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  • 99
    Gyros Protein Technologies symphony peptide synthesizer
    Symphony Peptide Synthesizer, supplied by Gyros Protein Technologies, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher smcc succinimidyl 4 n maleimidomethyl cyclohexane 1 carboxylate
    Smcc Succinimidyl 4 N Maleimidomethyl Cyclohexane 1 Carboxylate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Amgen cd3 melanoma ag
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    Cd3 Melanoma Ag, supplied by Amgen, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gemini Bio human ab serum
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    Human Ab Serum, supplied by Gemini Bio, used in various techniques. Bioz Stars score: 93/100, based on 899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Proimmune cmv495 503
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    Cmv495 503, supplied by Proimmune, 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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    90
    Beckman Coulter hla a 0201 melanoma antigen
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    Hla A 0201 Melanoma Antigen, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Roche 6000 iu
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    6000 Iu, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter h 2db human papillomavirus hpv 16
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    H 2db Human Papillomavirus Hpv 16, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Gemini Bio penicillin streptomycin
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
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    Thermo Fisher aimv
    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with <t>anti-CD3</t> plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p
    Aimv, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher granzyme b
    The superiority of CD40 over LOX-1 and Dectin-1 for boosting functional memory CD8 + CTLs. A–C . Purified CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of mAb-Flu.M1 58–66 conjugates or Flu.M1 58–66 peptide. CD8 + T cells were then stained with HLA-A*A0201-Flu.M1 58–66 tetramer. A . Frequencies of Flu.M1 58–66 -specific CD8 + T cells activated by Mo-DCs loaded with 0.1 μg/mL mAb-Flu.M1 58–66 conjugates. Dots represent data generated with cells from healthy donors (n = 5). B . Frequencies of Flu.M1 58–66 -specific CD8 + T cells elicited by Mo-DCs loaded with αCD40-Flu.M1 58–66 at 10, 1, 0.1 nM, or with Flu.M1 58–66 peptide at 20, 2, 0.2 nM. Each Flu.M1 58–66 conjugate molecule contains two molecules of Flu.M1 58–66 antigen. Representative flow cytometric data (left) and summarized data (mean ± SD) from five independent experiments (n = 6) are presented. C . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were further stained for <t>granzyme</t> B and perforin. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific granzyme B + or perforin + CD8 + T cells are shown. D . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were restimulated with 1 μM Flu.M1 peptide, and intracellular IFNγ expression was assessed. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific IFNγ + CD8 + T cells are shown. E . A 5 h 51 Cr release assay using T2 cells loaded with the indicated amounts of Flu.M1 58–66 peptide. CD8 + T cells activated with Mo-DCs loaded with 0.1 μg/mL αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 were used as effector cells. Error bars indicate SD of triplicate assays. Three independent experiments resulted in similar data. Significance in A , B and E was determined using an ANOVA test. *, P
    Granzyme B, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter fc500 flow cytometer
    The superiority of CD40 over LOX-1 and Dectin-1 for boosting functional memory CD8 + CTLs. A–C . Purified CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of mAb-Flu.M1 58–66 conjugates or Flu.M1 58–66 peptide. CD8 + T cells were then stained with HLA-A*A0201-Flu.M1 58–66 tetramer. A . Frequencies of Flu.M1 58–66 -specific CD8 + T cells activated by Mo-DCs loaded with 0.1 μg/mL mAb-Flu.M1 58–66 conjugates. Dots represent data generated with cells from healthy donors (n = 5). B . Frequencies of Flu.M1 58–66 -specific CD8 + T cells elicited by Mo-DCs loaded with αCD40-Flu.M1 58–66 at 10, 1, 0.1 nM, or with Flu.M1 58–66 peptide at 20, 2, 0.2 nM. Each Flu.M1 58–66 conjugate molecule contains two molecules of Flu.M1 58–66 antigen. Representative flow cytometric data (left) and summarized data (mean ± SD) from five independent experiments (n = 6) are presented. C . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were further stained for <t>granzyme</t> B and perforin. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific granzyme B + or perforin + CD8 + T cells are shown. D . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were restimulated with 1 μM Flu.M1 peptide, and intracellular IFNγ expression was assessed. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific IFNγ + CD8 + T cells are shown. E . A 5 h 51 Cr release assay using T2 cells loaded with the indicated amounts of Flu.M1 58–66 peptide. CD8 + T cells activated with Mo-DCs loaded with 0.1 μg/mL αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 were used as effector cells. Error bars indicate SD of triplicate assays. Three independent experiments resulted in similar data. Significance in A , B and E was determined using an ANOVA test. *, P
    Fc500 Flow Cytometer, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 99/100, based on 4479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter flow cytometry analysis
    The superiority of CD40 over LOX-1 and Dectin-1 for boosting functional memory CD8 + CTLs. A–C . Purified CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of mAb-Flu.M1 58–66 conjugates or Flu.M1 58–66 peptide. CD8 + T cells were then stained with HLA-A*A0201-Flu.M1 58–66 tetramer. A . Frequencies of Flu.M1 58–66 -specific CD8 + T cells activated by Mo-DCs loaded with 0.1 μg/mL mAb-Flu.M1 58–66 conjugates. Dots represent data generated with cells from healthy donors (n = 5). B . Frequencies of Flu.M1 58–66 -specific CD8 + T cells elicited by Mo-DCs loaded with αCD40-Flu.M1 58–66 at 10, 1, 0.1 nM, or with Flu.M1 58–66 peptide at 20, 2, 0.2 nM. Each Flu.M1 58–66 conjugate molecule contains two molecules of Flu.M1 58–66 antigen. Representative flow cytometric data (left) and summarized data (mean ± SD) from five independent experiments (n = 6) are presented. C . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were further stained for <t>granzyme</t> B and perforin. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific granzyme B + or perforin + CD8 + T cells are shown. D . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were restimulated with 1 μM Flu.M1 peptide, and intracellular IFNγ expression was assessed. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific IFNγ + CD8 + T cells are shown. E . A 5 h 51 Cr release assay using T2 cells loaded with the indicated amounts of Flu.M1 58–66 peptide. CD8 + T cells activated with Mo-DCs loaded with 0.1 μg/mL αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 were used as effector cells. Error bars indicate SD of triplicate assays. Three independent experiments resulted in similar data. Significance in A , B and E was determined using an ANOVA test. *, P
    Flow Cytometry Analysis, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 99/100, based on 3354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti pd1
    LILRB1 and <t>PD1</t> are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p
    Anti Pd1, supplied by BioLegend, used in various techniques. Bioz Stars score: 96/100, based on 166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher live dead fixable dead cell stain kit
    LILRB1 and <t>PD1</t> are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p
    Live Dead Fixable Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti lilrb1
    <t>LILRB1</t> and PD1 are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p
    Anti Lilrb1, supplied by BioLegend, used in various techniques. Bioz Stars score: 96/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Article Snippet: The BiTE molecule in this assay is CD3/melanoma Ag recognized by T cells 1 (MART-1) BiTE Ab construct (Amgen).

