Journal: Immunology

Article Title: Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

doi: 10.1111/imm.12663

Figure Lengend Snippet: Expression of Notch receptors on infiltrating regulatory T (Treg) cells in experimental autoimmune uveitis (EAU). (a) Representative histograms of Notch‐1, Notch‐2 and Notch‐3 on GFP + Treg cells in uveitic eyes. (b) Statistical analysis for the fluorescent intensity of Notch‐1, Notch‐2 and Notch‐3 on GFP + Treg cells in uveitic eyes. Isotype, isotype antibody control. ***P < 0·001. n = 3 per group. Each circle represents a mouse.

Article Snippet: Flow cytometry and cell sorting The following anti‐mouse antibodies were used for detection and sorting of Treg cells: allophycocyanin (APC) anti‐CD3 (17A2), APC‐Cy7 anti‐CD4 (GK1.5), Peridinin chlorophyll protein anti‐CD8a (53‐6.7), phycoerythrin (PE) anti‐Notch‐2 (16F11), PE anti‐DLL1 (30B11.1), PE‐Cy5 anti‐CD45 (30‐F11), Alexa Fluor ® 647 anti‐Foxp3 (R16‐715), PE anti‐CD45.1 (A20), PE‐Cy7 anti‐CD45.2 (104) and PE Annexin V were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Expressing

Journal: Immunology

Article Title: Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

doi: 10.1111/imm.12663

Figure Lengend Snippet: Notch signalling down‐regulates Foxp3 in infiltrating regulatory T (Treg) cells. (a) HES1 and HES5 expression in infiltrating Treg cells. Left panel: representative Immunoblot image. Right panel: statistical analysis for the expression of HES1 and HES5. (b, c) Staining of CD137, CD154 and Helios in infiltrating Treg cells. CD4+ GFP + Treg cells were sorted from uveitic eyes 21 days after experimental autoimmune uveitis (EAU) induction. Then expression of CD137, CD154 and Helios were evaluated in Treg cells. Representative dot plot and histograms are presented in (b), and percentages of Helios+ cells in Treg subpopulations are shown in (c). Numbers in (b) are percentages of gated populations. (d) Expression of HES1 in Treg subpopulations was quantified by real‐time PCR. Note that peripheral blood total Treg cells at day 14 were used as control. Day 14 PB total, peripheral blood total Treg cells at day 14. (e) Expression of Notch‐1 and Notch‐2 on Treg subpopulations. Left panel: representative histograms. Right panel: statistics for mean fluorescence intensity of Notch‐1 and Notch‐2. n = 3 per group. (f) The blocking effect of neutralizing antibodies against JAG1 and DLL1 on Notch signalling in Treg cells. Splenic Treg cells were cultured in a microplate pre‐coated with JAG1 and DLL1 in the presence or absence of mixed neutralizing antibodies against JAG1 and DLL1 for 12 hr. HES1 and HES5 were detected by Immunoblot. This is a representative image of two independent experiments. V, vehicle. Abs, mixed neutralizing antibodies. (g, h) Foxp3 expression in infiltrating Treg cells after in vitro culture for 24 and 48 hr. (g) Representative dot plots. The number in each plot is the percentage of GFP + cells. (h) Statistical analysis for the proportion of GFP + cells. (i) Apoptosis of Treg cells after culture. Fresh, freshly sorted Treg cells. Alone, Treg cells alone. Ligand, Treg cells cultured with immobilized JAG1 and DLL1. Ligand+Abs, Treg cells cultured with immobilized JAG1 and DLL1 in the presence of neutralizing antibodies against JAG1 and DLL1. *P < 0·05; **P < 0·01; ***P < 0·001. Each circle represents a mouse.

Article Snippet: Flow cytometry and cell sorting The following anti‐mouse antibodies were used for detection and sorting of Treg cells: allophycocyanin (APC) anti‐CD3 (17A2), APC‐Cy7 anti‐CD4 (GK1.5), Peridinin chlorophyll protein anti‐CD8a (53‐6.7), phycoerythrin (PE) anti‐Notch‐2 (16F11), PE anti‐DLL1 (30B11.1), PE‐Cy5 anti‐CD45 (30‐F11), Alexa Fluor ® 647 anti‐Foxp3 (R16‐715), PE anti‐CD45.1 (A20), PE‐Cy7 anti‐CD45.2 (104) and PE Annexin V were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Expressing, Western Blot, Staining, Real-time Polymerase Chain Reaction, Fluorescence, Blocking Assay, Cell Culture, In Vitro

Journal: Immunology

Article Title: Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

doi: 10.1111/imm.12663

Figure Lengend Snippet: Notch‐1 is crucial for regulatory T (Treg) cell function in experimental autoimmune uveitis (EAU). (a) Knockdown of Notch‐1 or Notch‐2 in Treg cells. Upper panel: representative Immunoblot image. Lower panel: statistical analysis for Notch‐1 and Notch‐2. (b) Experimental design for (c–e). (c) Representative dot plots of donor‐derived Treg cells in uveitic eyes. (d) Expression of Foxp3 and programmed cell death protein 1 (PD‐1) in donor‐derived Treg cells in uveitic eyes. Left panel: representative histograms. Right panel: statistical analysis. (e) mRNA levels of transforming growth factor‐β (TGF‐β), interleukin‐12a (IL‐12a), IL‐27b and interferon‐γ (IFN‐γ) in donor‐derived Treg cells in uveitic eyes. L‐SC, Treg cells transduced with L‐SC. L‐sh‐N‐1, Treg cells transduced with L‐sh‐N‐1. L‐sh‐N‐2, Treg cells transduced with L‐sh‐N‐2. *P < 0·05; **P < 0·01; ***P < 0·001. n = 3 or n = 5 per group. Each circle represents a mouse.

Article Snippet: Flow cytometry and cell sorting The following anti‐mouse antibodies were used for detection and sorting of Treg cells: allophycocyanin (APC) anti‐CD3 (17A2), APC‐Cy7 anti‐CD4 (GK1.5), Peridinin chlorophyll protein anti‐CD8a (53‐6.7), phycoerythrin (PE) anti‐Notch‐2 (16F11), PE anti‐DLL1 (30B11.1), PE‐Cy5 anti‐CD45 (30‐F11), Alexa Fluor ® 647 anti‐Foxp3 (R16‐715), PE anti‐CD45.1 (A20), PE‐Cy7 anti‐CD45.2 (104) and PE Annexin V were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Cell Function Assay, Western Blot, Derivative Assay, Expressing, Transduction