Journal:

Article Title: Increased resistance to CD4 + CD25 hi regulatory T cell-mediated suppression in patients with type 1 diabetes

doi: 10.1111/j.1365-2249.2008.03810.x

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMC) were stained using anti-CD3, anti-CD4, anti-CD25 and anti-forkhead box P3 (FoxP3) or anti-CD127 antibodies and analysed by fluorescence activated cell sorter (FACS). The CD25hi population was defined as those with a lowered expression of CD4 and this population was analysed for expression of FoxP3 or CD127. The percentage of FoxP3+ (a) or CD127lo/− (c) cells in the CD4+CD25hi population was determined in control subjects (squares) and patients with long-standing T1D (triangles). Each point represents one individual and mean values are indicated by a horizontal line. The percent of FoxP3-expressing cells (b) or CD127lo/− cells (d) in the control (squares, solid line) and patient (triangles, dotted line) groups within the top 10% of CD25 expression within the CD4+ T cell compartment is shown. Each point with error bars represents the mean and standard error of the mean of each group.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (clone SK7), phycoerythrin (PE)-conjugated anti-CD127, peridin-chlorophyll protein (PerCP)-conjugated anti-CD4 (clone L200) and allophycocyanin (APC)-labelled anti-CD4 (clone RPA-T4; BD Pharmingen, San Diego, CA, USA), PE-labelled anti-CD25 (clone MEM-181), Alexa Fluor 647-conjugated anti-CD25 (clone MEM-181) antibodies (Serotec, Oxford, UK), as well as APC-labelled anti-FoxP3 (clone PCH101; eBioscience, San Diego, CA, USA) and relevant isotype- and fluorochrome-matched control antibodies, were used in this study.

Techniques: Staining, Fluorescence, Expressing

Journal:

Article Title: Increased resistance to CD4 + CD25 hi regulatory T cell-mediated suppression in patients with type 1 diabetes

doi: 10.1111/j.1365-2249.2008.03810.x

Figure Lengend Snippet: Isolated populations of CD4-positive cells were stained for expression of CD3, CD4 and CD25 and analysed by flow cytometry. CD25hi T cells were defined as those with a slightly lowered expression of CD4 (a). The percentage of CD4+ T cells in control subjects (squares) and patients with long-standing T1D (triangles) which were CD25hi. Each point represents an individual and mean values are indicated with a horizontal line (b).

Article Snippet: Monoclonal antibodies Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (clone SK7), phycoerythrin (PE)-conjugated anti-CD127, peridin-chlorophyll protein (PerCP)-conjugated anti-CD4 (clone L200) and allophycocyanin (APC)-labelled anti-CD4 (clone RPA-T4; BD Pharmingen, San Diego, CA, USA), PE-labelled anti-CD25 (clone MEM-181), Alexa Fluor 647-conjugated anti-CD25 (clone MEM-181) antibodies (Serotec, Oxford, UK), as well as APC-labelled anti-FoxP3 (clone PCH101; eBioscience, San Diego, CA, USA) and relevant isotype- and fluorochrome-matched control antibodies, were used in this study.

Techniques: Isolation, Staining, Expressing, Flow Cytometry

Journal:

Article Title: Increased resistance to CD4 + CD25 hi regulatory T cell-mediated suppression in patients with type 1 diabetes

doi: 10.1111/j.1365-2249.2008.03810.x

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMC) were stained using anti-CD3, anti-CD4, anti-CD25 and anti-forkhead box P3 (FoxP3) or anti-CD127 antibodies and analysed by fluorescence activated cell sorter (FACS). The CD25hi population was defined as those with a lowered expression of CD4 and this population was analysed for expression of FoxP3 or CD127. The percentage of FoxP3+ (a) or CD127lo/− (c) cells in the CD4+CD25hi population was determined in control subjects (squares) and patients with long-standing T1D (triangles). Each point represents one individual and mean values are indicated by a horizontal line. The percent of FoxP3-expressing cells (b) or CD127lo/− cells (d) in the control (squares, solid line) and patient (triangles, dotted line) groups within the top 10% of CD25 expression within the CD4+ T cell compartment is shown. Each point with error bars represents the mean and standard error of the mean of each group.

Article Snippet: Monoclonal antibodies Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (clone SK7), phycoerythrin (PE)-conjugated anti-CD127, peridin-chlorophyll protein (PerCP)-conjugated anti-CD4 (clone L200) and allophycocyanin (APC)-labelled anti-CD4 (clone RPA-T4; BD Pharmingen, San Diego, CA, USA), PE-labelled anti-CD25 (clone MEM-181), Alexa Fluor 647-conjugated anti-CD25 (clone MEM-181) antibodies (Serotec, Oxford, UK), as well as APC-labelled anti-FoxP3 (clone PCH101; eBioscience, San Diego, CA, USA) and relevant isotype- and fluorochrome-matched control antibodies, were used in this study.

Techniques: Staining, Fluorescence, Expressing

Journal: Immunotherapy Advances

Article Title: α2-3 Sialic acid binding and uptake by human monocyte-derived dendritic cells alters metabolism and cytokine release and initiates tolerizing T cell programming

doi: 10.1093/immadv/ltab012

Figure Lengend Snippet: MoDCs stimulated with α2-3sia dendrimers induce tolerogenic T cell differentiation. (A) The workflow for the T helper differentiation assay is represented by the upper time line (Figure 6B, C). The workflow for the regulatory T cell induction and proliferation assay (Figure 6D, E) is represented by the lower time line. (B) Differentiation of naive CD4 + T cells was performed upon co-culture with LPS matured moDCs, and α2-3sia and LPS-stimulated moDCs. Flow cytometric analysis of the naive CD4 + T cells skewed toward T H 1 (IFNγ + cells) demonstrate that stimulation with α2-3sia and LPS resulted in reduced T H 1 skewing. (C) Cytokine secretion by CD4 + T cells was quantified in an ELISA assay after co-culture with LPS-matured moDCs stimulated with α2-3sia dendrimers. Stimulation with α2-3sia dendrimers resulted in a significant increase in TGFβ, and a trend was seen toward increased IL-10 production compared to the LPS control. (D) The induction of regulatory T cells by moDCs treated with α2-3sia dendrimers was measured by flow cytometry. A significant increase of regulatory T cell populations (FoxP3 + CD25 + CD127 - and FoxP3 + CD25 - CD127 - ) was observed compared to the medium control. (E) The proliferation of CD4 + T cells by moDCs treated with α2-3sia was quantified by CSFE staining and measured by flow cytometry. A significant decrease in proliferation of the FoxP3 - CD25 - CD127 - effector T cell population was measured. N ≥ 3; Groups were compared with a paired t -test or a Wilcoxon matched-pairs signed rank test depending on normality of the data. * = P ≤ 0.05.

Article Snippet: Samples were stained with CD4-PE (clone RPA-T4, BD Bioscience), CD25-APC (clone MEM-181, Immunotools), CD127-PE-Cy7 (clone A019D5, Biolegend), FOXP3-V450 (intracellular, clone 259D/C7, BD Bioscience) and fixable viability dye efluor 780 (Invitrogen) and subsequently measured with flow cytometry (LSRFortessa X-20, BD Bioscience) and analyzed with FlowJo (v10).

Techniques: Cell Differentiation, Differentiation Assay, Proliferation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining