Journal: Cancer immunology research

Article Title: Combined anti-VEGF and anti–CTLA-4 therapy elicits humoral immunity to galectin-1 which is associated with favorable clinical outcomes

doi: 10.1158/2326-6066.CIR-16-0385

Figure Lengend Snippet: Anti-Gal-1 antibodies isolated from an Ipi-Bev-treated patient abrogate Gal-1 binding to CD45. Anti-Gal-1 antibodies were affinity purified from plasma. Biotinylated HAS-Gal-1 (250 ng/ml) was incubated with a commercial anti-Gal-1 polyclonal antibody (Gal-1 Ab) or control antibody (10 μg/ml), enriched endogenous Gal-1 antibodies (Gal-1 Ig) or normal human IgG (1.98 μg/ml) prior to incubation with coated CD45. The binding of HAS-Gal-1 to CD45 was detected with streptavidin-HRP. Sucrose and lactose were added to the reaction at 5 mM. Data are presented as Mean ± SD of 3 experiments.

Article Snippet: Recombinant human CD45 (R&D Systems) was coated onto 96-well plates (25 ng/well) at 4°C overnight.

Techniques: Isolation, Binding Assay, Affinity Purification, Clinical Proteomics, Incubation, Control

Journal: PLoS ONE

Article Title: Human intrahepatic ILC2 are IL-13 positive amphiregulin positive and their frequency correlates with model of end stage liver disease score

doi: 10.1371/journal.pone.0188649

Figure Lengend Snippet: (A) ILC gating strategy. Intrahepatic lymphocytes were freshly isolated and gated on the CD45 pos CD3 neg population. The total ILC population was defined as lineage neg and CD127 pos . Of these, ILC1 were defined as CD117 neg CRTH2 neg , ILC2 as CD117 pos/neg CRTH2 pos and ILC3 as CD117 pos CRTH2 neg. (B) Frequency of the total ILC population among the CD3 neg CD45 pos population. (C) Frequencies of the three major ILC subsets in the total intrahepatic ILC in different inflammatory liver diseases. AILD: Autoimmune Liver Diseases (PSC, PBC, AIH); NASH: Nonalcoholic Steatohepatitis; ALD: Alcoholic Liver Disease. (* = p <0.05 by Kruskal Wallis test). (D) Distribution of NKp44 expression among intrahepatic ILC. ILC3 were NKp44 pos (NCR pos ) or NKp44 neg (NCR neg ). (E) Frequencies of NKp44 expression by intrahepatic ILC3 in normal and diseased livers. Summary data are median ± Interquartile range.

Article Snippet: Freshly isolated lymphocytes were stained on ice with antibodies against functional surface markers of interest together with antibodies to identify ILC subsets including: CD3 (UCHT1, BioLegend), CD45 (5B1, Miltenyi Biotec), CD127 (eBioRDR5, eBiosciences) and lineage markers (CD1a (HI149), CD11c (MJ4-27G12), CD14 (TUK4), CD16 (VEP13), CD19 (LT19), CD34 (AC136), CD94 (REA113), CD123 (AC145), BDCA2 (AC144), FceR1 (CRA1) (all from Miltenyi Biotec)) to identify total ILC, CRTH2 (BM16) and CD117 (104D2) (both from BioLegend) to distinguish ILC1 (CRTH2-CD117-), ILC2 (CRTH2+CD117+/-) and ILC3 (CRTH2-CD117+).

Techniques: Isolation, Expressing

Journal: PLoS ONE

Article Title: Human intrahepatic ILC2 are IL-13 positive amphiregulin positive and their frequency correlates with model of end stage liver disease score

doi: 10.1371/journal.pone.0188649

Figure Lengend Snippet: (A) The CD45 pos CD3 neg lineage neg CD127 pos CRTH2 pos ILC2 subset was gated and CD161 and CD69 expressions were analysed. CD161 and CD69 representative overlays and summary data are shown. (B) PGD2 production by human liver. The secretion of PGD2 by normal and inflamed human liver tissue was analysed by ELISA on 24-hour liver tissue supernatants prepared for 1g-tissue/1ml culture medium. Summary data are median ± Interquartile range. (C) Expression of the IL-33 receptor, ST2 was analysed on ILC2. Representative overlay and summary data are shown. In histogram overlays, dotted lines are isotype staining and shaded histograms marker expression.

Article Snippet: Freshly isolated lymphocytes were stained on ice with antibodies against functional surface markers of interest together with antibodies to identify ILC subsets including: CD3 (UCHT1, BioLegend), CD45 (5B1, Miltenyi Biotec), CD127 (eBioRDR5, eBiosciences) and lineage markers (CD1a (HI149), CD11c (MJ4-27G12), CD14 (TUK4), CD16 (VEP13), CD19 (LT19), CD34 (AC136), CD94 (REA113), CD123 (AC145), BDCA2 (AC144), FceR1 (CRA1) (all from Miltenyi Biotec)) to identify total ILC, CRTH2 (BM16) and CD117 (104D2) (both from BioLegend) to distinguish ILC1 (CRTH2-CD117-), ILC2 (CRTH2+CD117+/-) and ILC3 (CRTH2-CD117+).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining, Marker

Journal: PLoS ONE

Article Title: Human intrahepatic ILC2 are IL-13 positive amphiregulin positive and their frequency correlates with model of end stage liver disease score

doi: 10.1371/journal.pone.0188649

Figure Lengend Snippet: (A) The CD45 pos CD3 neg lineage neg CD127 pos CRTH2 pos ILC2 subset was gated and expression of the IL-2 receptor α-chain, CD25, was analysed. CD25 representative overlay and summary data in normal and diseased livers is shown. (** = p <0.01 by Mann Whitney test). In the histogram overlay the dotted line represents isotype staining and the shaded histogram reports CD25 staining. (B) Human inflamed liver supernatant was analysed for IL-2, IL-7, IL-9 and IFN-γ. The secretion of cytokines by normal and inflamed human liver tissue was analysed by luminex of 24-hour liver tissue supernatants prepared for 1g of tissue/1ml culture medium. (C) IL-33 production by human liver tissue (Normal and diseased) and (D) IL-33 production by Primary human biliary epithelial (BEC). IL-33 in 24-hour supernatants generated by 1g liver tissue/1ml medium or BEC cells unstimulated, stimulated with IFN-γ and TNF-α or with lipopolysaccharide (LPS) was analysed by ELISA. Summary data are median ± Interquartile range.

