anti-mouse perforin Search Results


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  • 85
    Thermo Fisher anti perforin monoclonal antibody mouse igg2b
    Colocalization of DsRed-FasL-pHluorin with <t>perforin</t> in NKL cells. ( a ) Confocal image of a paraformaldehyde-fixed NKL cell showing the distribution of DsRed, pHluorin and perforin. Human NKL cells were attached on the poly-coated slides for 10 min at 37 °C, fixed, permeabilized and stained with mouse monoclonal antibody <t>IgG2b</t> against human perforin and followed by goat anti-mouse IgG2b secondary antibody conjugated with Alexa Fluor 647 dye. The scale bar is 5.0 μm. ( b ) Confocal image of DsRed-FasL-pHluorin in a live cell after the addition of 500 m ammonium chloride (pH 7.4). The images are representative of at least 50 cells in two independent experiments. The scale bar is 5.0 μm.
    Anti Perforin Monoclonal Antibody Mouse Igg2b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc perforin 1
    Regulation of NFIL3 by IL-2-JAK1/3 signaling. ( A ) Average frequency of protein copy numbers per cell calculated from the proteomic datasets: The bin that contains NFIL3 is indicated in black. ( B ) Western blot analysis of NFIL3 abundance in CTLs following IL-2 deprivation or treatment with 100 nM Tofacitinib. Quantification is shown alongside as data normalized to SMC1 intensity and NFIL3 protein abundance in IL-2 maintained CTL. ( C ) Abundance of Nfil3 mRNA in CTLs. Abundance of NFIL3 protein ( D ) and mRNA ( E ) in naïve CD8 + T cells, TCR-activated CD8 + T cells, and CTLs activated for 48 hours and then maintained in IL-2 for 1 day, 3 days, or 5 days. ( F ) Analysis of NFIL3 protein abundance in HIF1α expressing ( Hif1a +/+ ) and HIF1α deficient ( Hif1a -/- ) CTLs maintained in normoxia (21% O 2 ) or switched into hypoxic (1% O 2 ) conditions for eight hours. Quantification of NFIL3 protein is shown alongside. Relative abundance of Nfil3 ( G ) and GLUT1 ( Slc2a1 ) ( H ) mRNA in CTLs maintained in normoxia or switched into 1% O 2 for four hours. Abundance of CD62L ( Sell ) mRNA ( I ) and cell-surface protein abundance ( J ) in IL-2-maintained Nfil3 +/+ and Nfil3 -/- CTL. In ( J ), the percentage of CD8 + CD62L high CTLs are shown alongside. Abundance of <t>perforin</t> ( Prf1 ) mRNA ( K ) and protein ( L ) in Nfil3 +/+ and Nfil3 -/- CTLs. In ( L ) quantification of NFIL3, normalized to SMC1 and perforin protein abundance in IL-2 maintained CTL, detected by Western blot is shown alongside. In ( C ), ( G ), and ( H ) the qPCR data were normalized to Tbp , and mRNA abundance in IL-2 maintained CTL in normoxia. In ( E ) qPCR data were normalized to Tbp and Cd8 and all are shown relative to day 5 IL-2-maintained CTLs in normoxia. In ( I ) and ( K ), mRNA was normalized to Hprt , and data are shown relative to the abundance in Nfil3 +/+ CTLs. Data in ( A )-( E ) and ( G )-( H ) data show, or are representative of, three biological replicates; in ( F ) data are representative of six Hif1a +/+ and five Hif1a -/- biological replicates, four independent experiments; in ( I )-( L ) data are representative of four Nfil3 +/+ and three Nfil3 -/- biological replicates. Bar charts show data from individual biological replicates, color matched where appropriate, the bar shows the mean and the errors bars show standard deviation. P values shown in ( B )-( H ) are two-tailed one sample Student’s t-tests and in ( I )-( L ) are two-tailed unpaired unequal variance Student’s t-tests.
    Perforin 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher mouse anti perforin
    Liver infiltration of CD4 + T cells and CD8 + T cells . CD8 (A: NC, B: CLF, C: ACLF) and CD4 (D: NC, E: CLF, F: ACLF) (×400) showed diffuse non-granular cytoplasmic staining while <t>perforin</t> showed granular cytoplasmic staining. The positive cells mainly infiltrated into the portal areas of the liver. NC, normal control; CLF, HBV-related chronic liver failure; ACLF, HBV-related acute on chronic liver failure.
    Mouse Anti Perforin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem anti mouse perforin
    Immunohistochemical staining (IHS) for <t>perforin</t> in heart tissue of C57BL/6 and pfp −/− mice. The figure shows representative pictures of IHS detection of perforin in the heart tissue sections of (a) non-infected C57BL/6 mouse, (b) Trypanosoma cruzi -infected pfp −/− , (c) T. cruzi -infected C57BL/6 in absence of the primary anti-perforin antibody and (d) T. cruzi -infected C57BL/6 mice at day 50 postinfection. (e) and (f) are representative heart tissue sections from infected C57BL/6 mice at day 150 postinfection. Perforin-expressing cells are mononuclear cells and perforin is not restricted to the cytoplasm but released onto the adjacent cardiomyocytes (d, arrows head). Panels a, b, c and e, 400×; panels d and f, 1000×. Each experimental group consisted of four analysed mice, in two independent experiments.
    Anti Mouse Perforin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam mouse monoclonal anti perforin antibody
    Images of representative histological and immunohistochemical. A. Unirradiated tumors (MC38 cells) in the group treated with or without radiotherapy (8 Gy × 3 fr) and/or anti-PD1 antibody (10 mg/kg) ([#1]: control group, [#2]: anti-PD1 antibody group, [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group) were stained with hematoxylin and eosin, anti-CD8 antibody, <t>anti-Perforin</t> antibody, and anti-ssDNA antibody. Scale bar = 150 µm. B. The index of CD8-, Perforin-, and ssDNA-positive stained cells. [#1]: control group, [#2]: anti-PD1 antibody group [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group. CD8-, Perforin-, and ssDNA-positive cells quantified by counting under high-power field with light microscopy (n = 5). **P
    Mouse Monoclonal Anti Perforin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam rat anti mouse perforin
    Small intestinal <t>granzyme-B/perforin</t> and TUNEL staining of the small intestine polyps
    Rat Anti Mouse Perforin, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson mouse anti human perforin
    Characterization of NK cells in the Nasal Lavage by Immunohistochemistry . NLF cells were characterized using immunohistochemistry. A) NLF cells are stained with anti-CD56-HRP to identify NK cells and B) NLF cells are stained with <t>anti-perforin-HRP</t> to identify cytotoxic NK cells. Bar = 10 μm.
    Mouse Anti Human Perforin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam monoclonal mouse anti human perforin 1
    T-cell–derived and –activating cytokines in calcified aortic valve cusps. Relative gene expression (normalized to noncalcified areas of stenotic valves) of <t>Perforin</t> 1 ( A ), Granzyme B ( C ), heat shock protein (Hsp)60 ( E ), CXCL9 ( G ), and major histocompatibility complex class II transactivator (CIITA; I ). Immunohistochemical localization of Perforin 1 ( B ), Granzyme B ( D ), Hsp60 ( F ), CXCL9 ( H ), and human leukocyte antigen (HLA)-DR ( J ). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions ( A , C , E , G , and I ); n = 5 ( B , D , F , H , and J ). ∗ P
    Monoclonal Mouse Anti Human Perforin 1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti mouse perforin
    T-cell–derived and –activating cytokines in calcified aortic valve cusps. Relative gene expression (normalized to noncalcified areas of stenotic valves) of <t>Perforin</t> 1 ( A ), Granzyme B ( C ), heat shock protein (Hsp)60 ( E ), CXCL9 ( G ), and major histocompatibility complex class II transactivator (CIITA; I ). Immunohistochemical localization of Perforin 1 ( B ), Granzyme B ( D ), Hsp60 ( F ), CXCL9 ( H ), and human leukocyte antigen (HLA)-DR ( J ). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions ( A , C , E , G , and I ); n = 5 ( B , D , F , H , and J ). ∗ P
    Anti Mouse Perforin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Abcam mouse anti human perforin mab
    TB granulomas in CD4-depleted macaques appeared to contain fewer IL-22+ and <t>perforin+</t> cells despite the presence of IL-17+ and IL-4+ cells
    Mouse Anti Human Perforin Mab, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Torrey Pines Biolabs rabbit anti mouse perforin antibody
    Western blots for <t>perforin</t> and granzyme B. CD8 + CD43 + T cells were purified from spleens of FV-infected mice at 2 weeks postinfection (acute) and 8 weeks postinfection (persistent), and analyzed by Western blot using anti-perforin and anti-granzyme
    Rabbit Anti Mouse Perforin Antibody, supplied by Torrey Pines Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend pe anti mouse perforin 1
    VAMP8 does not colocalize with cytotoxic granule constituents in human CTLs. (A and B) SIM images depicting VAMP8 localization in unconjugated (A) or SEA-target cell-conjugated (B) human CTLs stained with anti-VAMP8 and anti–granzyme B (GzmB). (C and D) SIM images depicting VAMP8 localization in unconjugated (C) or SEA-target cell-conjugated (D) CTLs stained with anti-VAMP8 and <t>anti-perforin.</t> (C) SIM images of resting human CTL stained with anti-VAMP8 and anti-perforin. For SEA-target cell-conjugated CTLs, the Pearson’s coefficient for VAMP8 versus perforin was r = 0.28 ( n = 10). Bars, 2.5 µm.
    Pe Anti Mouse Perforin 1, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Kamiya anti mouse perforin
    <t>Perforin</t> or Fas-L expression in CD8 + T cells in P-8-vaccinated and control mice at the site of infection. P-8-immunized and adjuvant control mice were infected with 10 5 L. amazonensis parasites in both ears. Pooled cells (groups of three mice), obtained from lesion sites at 9 weeks postinfection, were reactivated in vitro with Leishmania antigen for 5 h in the presence of brefeldin A and then processed for FACS analysis. Expression of Fas-L and intracellular perforin was determined for CD8 + lymphocytic cells by three-color flow cytometry. These results are representative of analyses obtained at 2, 5, and 9 weeks postinfection. The levels of Fas-L and perforin expression found for infection control mice were identical to that found for P. acnes control mice. —, P-8 plus P. acnes -vaccinated mice; - - - -, P. acnes control mice.
    Anti Mouse Perforin, supplied by Kamiya, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mab Technologies mouse anti human perforin pf 80
    <t>Perforin</t> or Fas-L expression in CD8 + T cells in P-8-vaccinated and control mice at the site of infection. P-8-immunized and adjuvant control mice were infected with 10 5 L. amazonensis parasites in both ears. Pooled cells (groups of three mice), obtained from lesion sites at 9 weeks postinfection, were reactivated in vitro with Leishmania antigen for 5 h in the presence of brefeldin A and then processed for FACS analysis. Expression of Fas-L and intracellular perforin was determined for CD8 + lymphocytic cells by three-color flow cytometry. These results are representative of analyses obtained at 2, 5, and 9 weeks postinfection. The levels of Fas-L and perforin expression found for infection control mice were identical to that found for P. acnes control mice. —, P-8 plus P. acnes -vaccinated mice; - - - -, P. acnes control mice.
    Mouse Anti Human Perforin Pf 80, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson mouse anti perforin antibody
    Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with <t>anti-perforin</t> (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.
    Mouse Anti Perforin Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pharmingen mouse anti perforin g9
    Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with <t>anti-perforin</t> (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.
    Mouse Anti Perforin G9, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Dianova mouse anti perforin antibody
    Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with <t>anti-perforin</t> (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.
    Mouse Anti Perforin Antibody, supplied by Dianova, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher pe conjugated rat anti mouse perforin
    <t>Perforin</t> and granzyme B levels in CD8 + T cells were not affected in mice lacking CD4 + T cells during primary responses to HSV-1 infection. Draining cells from CD4-depleted B6, MHC class II −/− , and WT mice were harvested
    Pe Conjugated Rat Anti Mouse Perforin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Colocalization of DsRed-FasL-pHluorin with perforin in NKL cells. ( a ) Confocal image of a paraformaldehyde-fixed NKL cell showing the distribution of DsRed, pHluorin and perforin. Human NKL cells were attached on the poly-coated slides for 10 min at 37 °C, fixed, permeabilized and stained with mouse monoclonal antibody IgG2b against human perforin and followed by goat anti-mouse IgG2b secondary antibody conjugated with Alexa Fluor 647 dye. The scale bar is 5.0 μm. ( b ) Confocal image of DsRed-FasL-pHluorin in a live cell after the addition of 500 m ammonium chloride (pH 7.4). The images are representative of at least 50 cells in two independent experiments. The scale bar is 5.0 μm.