    Techniques: Expressing, FACS, Activation Assay, Quantitation Assay, Purification, Recombinant, Staining

    The superiority of CD40 over LOX-1 and Dectin-1 for boosting functional memory CD8 + CTLs. A–C . Purified CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of mAb-Flu.M1 58–66 conjugates or Flu.M1 58–66 peptide. CD8 + T cells were then stained with HLA-A*A0201-Flu.M1 58–66 tetramer. A . Frequencies of Flu.M1 58–66 -specific CD8 + T cells activated by Mo-DCs loaded with 0.1 μg/mL mAb-Flu.M1 58–66 conjugates. Dots represent data generated with cells from healthy donors (n = 5). B . Frequencies of Flu.M1 58–66 -specific CD8 + T cells elicited by Mo-DCs loaded with αCD40-Flu.M1 58–66 at 10, 1, 0.1 nM, or with Flu.M1 58–66 peptide at 20, 2, 0.2 nM. Each Flu.M1 58–66 conjugate molecule contains two molecules of Flu.M1 58–66 antigen. Representative flow cytometric data (left) and summarized data (mean ± SD) from five independent experiments (n = 6) are presented. C . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were further stained for granzyme B and perforin. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific granzyme B + or perforin + CD8 + T cells are shown. D . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were restimulated with 1 μM Flu.M1 peptide, and intracellular IFNγ expression was assessed. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific IFNγ + CD8 + T cells are shown. E . A 5 h 51 Cr release assay using T2 cells loaded with the indicated amounts of Flu.M1 58–66 peptide. CD8 + T cells activated with Mo-DCs loaded with 0.1 μg/mL αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 were used as effector cells. Error bars indicate SD of triplicate assays. Three independent experiments resulted in similar data. Significance in A , B and E was determined using an ANOVA test. *, P

