouse isotype controls (BD Pharmingen); CD83 (Immunotech); and BDCA-2 and BDCA-4 (Miltenyi Biotec).

Techniques: Infection, Activation Assay, Cell Culture, Ligation, Staining, Proliferation Assay, Labeling, Incubation

Journal: Journal of leukocyte biology

Article Title: Adipose tissue of human omentum is a major source of dendritic cells, which lose MHC Class II and stimulatory function in Crohn's disease.

doi: 10.1189/jlb.0905501

Figure Lengend Snippet: Fig. 5. Morphology of omental DC. (a) A typical DC from a control omentum is shown. (b) An example of immunogold staining of the veils of a DC. The original bars indicate 1 m. In controls, gold labeling was always 5 gold grains per cell; 10 grains was considered positive. (c) The types of myeloid cells identified in the omental cells from two control patients; the proportion positive for HLA-DR staining is shown in the solid portion of the histograms, and those negative are hatched. M , Macrophage. (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. No evidence of DR-labeling was seen in either of the two Crohn’s patients studied. (e) A light microscopy picture of a cluster of putative DC immunostained with CD11c on a frozen section of omentum from a Crohn’s patient showing the dark, positive-peroxidase staining, which was absent from control slides. Cell nuclei, which were stained with hematoxylin, were distinguishable, showing that the veils were associated with mononuclear cells in the section. (f) Control for e using serum instead of specific antibody and with only hematoxylin staining visible. (g) Frozen section of control omentum, showing two cells specifically labeled for CD83 (TRITC-labeled). (h) The same section as g with label specific for CD86 (FITC-labeled).

Article Snippet: To block nonspecific binding sites, sections were incubated with normal mouse serum for 1 h and then incubated with FITC-conjugated primary antibody to CD86, CD80, or CD40 (Serotec) for 4 h, washed in PBS, and mounted under cover-slips using 4 ,6-diamidino-2-phenylindole-conjugated mounting medium (Vector Laboratories).

Techniques: Control, Staining, Labeling, Light Microscopy

Journal: Journal of viral hepatitis

Article Title: A class C CpG toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis C.

doi: 10.1111/j.1365-2893.2008.01011.x

Figure Lengend Snippet: Fig. 4 Immunophenotypic maturation of pDCs is not impaired in cHCV infection. After 20–24 h stimulation with TLR agonists, pDCs were processed for flow cytometry to identify immunophenotypic changes consistent with maturation. FACS histograms from a representative NHD and cHCV subject show (a) the rise in CD86 expression and (b) fall in BDCA-2 expression following stimulation with CpG-C (solid histogram) compared to unstimulated cells (open histogram overlay). The bar graphs show the average fold change in mean fluorescence intensity (MFI) ± SEM of (a) CD86 and (b) BDCA-2 on pDCs from 9 NHD (solid bars) and eight cHCV (open bars) subjects (seven NHD for TLR7 agonists) induced by the four TLR agonists. For each TLR agonist, the degree of (a) upregulation of CD86 and (b) downregulation of BDCA-2 did not differ significantly between NHD–pDC and cHCV–pDCs (n.s.). CpG-C was a significantly stronger stimulus for CD86 upregulation than CpG-A (NHD–pDCs, P < 0.001; cHCV–pDCs, P < 0.01) and for cHCV–pDCs loxoribine was also significantly more potent than CpG-A (P < 0.05). CpG-C caused significantly stronger downregulation of BDCA-2 than CpG-A (NHD–pDCs, P < 0.01; cHVC–pDCs, P < 0.01).

Article Snippet: Cells in PBS containing 0.1% NaN3, 1% BSA and 100 lg ⁄ mL human IgG (Jackson Immunoresearch, West Grove, PA, USA) were blocked for 15 min on ice then labelled with monoclonal antibodies against HLA-DR FITC (clone L243), CD86-FITC (clone FUN-1), CD83-APC (clone HB15e) (all from BD Biosciences) or CD303-APC (BDCA2, clone AC144; Miltenyi Biotec) or with isotype controls [murine IgG1 (clone MOPC 31, APC or FITC conjugated) or IgG2a FITC (clone G155-178) (all from BD Biosciences)].

Techniques: Infection, Cytometry, Expressing

Journal: Stem cell research

Article Title: Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells

doi: 10.1016/j.scr.2017.11.010

Figure Lengend Snippet: Effects of iMSC, Ad-MSC, and BM-MSC on T cell proliferation, PBMC IFN-γ production and DC maturation and activation. Peripheral blood mononuclear cells (PBMC) from healthy donor dogs (n = 3) were co-cultured with MSC at a ratio of 1:10 MSC:PBMC to assess the effects of MSC on T cell proliferation. Representative histograms of proliferating CD5+ T cells, either T cells alone, T cells co-cultured with iMSC, Ad-MSC or BM-MSC (the latter represented with shaded gray area) (5A). X-axis represents fluorescence intensity of CFSE (FITC) while the y-axis represents cell counts. Unstimulated, CFSE + CD5 + T cells shown in dotted line, while activated CFSE + CD5 + T cells are depicted in solid line, no shading. Effect of MSC on T cell cultures, each point represents average of technical replicates, using PBMC from 3 different unrelated donor dogs, and iMSC derived from a single iPS line. Percent T cell proliferation depicted on the y-axis (5B). IFN-γ concentrations were measured in supernatants collected from co-culture experiments at 96 h (5C). Statistical significance was evaluated using one-way ANOVA, with Tukey’s multiple means comparison test (*p < 0.05). Immature canine DC were treated with LPS to induce maturation; and expression of co-stimulatory molecules MHCII and CD86 was compared with DCs incubated with untreated or IFN-γ activated iMSCs, was measured by flow cytometry (5D, 5E). X axis represents normalized percentage mean florescence increase, with stimulated DC at 100%. MHCII expression differences between untreated DC (iDC), LPS-stimulated DCs, and DCs incubated with un-treated or IFN-γ activated iMSC (5D). CD86 expression (5E). Each point represents average of technical replicates of DC derived from 3 unrelated donor dogs. Statistical significance was determined using 1-way ANOVA with Tukey’s multiple means comparison (*p < 0.05).

Article Snippet: The impact of MSC on suppression of DC activation in response to LPS stimulation was assessed by measurement of MHCII and CD86 upregulation on DC using flow cytometry with the following antibodies: FITC-conjugated anti-canine MHCII (clone YKIX334.2 Bio-Rad, Hercules, CA) and PE-conjugated anti-human CD86 (clone IT2.2 eBioscience, San Diego, CA).

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