    Journal: Immunology and Cell Biology

    Article Title: Two modes of lytic granule fusion during degranulation by natural killer cells

    doi: 10.1038/icb.2010.167

    Figure Lengend Snippet: Colocalization of DsRed-FasL-pHluorin with perforin in NKL cells. ( a ) Confocal image of a paraformaldehyde-fixed NKL cell showing the distribution of DsRed, pHluorin and perforin. Human NKL cells were attached on the poly-coated slides for 10 min at 37 °C, fixed, permeabilized and stained with mouse monoclonal antibody IgG2b against human perforin and followed by goat anti-mouse IgG2b secondary antibody conjugated with Alexa Fluor 647 dye. The scale bar is 5.0 μm. ( b ) Confocal image of DsRed-FasL-pHluorin in a live cell after the addition of 500 m ammonium chloride (pH 7.4). The images are representative of at least 50 cells in two independent experiments. The scale bar is 5.0 μm.

    Article Snippet: Cells were stained with anti-perforin monoclonal antibody mouse IgG2b (clone δG9; Pierce Chemical Co.) for 60 min at RT.

    Techniques: Staining

    Regulation of NFIL3 by IL-2-JAK1/3 signaling. ( A ) Average frequency of protein copy numbers per cell calculated from the proteomic datasets: The bin that contains NFIL3 is indicated in black. ( B ) Western blot analysis of NFIL3 abundance in CTLs following IL-2 deprivation or treatment with 100 nM Tofacitinib. Quantification is shown alongside as data normalized to SMC1 intensity and NFIL3 protein abundance in IL-2 maintained CTL. ( C ) Abundance of Nfil3 mRNA in CTLs. Abundance of NFIL3 protein ( D ) and mRNA ( E ) in naïve CD8 + T cells, TCR-activated CD8 + T cells, and CTLs activated for 48 hours and then maintained in IL-2 for 1 day, 3 days, or 5 days. ( F ) Analysis of NFIL3 protein abundance in HIF1α expressing ( Hif1a +/+ ) and HIF1α deficient ( Hif1a -/- ) CTLs maintained in normoxia (21% O 2 ) or switched into hypoxic (1% O 2 ) conditions for eight hours. Quantification of NFIL3 protein is shown alongside. Relative abundance of Nfil3 ( G ) and GLUT1 ( Slc2a1 ) ( H ) mRNA in CTLs maintained in normoxia or switched into 1% O 2 for four hours. Abundance of CD62L ( Sell ) mRNA ( I ) and cell-surface protein abundance ( J ) in IL-2-maintained Nfil3 +/+ and Nfil3 -/- CTL. In ( J ), the percentage of CD8 + CD62L high CTLs are shown alongside. Abundance of perforin ( Prf1 ) mRNA ( K ) and protein ( L ) in Nfil3 +/+ and Nfil3 -/- CTLs. In ( L ) quantification of NFIL3, normalized to SMC1 and perforin protein abundance in IL-2 maintained CTL, detected by Western blot is shown alongside. In ( C ), ( G ), and ( H ) the qPCR data were normalized to Tbp , and mRNA abundance in IL-2 maintained CTL in normoxia. In ( E ) qPCR data were normalized to Tbp and Cd8 and all are shown relative to day 5 IL-2-maintained CTLs in normoxia. In ( I ) and ( K ), mRNA was normalized to Hprt , and data are shown relative to the abundance in Nfil3 +/+ CTLs. Data in ( A )-( E ) and ( G )-( H ) data show, or are representative of, three biological replicates; in ( F ) data are representative of six Hif1a +/+ and five Hif1a -/- biological replicates, four independent experiments; in ( I )-( L ) data are representative of four Nfil3 +/+ and three Nfil3 -/- biological replicates. Bar charts show data from individual biological replicates, color matched where appropriate, the bar shows the mean and the errors bars show standard deviation. P values shown in ( B )-( H ) are two-tailed one sample Student’s t-tests and in ( I )-( L ) are two-tailed unpaired unequal variance Student’s t-tests.