Article Snippet: Freshly isolated lymphocytes were stained on ice with antibodies against functional surface markers of interest together with antibodies to identify ILC subsets including: CD3 (UCHT1, BioLegend), CD45 (5B1, Miltenyi Biotec), CD127 (eBioRDR5, eBiosciences) and lineage markers (CD1a (HI149), CD11c (MJ4-27G12), CD14 (TUK4), CD16 (VEP13), CD19 (LT19), CD34 (AC136), CD94 (REA113), CD123 (AC145), BDCA2 (AC144), FceR1 (CRA1) (all from Miltenyi Biotec)) to identify total ILC, CRTH2 (BM16) and CD117 (104D2) (both from BioLegend) to distinguish ILC1 (CRTH2-CD117-), ILC2 (CRTH2+CD117+/-) and ILC3 (CRTH2-CD117+).

Techniques: Expressing, MANN-WHITNEY, Staining, Luminex, Generated, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Human intrahepatic ILC2 are IL-13 positive amphiregulin positive and their frequency correlates with model of end stage liver disease score

doi: 10.1371/journal.pone.0188649

Figure Lengend Snippet: (A) The CD45 pos CD3 neg lineage neg CD127 posi CRTH2 pos ILC2 subset was gated and expressions of CXCR3, CCR6, Very Late Antigen-5 (VLA-5) and Very Late Antigen-6 (VLA-6) were analysed. Representative overlays and summary data in normal and diseased livers are shown (normal livers = open squares; diseased livers = filled squares). Summary data are median ± Interquartile range. In histogram overlays, dotted lines represent isotype staining and shaded histograms show the marker expression. (B) Primary human biliary epithelial cells (BEC), stellate cells and fibroblasts were isolated and stimulated with IFN-γ and TNF-α. Interferon gamma-induced Protein-10 (IP-10) secretion was analysed by ELISA. (* = p<0.05, ** = <0.01 by Paired t- test). Summary data are mean ± SEM. (C) Expressions of CXCR3 and CCR6 by the peripheral blood ILC subsets of normal donors and autoimmune liver disease patients with the condition autoimmune hepatitis (AIH). Summary data are median ± interquartile range. (* = p<0.05 by Mann-Whitney test).

Article Snippet: Freshly isolated lymphocytes were stained on ice with antibodies against functional surface markers of interest together with antibodies to identify ILC subsets including: CD3 (UCHT1, BioLegend), CD45 (5B1, Miltenyi Biotec), CD127 (eBioRDR5, eBiosciences) and lineage markers (CD1a (HI149), CD11c (MJ4-27G12), CD14 (TUK4), CD16 (VEP13), CD19 (LT19), CD34 (AC136), CD94 (REA113), CD123 (AC145), BDCA2 (AC144), FceR1 (CRA1) (all from Miltenyi Biotec)) to identify total ILC, CRTH2 (BM16) and CD117 (104D2) (both from BioLegend) to distinguish ILC1 (CRTH2-CD117-), ILC2 (CRTH2+CD117+/-) and ILC3 (CRTH2-CD117+).

Techniques: Staining, Marker, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Human intrahepatic ILC2 are IL-13 positive amphiregulin positive and their frequency correlates with model of end stage liver disease score

doi: 10.1371/journal.pone.0188649

Figure Lengend Snippet: ( A) Intrahepatic ILC were freshly isolated and stimulated with PMA and Ionomycin for 4 hours and both CD45 pos CD3 neg lineage neg CD127 pos CRTH2 pos ILC2 and CD45 pos CD3 neg lineage neg CD127 pos CRTH2 neg populations were analysed for expressions of IL-4, IL-5, IL-13 and IFN-γ. Representative dot plots and summary data are shown. (B) Immunohistochemistry for amphiregulin in CD3 positive and negative intrahepatic immune cells in human liver. CD3 (Vector Red, red color); Amphiregulin (DAB, Brown color). (C) Amphiregulin expression by intrahepatic ILC2. Amphiregulin representative overlay and summary data in normal and diseased livers are shown. In the histogram overlay the dotted line represents isotype staining and the shaded histogram reports amphiregulin staining.

Article Snippet: Freshly isolated lymphocytes were stained on ice with antibodies against functional surface markers of interest together with antibodies to identify ILC subsets including: CD3 (UCHT1, BioLegend), CD45 (5B1, Miltenyi Biotec), CD127 (eBioRDR5, eBiosciences) and lineage markers (CD1a (HI149), CD11c (MJ4-27G12), CD14 (TUK4), CD16 (VEP13), CD19 (LT19), CD34 (AC136), CD94 (REA113), CD123 (AC145), BDCA2 (AC144), FceR1 (CRA1) (all from Miltenyi Biotec)) to identify total ILC, CRTH2 (BM16) and CD117 (104D2) (both from BioLegend) to distinguish ILC1 (CRTH2-CD117-), ILC2 (CRTH2+CD117+/-) and ILC3 (CRTH2-CD117+).

Techniques: Isolation, Immunohistochemistry, Plasmid Preparation, Expressing, Staining