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: FEBS letters

Article Title: Regulation of galectin-3-induced apoptosis of Jurkat cells by both O-glycans and N-glycans on CD45.

doi: 10.1016/j.febslet.2013.10.034

Figure Lengend Snippet: Fig. 2. Construction of CD45RO-J45.01 and CD45RABC-J45.01 cells and expression of galectin-3 receptors on the cells. (A) Schematic figure of CD45RABC isoform and CD45RO isoform. Compared to CD45RO, CD45RABC has all three alternatively spliced domain denoted A, B and C, which have heavily O-glycosylation. (B) CD45 expression was analyzed by RT-PCR. J45.01 cells were transfected with empty vector (mock control), CD45RO or CD45RABC. Total RNA was extracted followed by reverse transcription. PCR amplified bands were performed using the same CD45 upstream and downstream primers. (C) Western blotting analysis for CD45RO and CD45RABC. 1 107 J45.01 cells stably expressing mock, CD45RO or CD45RABC were lysed and the lysates were boiled in loading buffer. Then the supernatants were run 6% SDS–PAGE and transferred onto nitrocellulose membrane. CD45 was detected by rabbit anti-CD45 mAb followed by HRP-labeled goat anti-rabbit polyclonal antibody. (D) Flow cytometry analysis for expression of CD45 by anti-CD45 antibody on the JE6-1 cells and J45.01 cells transfected with mock, CD45RO or CD45RABC. (E) Polychromatic flow cytometry was performed to examine CD3, CD29, CD43 or CD71 expression on mock-, CD45RO- and CD45RABC-J45.01 cells stained with PerCP-CD3, PE-CD29, APC-CD43 and FITC-CD71 mAb. (F) Cells were incubated with 180 nM galectin-3 in the presence or absence of 0.1 M b-lactose. After washing, the cells were then stained with PE-labeled mouse anti-human galectin- 3 antibody for 30 min on ice. Galectin-3 binding to mock-, CD45RO-, CD45RABC-J45.01 cells and JE6-1 cells were examined by flow cytometry. Histograms are representative of three similar experiments.

Article Snippet: The template cDNA of CD45RO and CD45RABC were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Glycoproteomics, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot, Stable Transfection, SDS Page, Membrane, Labeling, Flow Cytometry, Cytometry, Staining, Incubation, Binding Assay

Journal: FEBS letters

Article Title: Regulation of galectin-3-induced apoptosis of Jurkat cells by both O-glycans and N-glycans on CD45.

doi: 10.1016/j.febslet.2013.10.034

Figure Lengend Snippet: Fig. 3. Regulation of galectin-3-induced apoptosis of Jurkat cells by O-glycans on CD45. (A) Aggregation of both CD45RO-J45.01 and CD45RABC-J45.01 cells induced by galectin-3. Cell apoptosis induced by galectin-3 (3 lM, 5.5 h) was examined by evaluation of mitochondrial membrane potential with JC-1 dye (B), or by Br-dUTP followed by incubation with FITC-labeled anti-BrdU mAb (C). FasL treatment (200 ng/ml, 18 h) was used as a positive control. (D) CD45RO- or CD45RABC-transfected J45.01 cells were treated with 3 lM galectin-3 for 5.5 h and subjected to Annexin-V/7-AAD binding assay by flow cytometry. CD45RABC-J45.01 cells were pretreated with O-glycosylation inhibitor (1 mM, Benzyl-GalNAc, BG) for 24 h before treatment with galectin-3. The percentage of apoptotic cells (right panel) induced by galectin-3 was significantly larger in CD45RABC-J45.01 cells than in mock-J45.01 cells, CD45RO-J45.01 cells and BG-treated CD45RABC-J45.01 cells (⁄P < 0.05). Gal3, BG and Con represent galectin-3, Benzyl- GalNAc and control, respectively. Dot plots and histograms are representative of three similar experiments. Data shown are the mean ± S.E.M. from three independent experiments.

Article Snippet: The template cDNA of CD45RO and CD45RABC were purchased from OriGene (Rockville, MD).

Techniques: Membrane, Incubation, Labeling, Positive Control, Transfection, Binding Assay, Cytometry, Glycoproteomics, Control