    Journal: Science signaling

    Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

    doi: 10.1126/scisignal.aap8112

    Figure Lengend Snippet: Regulation of NFIL3 by IL-2-JAK1/3 signaling. ( A ) Average frequency of protein copy numbers per cell calculated from the proteomic datasets: The bin that contains NFIL3 is indicated in black. ( B ) Western blot analysis of NFIL3 abundance in CTLs following IL-2 deprivation or treatment with 100 nM Tofacitinib. Quantification is shown alongside as data normalized to SMC1 intensity and NFIL3 protein abundance in IL-2 maintained CTL. ( C ) Abundance of Nfil3 mRNA in CTLs. Abundance of NFIL3 protein ( D ) and mRNA ( E ) in naïve CD8 + T cells, TCR-activated CD8 + T cells, and CTLs activated for 48 hours and then maintained in IL-2 for 1 day, 3 days, or 5 days. ( F ) Analysis of NFIL3 protein abundance in HIF1α expressing ( Hif1a +/+ ) and HIF1α deficient ( Hif1a -/- ) CTLs maintained in normoxia (21% O 2 ) or switched into hypoxic (1% O 2 ) conditions for eight hours. Quantification of NFIL3 protein is shown alongside. Relative abundance of Nfil3 ( G ) and GLUT1 ( Slc2a1 ) ( H ) mRNA in CTLs maintained in normoxia or switched into 1% O 2 for four hours. Abundance of CD62L ( Sell ) mRNA ( I ) and cell-surface protein abundance ( J ) in IL-2-maintained Nfil3 +/+ and Nfil3 -/- CTL. In ( J ), the percentage of CD8 + CD62L high CTLs are shown alongside. Abundance of perforin ( Prf1 ) mRNA ( K ) and protein ( L ) in Nfil3 +/+ and Nfil3 -/- CTLs. In ( L ) quantification of NFIL3, normalized to SMC1 and perforin protein abundance in IL-2 maintained CTL, detected by Western blot is shown alongside. In ( C ), ( G ), and ( H ) the qPCR data were normalized to Tbp , and mRNA abundance in IL-2 maintained CTL in normoxia. In ( E ) qPCR data were normalized to Tbp and Cd8 and all are shown relative to day 5 IL-2-maintained CTLs in normoxia. In ( I ) and ( K ), mRNA was normalized to Hprt , and data are shown relative to the abundance in Nfil3 +/+ CTLs. Data in ( A )-( E ) and ( G )-( H ) data show, or are representative of, three biological replicates; in ( F ) data are representative of six Hif1a +/+ and five Hif1a -/- biological replicates, four independent experiments; in ( I )-( L ) data are representative of four Nfil3 +/+ and three Nfil3 -/- biological replicates. Bar charts show data from individual biological replicates, color matched where appropriate, the bar shows the mean and the errors bars show standard deviation. P values shown in ( B )-( H ) are two-tailed one sample Student’s t-tests and in ( I )-( L ) are two-tailed unpaired unequal variance Student’s t-tests.

    Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr694 /pTyr699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R & D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer235 /pSer236 (CST #2211), S6 (CST #2217), S6K pThr389 (CST #9205), S6K (CST #9202), AKT pThr308 (CST #4056), AKT pSer473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr24 /pThr32 (CST #9464), and FOXO1 (CST #9454).

    Techniques: Western Blot, CTL Assay, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    IL-2 regulation of CTL cytolytic machinery. ( A  to  H ) Copy numbers estimated from the proteomic data for LAMP-1 (A), LAMP-2 (B), Perforin 1 (C), Granzyme A (D), Granzyme B (E), RAB27A (F), STXBP2 (G), and UNC13D (H). In graphs, the three biological replicates are color-matched, the bar shows the mean and errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.

    Journal: Science signaling

    Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

    doi: 10.1126/scisignal.aap8112

    Figure Lengend Snippet: IL-2 regulation of CTL cytolytic machinery. ( A to H ) Copy numbers estimated from the proteomic data for LAMP-1 (A), LAMP-2 (B), Perforin 1 (C), Granzyme A (D), Granzyme B (E), RAB27A (F), STXBP2 (G), and UNC13D (H). In graphs, the three biological replicates are color-matched, the bar shows the mean and errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.

    Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr694 /pTyr699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R & D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer235 /pSer236 (CST #2211), S6 (CST #2217), S6K pThr389 (CST #9205), S6K (CST #9202), AKT pThr308 (CST #4056), AKT pSer473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr24 /pThr32 (CST #9464), and FOXO1 (CST #9454).

    Techniques: CTL Assay, Standard Deviation, Two Tailed Test

    The Tofacitinib-regulated CTL proteome. ( A ) Schematic representation of selected molecules regulated by Tofacitinib. ( B ) Volcano plot showing the protein copy number ratio in IL-2-maintained CTLs treated with 100 nM Tofacitinib for 24 hours compared to IL-2-maintained CTLs, significantly regulated proteins are shown in dark grey and nutrient transporters are highlighted in red. Estimated copy numbers per cell are shown for SESTRIN2 ( C ), GLUT1 ( D ) and GLUT3 ( E ). ( F ) Uptake of a radiolabelled glucose analog shown relative to IL-2. ( G ) Lactate output in IL-2-maintained and Tofacitinib-treated CTLs. Estimated copy numbers per cell for perforin ( H ) and NFIL3 ( I ). ( J ) Western blot analysis of HIF1α abundance in response to 100 nM Tofacitinib. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. The data show, or are representative of, three biological replicates. In bar charts, data points from biological replicates are color matched, the bar shows the mean and the errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of IL-2+Tofacitinib/IL-2.

    Journal: Science signaling

    Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

    doi: 10.1126/scisignal.aap8112

    Figure Lengend Snippet: The Tofacitinib-regulated CTL proteome. ( A ) Schematic representation of selected molecules regulated by Tofacitinib. ( B ) Volcano plot showing the protein copy number ratio in IL-2-maintained CTLs treated with 100 nM Tofacitinib for 24 hours compared to IL-2-maintained CTLs, significantly regulated proteins are shown in dark grey and nutrient transporters are highlighted in red. Estimated copy numbers per cell are shown for SESTRIN2 ( C ), GLUT1 ( D ) and GLUT3 ( E ). ( F ) Uptake of a radiolabelled glucose analog shown relative to IL-2. ( G ) Lactate output in IL-2-maintained and Tofacitinib-treated CTLs. Estimated copy numbers per cell for perforin ( H ) and NFIL3 ( I ). ( J ) Western blot analysis of HIF1α abundance in response to 100 nM Tofacitinib. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. The data show, or are representative of, three biological replicates. In bar charts, data points from biological replicates are color matched, the bar shows the mean and the errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of IL-2+Tofacitinib/IL-2.

    Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr694 /pTyr699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R & D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer235 /pSer236 (CST #2211), S6 (CST #2217), S6K pThr389 (CST #9205), S6K (CST #9202), AKT pThr308 (CST #4056), AKT pSer473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr24 /pThr32 (CST #9464), and FOXO1 (CST #9454).

    Techniques: CTL Assay, Western Blot, Standard Deviation, Two Tailed Test

    Effect of miR-155 overexpression on NK-cell cytotoxic effector functions. (A) FACS-sorted NK1.1 + CD3 − NK cells from wt and miR-155 tg mice were used as effector cells in a 4-hour 51 Cr release assay using YAC-1 and RMA-Rae1β tumor cells as targets. (B) Eight days after expansion in IL-2, sorted wt and miR-155 tg NK cells were assayed for ADCC against 51 Cr-labeled P815 Ab-coated targets cells. (C) The wt and miR-155 tg NK cells were mixed with RMA-Rae1β tumor cells at a ratio of 2:1 and injected subcutaneously into the flank of Rag2 −/− xII2rg −/− recipient mice (left). RMA-Rae1β tumor cells alone were injected as control. Tumor volumes were calculated every 2 days. The graph summarizes mean ± SEM data from 2 experiments with a total of 7 mice for the wt and the miR-155 tg groups. The percent survival of mice that had been inoculated with either wt or miR-155 tg NK cells in combination with RMA-Rae1β tumor cells at a ratio of 2:1 or RMA-Rae1β tumor cells alone as control is shown (right). Data from 3 independent experiments using 11 mice for each group are summarized in the Kaplan-Meier survival plots. (D) Resting (left) and IL-2–activated (8 days; right) NK1.1 + CD3 − wt and miR-155 tg NK cells were analyzed for granzyme B, granzyme M, perforin, and actin protein levels by immunoblot. (E) The wt and miR-155 tg NK cells labeled with PE-conjugated anti-NK1.1 Ab were incubated with GFP + YAC-1 tumor cells (left). Conjugate formation was analyzed at time 0 and after 10 minutes of incubation by flow cytometry. NK-cell target cell conjugates were gated and identified as NK1.1 + GFP + cells. The percentages of conjugates are shown on top of the representative dot plots. The graph summarizes the data of conjugate formation obtained from 3 wt and 3 miR-155 tg NK cell samples coincubated with YAC-1 tumor cells (right). *Statistically significant; see text for details.

