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

Journal: International Journal of Rheumatic Diseases

Article Title: Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome

doi: 10.1111/1756-185x.12089

Figure Lengend Snippet: Figure 2 (a) Western blot showing reactivity to recombinant carbonic anhydrase III (CAIII) in serum from patients with vasculitis. Lane 1, serum from patient with polyarteritis nodosa (PAN), lane 2, serum from patient with Takayasu’s arteritis (TA), lane 3, serum from patient with Wegener’s granulomatosis (WG), lane 4, serum from patient with micro- scopic polyangiitis (MPA). Blot reactivity was observed in serum samples from patients with TA and MPA (lanes 2, 4). (b) Western blotting with non-treated serum and absorbed serum. Lane 1, Western blotting with serum incubated with recombinant CAIII to absorb anti-CAIII antibodies before blotting. Lane 2, Western blotting with serum from patient with untreated MPA.

Article Snippet: Western blotting using recombinant CAIII as antigen For detection of IgG antibodies to CAIII, 1 lg/lane recombinant human CAIII (R&D Systems, Inc., Minneapolis, MN, USA) was subjected to 15% SDS-PAGE under reducing conditions, and transferred onto PVDF membrane.

Techniques: Western Blot, Recombinant, Incubation

Journal: International Journal of Rheumatic Diseases

Article Title: Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome

doi: 10.1111/1756-185x.12089

Figure Lengend Snippet: Figure 3 Quantitative analysis of immunoglobulin G (IgG) reactivity against the recombinant protein carbonic anhydrase III (CAIII) in individual serum sample of patients with vasculi- tis. Serum samples from 23 patients with microscopic polyan- giitis (MPA), 15 with Wegener’s granulomatosis (WG), 10 with polyarteritis nodosa (PAN), nine with Takayasu’s arteritis (TA), four with allergic granulomatous angiitis (AGA), and 32 healthy controls were incubated with recombinant CAIII immobilized onto an enzyme-linked immunosorbent assay plate. The mean optical density (OD) values for the recombi- nant protein were normalized by mean OD values of the back- ground immunoreactivity and expressed as arbitrary units (AU) based on the positive and negative reference sera. Hori- zontal lines indicate the mean for each group and the broken line indicates the cutoff level at the mean + 2 standard devia- tions (SD) of the healthy control. In patients with MPA, 11 of 23 serum samples were positive for CAIII. In contrast, of the 32 healthy controls, two were positive for CAIII reactivity. The difference between patients with MPA and healthy controls was statistically significant (P < 0.001).

Article Snippet: Western blotting using recombinant CAIII as antigen For detection of IgG antibodies to CAIII, 1 lg/lane recombinant human CAIII (R&D Systems, Inc., Minneapolis, MN, USA) was subjected to 15% SDS-PAGE under reducing conditions, and transferred onto PVDF membrane.

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Rheumatic Diseases

Article Title: Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome

doi: 10.1111/1756-185x.12089

Figure Lengend Snippet: Figure 4 Comparison between carbonic anhydrase III (CAIII) enzyme-linked immunosorbent assay values and vasculitis disease activity (Birmingham vasculitis activity scores, BVAS). Patients with microscopic polyangiitis (MPA) were divided into two groups, the anti-CAIII-positive group and anti- CAIII-negative group. BVAS were significantly higher in the anti-CAIII-positive group compared to the anti-CAIII-negative group. Horizontal bars indicate the median BVAS in each group.

Article Snippet: Western blotting using recombinant CAIII as antigen For detection of IgG antibodies to CAIII, 1 lg/lane recombinant human CAIII (R&D Systems, Inc., Minneapolis, MN, USA) was subjected to 15% SDS-PAGE under reducing conditions, and transferred onto PVDF membrane.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Frontiers in Medicine

Article Title: Urinary CD8+HLA-DR+ T Cell Abundance Non-invasively Predicts Kidney Transplant Rejection

doi: 10.3389/fmed.2022.928516

Figure Lengend Snippet: Gating strategies for T cell subsets (A) and tubular epithelial cells (TEC) (B) . Isotype controls are displayed as blue, while full stains are represented in red. (C.1) Schematic overview of investigated subsets. Proximal TECs were defined CD10+ and CD13+, while distal TECs were characterized being CD227+ and CD326(EpCAM)+. (C.2) Maturation of naïve T cells into memory T cells. (D) Workflow for epigenetic analysis of urine samples. SSC, side scatter; FSC, forward scatter; TNV, naïve T cells; TEM, T effector memory cells; TCM, T central memory cells; TEMRA, T effector memory cells re-expressing CD45RA.

Article Snippet: The following antibodies were used: for T cells anti-CD3-APCeF780 (eBioscience, SK7, mo IgG1k), -CD4-PEVio770 (Miltenyi Biotec, REA623, REA) -CD8-APC (Biolegend, SK1, mo IgG1k) -CD45RO-PE (Biolegend, UCHL1, mo IgG1k2), -CD45-BUV805 (BD, 3D12, rat IgG1ak), -CCR7-BV421 (Biolegend, G043H7, mo IgG2ak), -HLA-DR-BUV395 (BD, G46-6, mo IgG2ak), -CD28-FITC (Biolegend, CD28.2, mo IgG1k) and for tubular epithelial cells anti-Cytokeratin-FITC (Miltenyi Biotec, CK3-6H5, mo IgG1k), -Vimentin-APC (Miltenyi Biotec, REA409, REA), -CD10-PeVio770 (Miltenyi Biotec, REA877, REA), -CD13-APCVio770 (Miltenyi Biotec, REA263, REA), -CD227-PE (Miltenyi Biotec, REA448, REA), -CD326-BV711 (Biolegend, 9C4, mo IgG2b).

Techniques: Expressing

Journal: Cell

Article Title: HIF Regulates Multiple Translated Endogenous Retroviruses: Implications for Cancer Immunotherapy

doi: 10.1016/j.cell.2025.01.046

Figure Lengend Snippet: Key resource table

Article Snippet: Carbonic Anhydrase 9 antibody, antihuman, PE, REAfinity , Miltenyi Biotec , Cat#130–123-299, RRID: AB_2857582.

Techniques: Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Purification, Sample Prep, Sequencing, Amplification, Software, Binding Assay, Transferring, Reverse Transcription, Low Protein Binding, Antibody Purification, Adjuvant, Enzyme-linked Immunospot