Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: ITIM motif of the cytoplasmic tail domain in BST2.

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Sequencing, Residue

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: PolyI:C stimulated Bst2 -/- NK cells have a higher cytotoxicity to tumor cells than Bst2 +/+ NK cells. B6-origin Bst2 wildtype (+/+) and Bst2 knockout (-/-) mice were intraperitoneally injected with polyI:C. At 16 hrs after injections, splenic NK cells were isolated using DX-5 microbeads. ( A ) Bst2 +/+ and Bst2 -/- NK cells were stained with anti-BST2 antibody and analyzed by flow cytometry. NK1.1 + TCRβ - cells were indicated as NK cells. Data are representative of two independent experiments. ( B ) Calcein-AM stained target cells (YAC-1 and RMA-S) were co-cultured with Bst2 +/+ and Bst2 -/- NK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured by SpectraMAX with excitation/emission wavelengths at 485 nm/535 nm. Graphs showed a mean ± S.D. ( n = 3 per group). Data are representative of three independent experiments. (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Knock-Out, Injection, Isolation, Staining, Flow Cytometry, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Bst2 -/- lymphokine activated killer cells have higher cytotoxicity to tumor cells than Bst2 +/+ LAK cells. Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated with IL-2 for seven days to generate LAK cells. ( A ) Bst2 +/+ and Bst2 -/- LAK cells were stained with anti-BST2 antibody and analyzed by flow cytometry. ( B ) Calcein-AM stained target cells (YAC-1) were co-cultured with Bst2 +/+ and Bst2 -/- LAK cells for 1 hr at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 3–4 per group). Data are representative of three independent experiments. ( C ) After detecting calcein released in media, released granzyme B in the same media was measured by enzyme-linked immunosorbent assay (ELISA). Graphs showed a mean ± S.D. ( n = 3–4 per group). Data are representative of two independent experiments. ( D ) Bst2 +/+ and Bst2 -/- LAK cells were co-cultured with target cells (YAC-1) in culture media containing Golgistop and anti-CD107a antibody. NK1.1 and TCRβ were stained after 1 hr of cultivation at 37 °C in a humidified incubator. Percentages of CD107a positive cells from LAK cells (NK1.1 + TCRβ - ) were indicated. Graphs showed a mean ± S.D. ( n = 3 per group). Data are representative of two independent experiments. ( E ) CFSE stained Bst2 +/+ LAK cells were treated with isotype control or anti-BST2 antibodies and co-cultured with target cells (RMA-S and B16) for 4 hrs at 37 °C in a humidified incubator. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry. CFSE - , 7-AAD + population is designated as lysed target cells. The word ‘iso’ indicates isotype control antibody treatment and ‘αBST2′ indicates anti-BST2 antibody treatment. Graphs showed a mean ± S.D. ( n = 3–5 per group). (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Galectin-9 is associated with BST2-dependent NK cell activity. ( A ) Recombinant BST2 protein was pulled down with GST-tagged Gal-3 (GST-Gal3), as well as the GST-tagged long isoform of Gal-9 (GST-Gal9L). Precipitates were then analyzed by western blotting with anti-BST-2 antibody. ( B ) Recombinant BST2 protein was pulled down with either GST-tagged NCRD or CCRD of Gal9L (GST-Gal9L-NCRD or GST-Gal9L-CCRD). Precipitates were then analyzed by western blotting with anti-BST-2 antibody. ( C ) Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. CFSE stained Bst2 +/+ and Bst2 -/- LAK cells were co-cultured with target cells (B16) for 4 hrs at 37 °C in a humidified incubator in the presence of isotype control antibody or anti-Gal-9 antibody. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry. CFSE - , 7-AAD + population is designated as lysed target cells. Graphs showed a mean ± S.D. ( n = 2–4 per group). ( D ) Naïve splenocytes of B6-origin Bst2 +/+ and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Calcein-AM stained target cells (Wild type (WT) YAC-1 and Lgals9 -/- YAC-1) were co-cultured with Bst2 +/+ and Bst2 -/- LAK cells for 3 hrs in a 96-well round plate. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 4–6 per group). Data are representative of two independent experiments. ( E ) Western blotting was performed on whole cell lysates of tumor target cell lines with anti-galectin-9 antibody and anti-GAPDH antibody. GAPDH was used as a loading control. The upper panel showed the result of wildtype tumor cell lines (RMA-S, B16, and YAC-1) and the lower panel showed the comparison of wildtype YAC-1 and Lgals9 -/- YAC-1 clone used in this study. (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Activity Assay, Recombinant, Western Blot, Staining, Cell Culture, Control, Flow Cytometry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: Long isoform, but not short isoform, of BST2 on NK cells impedes cytotoxicity. ( A ) F1 hybrids were generated by crossing B6-origin Bst2 +/- mice and NZW mice ( Bst2 S/S ). ( B ) Naïve splenocytes of F1 hybrids and B6-origin Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Expression of BST2 was detected through flow cytometry staining. ( C ) CFSE stained Bst2 S/+ and Bst2 S/- LAK cells were co-cultured with target cells (YAC-1, RMA-S, and B16) for 4 hrs at 37 °C in a humidified incubator. Cells were stained with 7-AAD after cultivation. These cells were then fixed with 1% paraformaldehyde and analyzed by FACS. CFSE-, 7-AAD+ population is designated as lysed target cells. Graphs showed a mean ± S.D. ( n = 4 per group). ( D ) CFSE stained Bst2 S/+ and Bst2 S/- LAK cells from F1 hybrids were co-cultured with target cells (RMA-S) cells for 4 hrs at 37 °C in a humidified incubator in absence or presence of anti-BST2 antibody. Graphs showed mean ± S.D. ( n = 3–4 per group). (**, p < 0.01; ****, p < 0.0001; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Generated, Expressing, Flow Cytometry, Staining, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain

doi: 10.3390/ijms231911395

Figure Lengend Snippet: NK cells with short isoform of BST2 show enhanced cytotoxicity as well as BST2 deficient NK cells. ( A ) Backcross from NZW ( Bst2 S/S ) background to B6 background. ( B ) BST2 expression of LAK cells generated from splenocytes of B6-background Bst2 +/- , Bst2 S/- , and Bst2 -/- mice. ( C ) Calcein-AM stained target cells (YAC-1, RMA-S, and B16) were co-cultured with Bst2 +/- , Bst2 S/- , and Bst2 -/- LAK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Data are representative of two independent experiments. Graphs showed mean ± S.D. ( n = 4 per group). ( D ) Naïve splenocytes of B6-origin Bst2 +/+ , Bst2 S/- , and Bst2 -/- mice were stimulated by IL-2 for seven days to generate LAK cells. Expression level of BST2 was measured through flow cytometry staining. ( E ) Calcein-AM stained target cells (YAC-1 and Lgals9 -/- YAC-1) were co-cultured with Bst2 +/+ , Bst2 S/- , and Bst2 -/- LAK cells for 4 hrs at 37 °C in a humidified incubator. Released calcein was measured to analyze cytotoxicity of effector cells as described in . Graphs showed a mean ± S.D. ( n = 6 per group). (*, p < 0.05; **, p < 0.01; n.s., not significant).

Article Snippet: NZW/N mice expressing cytoplasmic tail deficient, short isoform of BST2 ( Bst2 S/S ) were purchased from Japan SLC (Hamamatsu, Japan) and crossed with Bst2 knockout C57BL/6 (B6) mice to generate B6/NZW F1 mice.

Techniques: Expressing, Generated, Staining, Cell Culture, Flow Cytometry