    Journal: Blood

    Article Title: Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells

    doi: 10.1182/blood-2012-12-467597

    Figure Lengend Snippet: Effect of miR-155 overexpression on NK-cell cytotoxic effector functions. (A) FACS-sorted NK1.1 + CD3 − NK cells from wt and miR-155 tg mice were used as effector cells in a 4-hour 51 Cr release assay using YAC-1 and RMA-Rae1β tumor cells as targets. (B) Eight days after expansion in IL-2, sorted wt and miR-155 tg NK cells were assayed for ADCC against 51 Cr-labeled P815 Ab-coated targets cells. (C) The wt and miR-155 tg NK cells were mixed with RMA-Rae1β tumor cells at a ratio of 2:1 and injected subcutaneously into the flank of Rag2 −/− xII2rg −/− recipient mice (left). RMA-Rae1β tumor cells alone were injected as control. Tumor volumes were calculated every 2 days. The graph summarizes mean ± SEM data from 2 experiments with a total of 7 mice for the wt and the miR-155 tg groups. The percent survival of mice that had been inoculated with either wt or miR-155 tg NK cells in combination with RMA-Rae1β tumor cells at a ratio of 2:1 or RMA-Rae1β tumor cells alone as control is shown (right). Data from 3 independent experiments using 11 mice for each group are summarized in the Kaplan-Meier survival plots. (D) Resting (left) and IL-2–activated (8 days; right) NK1.1 + CD3 − wt and miR-155 tg NK cells were analyzed for granzyme B, granzyme M, perforin, and actin protein levels by immunoblot. (E) The wt and miR-155 tg NK cells labeled with PE-conjugated anti-NK1.1 Ab were incubated with GFP + YAC-1 tumor cells (left). Conjugate formation was analyzed at time 0 and after 10 minutes of incubation by flow cytometry. NK-cell target cell conjugates were gated and identified as NK1.1 + GFP + cells. The percentages of conjugates are shown on top of the representative dot plots. The graph summarizes the data of conjugate formation obtained from 3 wt and 3 miR-155 tg NK cell samples coincubated with YAC-1 tumor cells (right). *Statistically significant; see text for details.

    Article Snippet: The Abs used included the following: the anti-SHIP1, anti-actin, and anti-granzyme M (Santa Cruz Biotechnology); anti-granzyme B, anti-perforin, anti-phospho-AKTSer473 , and anti-phospho-extracellular signal-regulated kinaseThr202/Tyr204 (Cell Signaling Technology).

    Techniques: Over Expression, FACS, Mouse Assay, Release Assay, Labeling, Injection, Incubation, Flow Cytometry, Cytometry

    NK cell activation and conjugation to EGFR + target cells under ADCC conditions. A, Representative dot plots of NK activation measured by flow cytometry analysis using CD107α (APC) and GFP + NK92-CD16V cells at a 1:1 E:T for 2 hours as described in Materials and Methods. ADCCS1 (top row) and ADCCR1 cells (bottom row) were incubated with NK92-CD16V cells in the absence (middle panels) or in the presence of cetuximab (1 µg/mL; right panel) for 2 hours. B, ELISA measuring IFNγ levels in the media of ADCCS1 (blue bars) and ADCCR1 (red bars) 4 hours post exposure to ADCC conditions (cetuximab [1 µg/mL] plus NK92-CD16V cells) at E:T ratios of 0-4:1 and NK92-CD16V cells in the absence of cetuximab. C, Western blot analysis of granzyme B and perforin protein expression in target cells two hours after exposure to T (media control), Ab (cetuximab only at 1µg/mL), NK (NK92-CD16V cells only at 1:1 E:T) and ADCC (NK92-CD16V cells at 1:1 E:T plus 1 µg/mL cetuximab). D, Percentage of NK cells conjugated to target was measured by a multi-well conjugation assay as described in Materials and Methods. ADCCS1 (blue) and ADCCR1 (red) were incubated with NK92-CD16V cells at a 1:1 E:T ratio in the absence (NK, checkered bar) or in the presence of cetuximab (1 µg/mL; ADCC, solid bar) for 2 hours. *, p

    Journal: Cancer immunology research

    Article Title: A Mechanism of Resistance to Antibody-Targeted Immune Attack

    doi: 10.1158/2326-6066.CIR-18-0266

    Figure Lengend Snippet: NK cell activation and conjugation to EGFR + target cells under ADCC conditions. A, Representative dot plots of NK activation measured by flow cytometry analysis using CD107α (APC) and GFP + NK92-CD16V cells at a 1:1 E:T for 2 hours as described in Materials and Methods. ADCCS1 (top row) and ADCCR1 cells (bottom row) were incubated with NK92-CD16V cells in the absence (middle panels) or in the presence of cetuximab (1 µg/mL; right panel) for 2 hours. B, ELISA measuring IFNγ levels in the media of ADCCS1 (blue bars) and ADCCR1 (red bars) 4 hours post exposure to ADCC conditions (cetuximab [1 µg/mL] plus NK92-CD16V cells) at E:T ratios of 0-4:1 and NK92-CD16V cells in the absence of cetuximab. C, Western blot analysis of granzyme B and perforin protein expression in target cells two hours after exposure to T (media control), Ab (cetuximab only at 1µg/mL), NK (NK92-CD16V cells only at 1:1 E:T) and ADCC (NK92-CD16V cells at 1:1 E:T plus 1 µg/mL cetuximab). D, Percentage of NK cells conjugated to target was measured by a multi-well conjugation assay as described in Materials and Methods. ADCCS1 (blue) and ADCCR1 (red) were incubated with NK92-CD16V cells at a 1:1 E:T ratio in the absence (NK, checkered bar) or in the presence of cetuximab (1 µg/mL; ADCC, solid bar) for 2 hours. *, p

    Article Snippet: Western blots were conducted using: the Abcam antibodies to EGFR (Cat. No. 52892) and perforin (Cat. No. ab180773), and Cell Signaling Technology (CST) antibodies to GAPDH (Cat. No. 5174), JAK1 (Cat. No. 3332), STAT1 (Cat. No. 14994), p-STAT1 Y701 (Cat. No. 9167), PCAF/KA2B (Cat. No. 3378), Granzyme B (Cat. No. 4275), NFKB p65 (Cat. No. 82420), p-NFKB P65 (Cat. No. 3031S), and HSPB1 (Cat. No. 2402S).

    Techniques: Activation Assay, Conjugation Assay, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

    Liver infiltration of CD4 + T cells and CD8 + T cells . CD8 (A: NC, B: CLF, C: ACLF) and CD4 (D: NC, E: CLF, F: ACLF) (×400) showed diffuse non-granular cytoplasmic staining while perforin showed granular cytoplasmic staining. The positive cells mainly infiltrated into the portal areas of the liver. NC, normal control; CLF, HBV-related chronic liver failure; ACLF, HBV-related acute on chronic liver failure.

    Journal: BMC Immunology

    Article Title: The balance between intrahepatic IL-17+ T cells and Foxp3+ regulatory T cells plays an important role in HBV-related end-stage liver disease

    doi: 10.1186/1471-2172-12-47

    Figure Lengend Snippet: Liver infiltration of CD4 + T cells and CD8 + T cells . CD8 (A: NC, B: CLF, C: ACLF) and CD4 (D: NC, E: CLF, F: ACLF) (×400) showed diffuse non-granular cytoplasmic staining while perforin showed granular cytoplasmic staining. The positive cells mainly infiltrated into the portal areas of the liver. NC, normal control; CLF, HBV-related chronic liver failure; ACLF, HBV-related acute on chronic liver failure.

    Article Snippet: Rat mAbs for anti-human Foxp3 (eBiosciences, San Diego, CA), rabbit anti-human IL-17 (Beijing Bioss Biotech, Beijing, China), rabbit anti-human CD4, CD8 and mouse anti-perforin (Zymed, San Diego, CA) were used for the immunochemistry staining of Foxp3, IL-17, CD4, CD8 and perforin, respectively.

    Techniques: Staining

    Liver infiltration of perforin + cells . Perforin showed granular cytoplastic staining (A: NC, B: CLF, C: ACLF) (×400). NC, normal control; CLF, HBV-related chronic liver failure; ACLF, HBV-related acute on chronic liver failure.

