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: Infection and Immunity

Article Title: Limited Pulmonary Mucosal-Associated Invariant T Cell Accumulation and Activation during Mycobacterium tuberculosis Infection in Rhesus Macaques

doi: 10.1128/IAI.00431-18

Figure Lengend Snippet: Abundance of MAIT versus peptide-specific Th1 cells in M. tuberculosis-infected tissues of rhesus macaques. Animals were euthanized 6 to 7 weeks postinfection for analysis of MAIT and M. tuberculosis-specific CD4 T cells in infected tissues. Animals were intravenously injected with biotinylated anti-CD45 just prior to euthanasia to allow for identification of parenchymal T cells. (A) Sample FACS plots of MAITs and MTB300-specific CD4 T cells in different tissues of a rhesus macaque 6 weeks postinfection. Both MAITs and peptide-specific CD4 T cells are gated on intravascular stain-negative cells. (B) The frequencies of MAIT and MTB300-specific CD4 T cells among total CD3+ cells were compared in various tissues. Connecting lines indicate that both MAITs and MTB300-specific CD4 T cells were measured in the same samples from spleen, peripheral lymph nodes, pulmonary lymph nodes, PBMCs, BAL, and large instillation site lesions. Paired t tests were used to calculate statistical difference for these tissues. For individual granulomas, either MAITs or MTB300-specific T cells were measured in different lesions, so an unpaired t test was used to calculate statistical difference. (C and D) Bacterial burdens in individual granulomas (C) or lymph nodes (D) from each animal in this study. (E and F) Correlation between bacterial burdens and MAITs (E) or MTB300-specific CD4 T cells (F) in individual granulomas.

Article Snippet: Briefly, prior to euthanasia, animals were anesthetized, a BAL sample collected, and animals were injected with 50 μg/kg of body weight of anti-nonhuman primate CD45 (clone MB4-6D6; Miltenyi Biotec, San Diego, CA).

Techniques: Infection, Injection, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-induced lymphangiogenesis in cervical lymph nodes in oral melanoma-bearing mice

doi: 10.1186/1756-9966-31-83

Figure Lengend Snippet: Lymphangiogenesis in nonmetastatic sentinel lymph nodes. ( A ), ( B ) Double immunofluorescent images of CD45RB (green) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1; red) in nonmetastatic sentinel lymph nodes (SLN). Increase in LYVE-1-positive lymphatic sinuses is evident in both subcapsular margins ( A ) and medulla ( B ). sm, subcapsular margins; Me, medulla; f, follicle; pc, paracortex. Scale bar = 50 μm. ( C ) Measurement of LYVE-1-positive lymphatic sinus area in control LNs and nonmetastatic SLNs. A significant increase was observed in non-metastatic SLNs, compared with untreated controls. Columns, mean; bar, standard error. *, P<0.001 relative to controls.

Article Snippet: To evaluate lymphangiogenesis in tumor-associated LNs, we simultaneously performed three types of double immunofluorescent staining on frozen sections comprising two mixtures of two primary antibodies, goat anti-mouse/rat tyrosinase-related protein 1 (TRP-1, 1:100; Santa Cruz Biotechnology, Inc., Sata Cruz, CA, USA) and biotinylated anti-mouse LYVE-1 (1:200; R&D Systems, Minneapolis, MN, USA) and rat anti-mouse CD45RB (1:100; Acris Antibodies, Herford, Germany) and biotinylated anti-mouse LYVE-1 and a mixture of rat anti-mouse CD31 (1:100; Becton Dickinson and Co., Franklin Lakes, NJ, USA) and biotinylated anti-mouse LYVE-1 for 2 h at room temperature.

Techniques: Control