    Journal: BMC Immunology

    Article Title: The balance between intrahepatic IL-17+ T cells and Foxp3+ regulatory T cells plays an important role in HBV-related end-stage liver disease

    doi: 10.1186/1471-2172-12-47

    Figure Lengend Snippet: Liver infiltration of perforin + cells . Perforin showed granular cytoplastic staining (A: NC, B: CLF, C: ACLF) (×400). NC, normal control; CLF, HBV-related chronic liver failure; ACLF, HBV-related acute on chronic liver failure.

    Article Snippet: Rat mAbs for anti-human Foxp3 (eBiosciences, San Diego, CA), rabbit anti-human IL-17 (Beijing Bioss Biotech, Beijing, China), rabbit anti-human CD4, CD8 and mouse anti-perforin (Zymed, San Diego, CA) were used for the immunochemistry staining of Foxp3, IL-17, CD4, CD8 and perforin, respectively.

    Techniques: Staining

    Highly activated hepatic NK cells and increased levels of cytokines act synergistically to amplify ConA-induced liver injury in HCV mice A . Enhanced production of IFN-γ, TNF-α and perforin from hepatic NK cells in HCV mice. Hepatic lymphocytes from p att B-HCV-Fluc-injected mice or p att B–Fluc-injected mice were prepared and examined by FACS using FITC-conjugated anti-NK1.1, PE-Cy TM 5-conjugated anti-CD3e, PE-conjugated anti-IFN-γ, PE-conjugated anti-TNF-α, and PE-conjugated anti-perforin. The percentage of hepatic NK cells secreting IFN-γ, TNF-α and perforin at 24 h after ConA treatment is shown. The results represent the mean ±SD of triplicate samples. *P

    Journal: Oncotarget

    Article Title: Hepatic NK cell-mediated hypersensitivity to ConA-induced liver injury in mouse liver expressing hepatitis C virus polyprotein

    doi: 10.18632/oncotarget.11052

    Figure Lengend Snippet: Highly activated hepatic NK cells and increased levels of cytokines act synergistically to amplify ConA-induced liver injury in HCV mice A . Enhanced production of IFN-γ, TNF-α and perforin from hepatic NK cells in HCV mice. Hepatic lymphocytes from p att B-HCV-Fluc-injected mice or p att B–Fluc-injected mice were prepared and examined by FACS using FITC-conjugated anti-NK1.1, PE-Cy TM 5-conjugated anti-CD3e, PE-conjugated anti-IFN-γ, PE-conjugated anti-TNF-α, and PE-conjugated anti-perforin. The percentage of hepatic NK cells secreting IFN-γ, TNF-α and perforin at 24 h after ConA treatment is shown. The results represent the mean ±SD of triplicate samples. *P

    Article Snippet: Anti-mouse-TNF-α-PE, anti-mouse-IFN-γ-PE, anti-mouse-CD335(NKp46)-PE, anti-mouse-TRAIL-PE, anti-mouse-CD178(FasL)-PE, and anti-mouse-perforin-PE were obtained from eBioscience (San Diego, CA).

    Techniques: Activated Clotting Time Assay, Mouse Assay, Injection, FACS

    IL-2 signals at priming program CD8 + T-cell differentiation. (A) At 48 hours after anti-CD3/CD28 antibodies stimulation, IFNγ level was checked by ELISA. Naïve CD8 + T cells were stimulated by anti-CD3/CD28 in the presence or absence of rhIL-2 (5 ng/mL and 0.5 ng/mL) treatment at 0 hour and 24 hours, respectively. (B) At 24 and 48 hours after anti-CD3/CD28 stimulation, production of IFNγ in CD8 + T cells was checked by intracellular staining and analyzed by flow cytometry. rhIL-2 (5 ng/mL) was added at 0, 12 and 24 hours after stimulation, respectively. Percentage of IFNγ-producing cells was shown. (C) At 48 hours after anti-CD3/CD28 stimulation, granzyme B production was checked by intracellular staining and analyzed by flow cytometry. Percentage of granzyme B-producing cells was shown. (D) At 24 hours after anti-CD3/CD28 stimulation, perforin production was checked by intracellular staining and analyzed by flow cytometry. Percentage of perforin-producing cells was shown. Similar results were obtained in three independent experiments.

    Journal: PLoS ONE

    Article Title: CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

    doi: 10.1371/journal.pone.0007766

    Figure Lengend Snippet: IL-2 signals at priming program CD8 + T-cell differentiation. (A) At 48 hours after anti-CD3/CD28 antibodies stimulation, IFNγ level was checked by ELISA. Naïve CD8 + T cells were stimulated by anti-CD3/CD28 in the presence or absence of rhIL-2 (5 ng/mL and 0.5 ng/mL) treatment at 0 hour and 24 hours, respectively. (B) At 24 and 48 hours after anti-CD3/CD28 stimulation, production of IFNγ in CD8 + T cells was checked by intracellular staining and analyzed by flow cytometry. rhIL-2 (5 ng/mL) was added at 0, 12 and 24 hours after stimulation, respectively. Percentage of IFNγ-producing cells was shown. (C) At 48 hours after anti-CD3/CD28 stimulation, granzyme B production was checked by intracellular staining and analyzed by flow cytometry. Percentage of granzyme B-producing cells was shown. (D) At 24 hours after anti-CD3/CD28 stimulation, perforin production was checked by intracellular staining and analyzed by flow cytometry. Percentage of perforin-producing cells was shown. Similar results were obtained in three independent experiments.

    Article Snippet: Anti-mouse IL-2 antibody (clone S4B6), isotype control rat IgG2a , anti-mouse IFNγ (clone XMG1.2), FITC anti-mouse granzyme B (clone 16G6), PE anti-mouse perforin (clone eBioOMAK-D, eBioscience, San Diego, CA, USA), FITC anti-mouse CD3, PE anti-mouse CD4, PE-Cy5 anti-mouse CD8, FITC anti-mouse CD25, PE anti-mouse CD69, FITC anti-CD122, FITC Annexin V, mouse anti–p27 antibodies and BrdU kit (BD Pharmingen, San Diego, CA, USA), rat anti-CD122 antibody (TM-β1) and isotype control rat IgG2b (eBioscience, San Diego, CA, USA), rabbit polyclonal anti-HIg antibody (R anti-H, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), mouse anti-cyclin E antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-actin (Chemicon, Temecula, CA, USA) were used in the experiments.

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

    Immunohistochemical staining (IHS) for perforin in heart tissue of C57BL/6 and pfp −/− mice. The figure shows representative pictures of IHS detection of perforin in the heart tissue sections of (a) non-infected C57BL/6 mouse, (b) Trypanosoma cruzi -infected pfp −/− , (c) T. cruzi -infected C57BL/6 in absence of the primary anti-perforin antibody and (d) T. cruzi -infected C57BL/6 mice at day 50 postinfection. (e) and (f) are representative heart tissue sections from infected C57BL/6 mice at day 150 postinfection. Perforin-expressing cells are mononuclear cells and perforin is not restricted to the cytoplasm but released onto the adjacent cardiomyocytes (d, arrows head). Panels a, b, c and e, 400×; panels d and f, 1000×. Each experimental group consisted of four analysed mice, in two independent experiments.

    Journal: International Journal of Experimental Pathology

    Article Title: Perforin-expressing cytotoxic cells contribute to chronic cardiomyopathy in Trypanosoma cruzi infection

    doi: 10.1111/j.1365-2613.2009.00670.x

    Figure Lengend Snippet: Immunohistochemical staining (IHS) for perforin in heart tissue of C57BL/6 and pfp −/− mice. The figure shows representative pictures of IHS detection of perforin in the heart tissue sections of (a) non-infected C57BL/6 mouse, (b) Trypanosoma cruzi -infected pfp −/− , (c) T. cruzi -infected C57BL/6 in absence of the primary anti-perforin antibody and (d) T. cruzi -infected C57BL/6 mice at day 50 postinfection. (e) and (f) are representative heart tissue sections from infected C57BL/6 mice at day 150 postinfection. Perforin-expressing cells are mononuclear cells and perforin is not restricted to the cytoplasm but released onto the adjacent cardiomyocytes (d, arrows head). Panels a, b, c and e, 400×; panels d and f, 1000×. Each experimental group consisted of four analysed mice, in two independent experiments.

    Article Snippet: In our IHS studies, monoclonal antibodies, anti-mouse perforin (CB5.4; Alexis Biochemicals, Plymouth Meeting, PA, USA), anti-IFN-γ (R4-6A2; BD PharMingen, San Jose, CA, USA) and anti-interleukin (IL)-4 (BVDG-24G2; CALTAG) as well as the purified polyclonal antibody recognizing inducible nitric oxide synthase (iNOS) from mouse macrophages (RAW 264.7; Cayman Chemical, Ann Arbor, MI, USA) were applied; the polyclonal antibody recognizing connexin 43 was purchased from Sigma (St. Louis, MO, USA); the biotinylated anti-rat immunoglobulin (Ig) from DAKO (Glostrup, Denmark) and the biotinylated anti-rabbit immunoglobulin and peroxidase–streptavidin complex from Amersham (England).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Infection, Expressing

    Cardiomyocyte damage and inflammatory cells in the heart tissue of acute (50 days postinfection) and chronically (150 days postinfection) Trypanosoma cruzi -infected C57BL/6 mice. (a) Cardiomyocyte lesion was evaluated by creatine kinase cardiac isoenzyme MB (CK-MB) activity in serum samples from C57BL/6 mice (black circle) in comparison with the non-infected controls (black triangle). CD8 + , perforin-positive, interferon (IFN)-γ + and interleukin (IL)-4 + cells were immunohistochemically analysed in serial sections of heart tissue. (b) Total inflammatory mononuclear cells in the cardiac tissue of infected mice. (c) IFN-γ + and IL-4 + as well as (d) CD8 + cells invading heart tissue during T. cruzi infection. The relative frequency of (e) perforin-positive or (f) IFN-γ + cells among CD8 + cells in the heart tissue of acute and chronically infected mice was estimated. At least 200 fields (four sections per analysed mouse) were counted for each parameter at each time point studied. Each value represents an individual mouse. ** P

    Journal: International Journal of Experimental Pathology

    Article Title: Perforin-expressing cytotoxic cells contribute to chronic cardiomyopathy in Trypanosoma cruzi infection

    doi: 10.1111/j.1365-2613.2009.00670.x

    Figure Lengend Snippet: Cardiomyocyte damage and inflammatory cells in the heart tissue of acute (50 days postinfection) and chronically (150 days postinfection) Trypanosoma cruzi -infected C57BL/6 mice. (a) Cardiomyocyte lesion was evaluated by creatine kinase cardiac isoenzyme MB (CK-MB) activity in serum samples from C57BL/6 mice (black circle) in comparison with the non-infected controls (black triangle). CD8 + , perforin-positive, interferon (IFN)-γ + and interleukin (IL)-4 + cells were immunohistochemically analysed in serial sections of heart tissue. (b) Total inflammatory mononuclear cells in the cardiac tissue of infected mice. (c) IFN-γ + and IL-4 + as well as (d) CD8 + cells invading heart tissue during T. cruzi infection. The relative frequency of (e) perforin-positive or (f) IFN-γ + cells among CD8 + cells in the heart tissue of acute and chronically infected mice was estimated. At least 200 fields (four sections per analysed mouse) were counted for each parameter at each time point studied. Each value represents an individual mouse. ** P

    Article Snippet: In our IHS studies, monoclonal antibodies, anti-mouse perforin (CB5.4; Alexis Biochemicals, Plymouth Meeting, PA, USA), anti-IFN-γ (R4-6A2; BD PharMingen, San Jose, CA, USA) and anti-interleukin (IL)-4 (BVDG-24G2; CALTAG) as well as the purified polyclonal antibody recognizing inducible nitric oxide synthase (iNOS) from mouse macrophages (RAW 264.7; Cayman Chemical, Ann Arbor, MI, USA) were applied; the polyclonal antibody recognizing connexin 43 was purchased from Sigma (St. Louis, MO, USA); the biotinylated anti-rat immunoglobulin (Ig) from DAKO (Glostrup, Denmark) and the biotinylated anti-rabbit immunoglobulin and peroxidase–streptavidin complex from Amersham (England).

    Techniques: Infection, Mouse Assay, Activity Assay

    Images of representative histological and immunohistochemical. A. Unirradiated tumors (MC38 cells) in the group treated with or without radiotherapy (8 Gy × 3 fr) and/or anti-PD1 antibody (10 mg/kg) ([#1]: control group, [#2]: anti-PD1 antibody group, [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group) were stained with hematoxylin and eosin, anti-CD8 antibody, anti-Perforin antibody, and anti-ssDNA antibody. Scale bar = 150 µm. B. The index of CD8-, Perforin-, and ssDNA-positive stained cells. [#1]: control group, [#2]: anti-PD1 antibody group [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group. CD8-, Perforin-, and ssDNA-positive cells quantified by counting under high-power field with light microscopy (n = 5). **P

    Journal: American Journal of Cancer Research

    Article Title: Experimental model for the irradiation-mediated abscopal effect and factors influencing this effect

    doi:

    Figure Lengend Snippet: Images of representative histological and immunohistochemical. A. Unirradiated tumors (MC38 cells) in the group treated with or without radiotherapy (8 Gy × 3 fr) and/or anti-PD1 antibody (10 mg/kg) ([#1]: control group, [#2]: anti-PD1 antibody group, [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group) were stained with hematoxylin and eosin, anti-CD8 antibody, anti-Perforin antibody, and anti-ssDNA antibody. Scale bar = 150 µm. B. The index of CD8-, Perforin-, and ssDNA-positive stained cells. [#1]: control group, [#2]: anti-PD1 antibody group [#3]: RT alone group, [#4]: RT+anti-PD1 antibody group. CD8-, Perforin-, and ssDNA-positive cells quantified by counting under high-power field with light microscopy (n = 5). **P

    Article Snippet: The primary antibody, a mouse monoclonal anti-CD8 alpha antibody ( ; Abcam, Cambridge, UK) at 1:2000 dilution, a mouse monoclonal anti-Perforin antibody (5B10; Abcam) at 1:20 dilution, and a mouse monoclonal anti-single stranded DNA (ssDNA) (F7-26; ELS, New York, NY, USA) at 1:100 dilution were subsequently applied overnight at 4°C.

    Techniques: Immunohistochemistry, Staining, Light Microscopy

    Small intestinal granzyme-B/perforin and TUNEL staining of the small intestine polyps

    Journal: Cancer immunology research

    Article Title: T-cell expression of IL10 is essential for tumor immune surveillance in the small intestine

    doi: 10.1158/2326-6066.CIR-14-0169

    Figure Lengend Snippet: Small intestinal granzyme-B/perforin and TUNEL staining of the small intestine polyps

    Article Snippet: Rabbit anti-mouse granzyme-B or rabbit IgG control and rat anti-mouse perforin (Abcam) were incubated at 1:100 overnight at +4°C, and then visualized using secondary antibodies anti-rabbit AlexaFluor-594 and anti-rat AlexaFluor-488 (Invitrogen).

    Techniques: TUNEL Assay, Staining

    IL10 deficiency aborts perforin and granzyme-B activities

    Journal: Cancer immunology research

    Article Title: T-cell expression of IL10 is essential for tumor immune surveillance in the small intestine

    doi: 10.1158/2326-6066.CIR-14-0169

    Figure Lengend Snippet: IL10 deficiency aborts perforin and granzyme-B activities

    Article Snippet: Rabbit anti-mouse granzyme-B or rabbit IgG control and rat anti-mouse perforin (Abcam) were incubated at 1:100 overnight at +4°C, and then visualized using secondary antibodies anti-rabbit AlexaFluor-594 and anti-rat AlexaFluor-488 (Invitrogen).

    Techniques:

    Characterization of NK cells in the Nasal Lavage by Immunohistochemistry . NLF cells were characterized using immunohistochemistry. A) NLF cells are stained with anti-CD56-HRP to identify NK cells and B) NLF cells are stained with anti-perforin-HRP to identify cytotoxic NK cells. Bar = 10 μm.

    Journal: Respiratory Research

    Article Title: Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus

    doi: 10.1186/1465-9921-12-102

    Figure Lengend Snippet: Characterization of NK cells in the Nasal Lavage by Immunohistochemistry . NLF cells were characterized using immunohistochemistry. A) NLF cells are stained with anti-CD56-HRP to identify NK cells and B) NLF cells are stained with anti-perforin-HRP to identify cytotoxic NK cells. Bar = 10 μm.

    Article Snippet: Slides were then incubated with the following primary antibodies: mouse anti-human CD56 antibody (MAB24081 RnD, Minneapolis, MN) or mouse anti-human perforin (BD Biosciences) overnight at 4°C.

    Techniques: Immunohistochemistry, Staining

    TOX2-KD effects on receptor acquisition, degranulation function, and perforin expression. (A) Control or TOX2-KD–transduced CD34 + cells were subjected to in vitro NK cell differentiation. Flow cytometry was performed on day 17 to analyze the expression

    Journal: Blood

    Article Title: TOX2 regulates human natural killer cell development by controlling T-BET expression

    doi: 10.1182/blood-2014-06-582965

    Figure Lengend Snippet: TOX2-KD effects on receptor acquisition, degranulation function, and perforin expression. (A) Control or TOX2-KD–transduced CD34 + cells were subjected to in vitro NK cell differentiation. Flow cytometry was performed on day 17 to analyze the expression

    Article Snippet: Intracellular perforin was stained with mouse anti-human perforin-PE (BD Pharmingen, San Diego, CA) and analyzed by flow cytometry.

    Techniques: Expressing, In Vitro, Cell Differentiation, Flow Cytometry, Cytometry

    T-cell–derived and –activating cytokines in calcified aortic valve cusps. Relative gene expression (normalized to noncalcified areas of stenotic valves) of Perforin 1 ( A ), Granzyme B ( C ), heat shock protein (Hsp)60 ( E ), CXCL9 ( G ), and major histocompatibility complex class II transactivator (CIITA; I ). Immunohistochemical localization of Perforin 1 ( B ), Granzyme B ( D ), Hsp60 ( F ), CXCL9 ( H ), and human leukocyte antigen (HLA)-DR ( J ). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions ( A , C , E , G , and I ); n = 5 ( B , D , F , H , and J ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Interferon-γ Released by Activated CD8+ T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves

    doi: 10.1016/j.ajpath.2017.02.012

    Figure Lengend Snippet: T-cell–derived and –activating cytokines in calcified aortic valve cusps. Relative gene expression (normalized to noncalcified areas of stenotic valves) of Perforin 1 ( A ), Granzyme B ( C ), heat shock protein (Hsp)60 ( E ), CXCL9 ( G ), and major histocompatibility complex class II transactivator (CIITA; I ). Immunohistochemical localization of Perforin 1 ( B ), Granzyme B ( D ), Hsp60 ( F ), CXCL9 ( H ), and human leukocyte antigen (HLA)-DR ( J ). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions ( A , C , E , G , and I ); n = 5 ( B , D , F , H , and J ). ∗ P

    Article Snippet: For immunohistochemical analysis the following antibodies were used: polyclonal rabbit anti-human CXCL9 (dilution 1:50; NBP1-31155; Novus Biologicals, Littleton, CO), monoclonal mouse anti-human Perforin 1 (dilution 1:25; ab47225; Abcam), polyclonal rabbit anti-human GZMB (dilution 1:25; ab4059; Abcam), monoclonal mouse anti-human TRAP (ready-to-use; PA0093; Novocastra Leica, Nussloch, Germany), polyclonal rabbit anti-human Cathepsin K (dilution 1:100; ab19027; Abcam), polyclonal rabbit anti-human Hsp60 antibody (dilution 1:200; NBP1-04303; Novus Biologicals), monoclonal mouse anti-human HLA-DR antibody (dilution 1:200; M 0746; Dako, Santa Clara, CA), monoclonal mouse anti-human RANKL (dilution 1:25; NBP100-56512; Novus Biologicals), and polyclonal rabbit anti-human OSCAR (dilution 1:100; ab93817; Abcam).

    Techniques: Derivative Assay, Expressing, Immunohistochemistry

    TB granulomas in CD4-depleted macaques appeared to contain fewer IL-22+ and perforin+ cells despite the presence of IL-17+ and IL-4+ cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    doi: 10.4049/jimmunol.1301373

    Figure Lengend Snippet: TB granulomas in CD4-depleted macaques appeared to contain fewer IL-22+ and perforin+ cells despite the presence of IL-17+ and IL-4+ cells

    Article Snippet: Briefly, lung tissue sections in Trilogy™ solution were heated for epitope retrieval at 121 °C for 15 min, and incubated at room temperature for 1hr with rabbit anti-human IL-17 (sc-7927, Santa Cruzbiotechnology), anti-human IL-22 (CI0144, Capralogics), anti-human IL-4(RPA05112, Reprokine), or mouse anti-human perforin mAb (5B10, abcam) at 4 ug/ml concentration each.

    Techniques:

    CD4 depletion during Mtb infection depressed the ability of CD3- lymphocytes to mount CD3-IL-22+ and CD3-perforin+ effector responses, but left CD3-IL-17+ effectors unaffected

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    doi: 10.4049/jimmunol.1301373

    Figure Lengend Snippet: CD4 depletion during Mtb infection depressed the ability of CD3- lymphocytes to mount CD3-IL-22+ and CD3-perforin+ effector responses, but left CD3-IL-17+ effectors unaffected

    Article Snippet: Briefly, lung tissue sections in Trilogy™ solution were heated for epitope retrieval at 121 °C for 15 min, and incubated at room temperature for 1hr with rabbit anti-human IL-17 (sc-7927, Santa Cruzbiotechnology), anti-human IL-22 (CI0144, Capralogics), anti-human IL-4(RPA05112, Reprokine), or mouse anti-human perforin mAb (5B10, abcam) at 4 ug/ml concentration each.

    Techniques: Infection

    CD4-depleted macaques exhibited no or few pulmonary T effector cells capable of constitutively produced IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    doi: 10.4049/jimmunol.1301373

    Figure Lengend Snippet: CD4-depleted macaques exhibited no or few pulmonary T effector cells capable of constitutively produced IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint

    Article Snippet: Briefly, lung tissue sections in Trilogy™ solution were heated for epitope retrieval at 121 °C for 15 min, and incubated at room temperature for 1hr with rabbit anti-human IL-17 (sc-7927, Santa Cruzbiotechnology), anti-human IL-22 (CI0144, Capralogics), anti-human IL-4(RPA05112, Reprokine), or mouse anti-human perforin mAb (5B10, abcam) at 4 ug/ml concentration each.

    Techniques: Produced

    CD4-depleted macaques showed no or few CD3-perforin+ and CD3-IL-22+ cells in the pulmonary compartment at the endpoint, despite presence of CD3-IL-17+ and CD3-IL-4+ cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    doi: 10.4049/jimmunol.1301373

    Figure Lengend Snippet: CD4-depleted macaques showed no or few CD3-perforin+ and CD3-IL-22+ cells in the pulmonary compartment at the endpoint, despite presence of CD3-IL-17+ and CD3-IL-4+ cells

    Article Snippet: Briefly, lung tissue sections in Trilogy™ solution were heated for epitope retrieval at 121 °C for 15 min, and incubated at room temperature for 1hr with rabbit anti-human IL-17 (sc-7927, Santa Cruzbiotechnology), anti-human IL-22 (CI0144, Capralogics), anti-human IL-4(RPA05112, Reprokine), or mouse anti-human perforin mAb (5B10, abcam) at 4 ug/ml concentration each.

    Techniques:

    Western blots for perforin and granzyme B. CD8 + CD43 + T cells were purified from spleens of FV-infected mice at 2 weeks postinfection (acute) and 8 weeks postinfection (persistent), and analyzed by Western blot using anti-perforin and anti-granzyme

    Journal: Journal of Virology

    Article Title: CD8+ T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection

    doi: 10.1128/JVI.79.16.10619-10626.2005

    Figure Lengend Snippet: Western blots for perforin and granzyme B. CD8 + CD43 + T cells were purified from spleens of FV-infected mice at 2 weeks postinfection (acute) and 8 weeks postinfection (persistent), and analyzed by Western blot using anti-perforin and anti-granzyme

    Article Snippet: The membranes were probed with rabbit anti-mouse perforin antibody (Torrey Pines Biolabs, Houston, Texas), then with horseradish peroxidase-conjugated antibody to rabbit IgG (Pierce) and revealed by chemiluminescence (SuperSignalWest Pico, Pierce).

    Techniques: Western Blot, Purification, Infection, Mouse Assay

    mRNA levels for cytotoxic molecules in activated CD8 + T cells. Levels of transcripts for perforin, granzyme A, and granzyme B from effector (CD43 + ) CD8 + T cells isolated from acutely (2 weeks postinfection) and persistently (8

    Journal: Journal of Virology

    Article Title: CD8+ T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection

    doi: 10.1128/JVI.79.16.10619-10626.2005

    Figure Lengend Snippet: mRNA levels for cytotoxic molecules in activated CD8 + T cells. Levels of transcripts for perforin, granzyme A, and granzyme B from effector (CD43 + ) CD8 + T cells isolated from acutely (2 weeks postinfection) and persistently (8

    Article Snippet: The membranes were probed with rabbit anti-mouse perforin antibody (Torrey Pines Biolabs, Houston, Texas), then with horseradish peroxidase-conjugated antibody to rabbit IgG (Pierce) and revealed by chemiluminescence (SuperSignalWest Pico, Pierce).

    Techniques: Isolation

    VAMP8 does not colocalize with cytotoxic granule constituents in human CTLs. (A and B) SIM images depicting VAMP8 localization in unconjugated (A) or SEA-target cell-conjugated (B) human CTLs stained with anti-VAMP8 and anti–granzyme B (GzmB). (C and D) SIM images depicting VAMP8 localization in unconjugated (C) or SEA-target cell-conjugated (D) CTLs stained with anti-VAMP8 and anti-perforin. (C) SIM images of resting human CTL stained with anti-VAMP8 and anti-perforin. For SEA-target cell-conjugated CTLs, the Pearson’s coefficient for VAMP8 versus perforin was r = 0.28 ( n = 10). Bars, 2.5 µm.

    Journal: The Journal of Cell Biology

    Article Title: VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

    doi: 10.1083/jcb.201411093

    Figure Lengend Snippet: VAMP8 does not colocalize with cytotoxic granule constituents in human CTLs. (A and B) SIM images depicting VAMP8 localization in unconjugated (A) or SEA-target cell-conjugated (B) human CTLs stained with anti-VAMP8 and anti–granzyme B (GzmB). (C and D) SIM images depicting VAMP8 localization in unconjugated (C) or SEA-target cell-conjugated (D) CTLs stained with anti-VAMP8 and anti-perforin. (C) SIM images of resting human CTL stained with anti-VAMP8 and anti-perforin. For SEA-target cell-conjugated CTLs, the Pearson’s coefficient for VAMP8 versus perforin was r = 0.28 ( n = 10). Bars, 2.5 µm.

    Article Snippet: For confocal microscopy, mouse monoclonal anti-perforin (δG9; BioLegend) and anti–granzyme B (GB11; BioLegend) as well as rabbit polyclonal anti-VAMP8 (W. Hong) antibodies were used.

    Techniques: Staining, CTL Assay

    VAMP8-carrying cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8 + T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell ( n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 18). Error bars show means and SDs.

    Journal: The Journal of Cell Biology

    Article Title: VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

    doi: 10.1083/jcb.201411093

    Figure Lengend Snippet: VAMP8-carrying cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8 + T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell ( n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell ( n = 18). Error bars show means and SDs.

    Article Snippet: For confocal microscopy, mouse monoclonal anti-perforin (δG9; BioLegend) and anti–granzyme B (GB11; BioLegend) as well as rabbit polyclonal anti-VAMP8 (W. Hong) antibodies were used.

    Techniques: Transfection, Construct, Microscopy, Fluorescence

    Perforin or Fas-L expression in CD8 + T cells in P-8-vaccinated and control mice at the site of infection. P-8-immunized and adjuvant control mice were infected with 10 5 L. amazonensis parasites in both ears. Pooled cells (groups of three mice), obtained from lesion sites at 9 weeks postinfection, were reactivated in vitro with Leishmania antigen for 5 h in the presence of brefeldin A and then processed for FACS analysis. Expression of Fas-L and intracellular perforin was determined for CD8 + lymphocytic cells by three-color flow cytometry. These results are representative of analyses obtained at 2, 5, and 9 weeks postinfection. The levels of Fas-L and perforin expression found for infection control mice were identical to that found for P. acnes control mice. —, P-8 plus P. acnes -vaccinated mice; - - - -, P. acnes control mice.

    Journal: Infection and Immunity

    Article Title: Perforin and Gamma Interferon Are Critical CD8+ T-Cell-Mediated Responses in Vaccine-Induced Immunity against Leishmania amazonensis Infection

    doi: 10.1128/IAI.71.6.3172-3182.2003

    Figure Lengend Snippet: Perforin or Fas-L expression in CD8 + T cells in P-8-vaccinated and control mice at the site of infection. P-8-immunized and adjuvant control mice were infected with 10 5 L. amazonensis parasites in both ears. Pooled cells (groups of three mice), obtained from lesion sites at 9 weeks postinfection, were reactivated in vitro with Leishmania antigen for 5 h in the presence of brefeldin A and then processed for FACS analysis. Expression of Fas-L and intracellular perforin was determined for CD8 + lymphocytic cells by three-color flow cytometry. These results are representative of analyses obtained at 2, 5, and 9 weeks postinfection. The levels of Fas-L and perforin expression found for infection control mice were identical to that found for P. acnes control mice. —, P-8 plus P. acnes -vaccinated mice; - - - -, P. acnes control mice.

    Article Snippet: Other antibodies used were anti-mouse perforin (clone P1.8; Kamiya Biomedical, Seattle, Wash.) and polyclonal goat anti-rat Ig (FITC conjugated; Kirkegaard & Perry Laboratories).

    Techniques: Expressing, Mouse Assay, Infection, In Vitro, FACS, Flow Cytometry, Cytometry

    Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with anti-perforin (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.

    Journal: Blood

    Article Title: Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity

    doi: 10.1182/blood-2011-03-344846

    Figure Lengend Snippet: Single molecules of myosin IIA associate with NK-cell lytic granules. Lytic granules isolated from resting YTS cells were evaluated by platinum rotary shadowing electron microscopy (A). Whole resting YTS cells or lytic granules isolated from resting YTS cells were evaluated by STED microscopy (B-E). (A) YTS cell lytic granule with emanating structures resembling myosin IIA, which are highlighted in color in the image at right. Scale bar indicates 200 nm. (B) YTS cell isolated lytic granule visualized with an antibody against the myosin IIA tailpiece (green, left). (C) Lytic granule within fixed YTS cell visualized with anti-perforin (red, confocal) and anti-myosin (green, STED) antibodies. The distance between myosin IIA antibody molecules was measured using a line drawn around the periphery of the lytic granules (B-C, white, right). Scale bar indicates 500 nm. (D-E) Normalized intensity plots along the line drawn at the periphery of the lytic granules shown in panels B-C. Values are normalized such that the maximum intensity in the plot is equal to 1. Numbers at the top indicate the distance between adjacent antibody molecules. Results are representative of > 10 (A), 3 (B,D), or > 30 (C,E) images. (F) Proposed model of myosin IIA interaction with NK-cell lytic granules. Unphosphorylated myosin IIA tailpiece obscures the binding site for lytic granules (highlighted in turquoise). Myosin IIA tailpiece phosphorylated at S1943 bends backward onto the rod, revealing the putative granule binding site. 1933x myosin IIA is missing a large portion of the granule-binding site, and therefore cannot sustain an interaction with granules under force. S1943A myosin IIA has an intact tailpiece, but cannot be phosphorylated and therefore cannot reveal its putative granule-binding site. Myosin molecules containing one 1933x and one WT myosin are able to bind to granules with a weaker interaction, whereas molecules containing 1 S1943A and 1 wild-type myosin are sterically hindered from binding.

    Article Snippet: Alexa Fluor 568 phalloidin (Invitrogen) and mouse anti–perforin antibody (clone δG9; BD Biosciences), followed by Pacific Blue conjugated goat anti–mouse antibody (Invitrogen), were used for visualization of F-actin and perforin, respectively.

    Techniques: Isolation, Electron Microscopy, Microscopy, Binding Assay

    Perforin and granzyme B levels in CD8 + T cells were not affected in mice lacking CD4 + T cells during primary responses to HSV-1 infection. Draining cells from CD4-depleted B6, MHC class II −/− , and WT mice were harvested

    Journal: Journal of Virology

    Article Title: CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1 ▿ T Cells following Infection with Herpes Simplex Virus Type 1 ▿ †

    doi: 10.1128/JVI.01997-08

    Figure Lengend Snippet: Perforin and granzyme B levels in CD8 + T cells were not affected in mice lacking CD4 + T cells during primary responses to HSV-1 infection. Draining cells from CD4-depleted B6, MHC class II −/− , and WT mice were harvested

    Article Snippet: Cells were then washed, fixed, permeabilized, and stained for perforin and granzyme B using PE-conjugated rat anti-mouse perforin (JAW246; eBioscience) and FITC-conjugated rat anti-mouse granzyme B (16G6; eBioscience).

    Techniques: Mouse Assay, Infection