    Journal: EBioMedicine

    Article Title: Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8+ and CD4+ T Cells

    doi: 10.1016/j.ebiom.2016.01.029

    Figure Lengend Snippet: The superiority of CD40 over LOX-1 and Dectin-1 for boosting functional memory CD8 + CTLs. A–C . Purified CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of mAb-Flu.M1 58–66 conjugates or Flu.M1 58–66 peptide. CD8 + T cells were then stained with HLA-A*A0201-Flu.M1 58–66 tetramer. A . Frequencies of Flu.M1 58–66 -specific CD8 + T cells activated by Mo-DCs loaded with 0.1 μg/mL mAb-Flu.M1 58–66 conjugates. Dots represent data generated with cells from healthy donors (n = 5). B . Frequencies of Flu.M1 58–66 -specific CD8 + T cells elicited by Mo-DCs loaded with αCD40-Flu.M1 58–66 at 10, 1, 0.1 nM, or with Flu.M1 58–66 peptide at 20, 2, 0.2 nM. Each Flu.M1 58–66 conjugate molecule contains two molecules of Flu.M1 58–66 antigen. Representative flow cytometric data (left) and summarized data (mean ± SD) from five independent experiments (n = 6) are presented. C . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were further stained for granzyme B and perforin. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific granzyme B + or perforin + CD8 + T cells are shown. D . CD8 + T cells activated with Mo-DCs loaded with αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 in A were restimulated with 1 μM Flu.M1 peptide, and intracellular IFNγ expression was assessed. Three independent experiments showed similar results. Representative flow cytometric data on the frequencies of Flu.M1 58–66 -specific IFNγ + CD8 + T cells are shown. E . A 5 h 51 Cr release assay using T2 cells loaded with the indicated amounts of Flu.M1 58–66 peptide. CD8 + T cells activated with Mo-DCs loaded with 0.1 μg/mL αCD40-Flu.M1 58–66 or IgG4-Flu.M1 58–66 were used as effector cells. Error bars indicate SD of triplicate assays. Three independent experiments resulted in similar data. Significance in A , B and E was determined using an ANOVA test. *, P

    Article Snippet: LIVE/DEAD fixable dead cell stain kit and mAbs to granzyme B were from Invitrogen.

    Techniques: Functional Assay, Purification, Cell Culture, Staining, Generated, Flow Cytometry, Expressing, Release Assay

    CD8 + CTLs primed with CD40-targeted DCs are functional. A . Purified naïve CD8 T cells were co-cultured with Mo-DCs loaded with the indicated amounts of αCD40-MART-1 26–35 (27L) or IgG4-MART-1 26–35 (27L) conjugates for 9 days. CD8 + T cells were then stained with HLA-A*A0201-MART-1 26–35 tetramer. Representative flow cytometric data (left) and donor-matched frequencies of MART-1 26–35 -specific CD8 + T cells induced with αCD40-MART-1 26–35 (27L) - or IgG4-MART-1 26–35 (27L) -loaded Mo-DCs are shown (right). Dots represent data generated with cells from individual healthy donors (n = 13). Significance was determined using a paired t -test. B . As in A , purified naïve CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of αCD40-MART-1 26–35 (27L) conjugate or MART-1 26–35 (27L) peptide. CD8 + T cells were stained with HLA-A*A0201-MART-1 26–35 tetramer. Representative flow cytometric data (left) and summarized data (right). Dots represent data generated with cells from individual healthy donors (n = 6). Data are presented as mean ± SD. Significance was determined using an ANOVA test. C . CD8 + T cells in A primed with Mo-DCs loaded with 1 μg/mL mAb-MART-1 26–35 (27L) were stained for granzyme B and perforin. D . A 5 h 51 Cr release assay using T2 cells loaded with 10 μM MART-1 26–35 peptide were used as target cells. CD8 + T cells primed with Mo-DCs loaded with 1 μg/mL αCD40-MART-1 26–35 (27L) or IgG4-MART-1 26–35 (27L) were used as effector cells. E . A 5 h 51 Cr release assay using MEL290 and control K562 cell lines as target cells. CD8 + T cells primed with Mo-DCs loaded with 1 μg/mL αCD40-MART-1 26–35 (27L) (left) or IgG4-MART-1 26–35 (27L) (right) were used as effector cells. Error bars in D and E indicate SD of triplicate assays. Significance was determined using an ANOVA test. Two independent experiments resulted in similar data. *, P

    Journal: EBioMedicine

    Article Title: Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8+ and CD4+ T Cells

    doi: 10.1016/j.ebiom.2016.01.029

    Figure Lengend Snippet: CD8 + CTLs primed with CD40-targeted DCs are functional. A . Purified naïve CD8 T cells were co-cultured with Mo-DCs loaded with the indicated amounts of αCD40-MART-1 26–35 (27L) or IgG4-MART-1 26–35 (27L) conjugates for 9 days. CD8 + T cells were then stained with HLA-A*A0201-MART-1 26–35 tetramer. Representative flow cytometric data (left) and donor-matched frequencies of MART-1 26–35 -specific CD8 + T cells induced with αCD40-MART-1 26–35 (27L) - or IgG4-MART-1 26–35 (27L) -loaded Mo-DCs are shown (right). Dots represent data generated with cells from individual healthy donors (n = 13). Significance was determined using a paired t -test. B . As in A , purified naïve CD8 + T cells were co-cultured with Mo-DCs loaded with the indicated amounts of αCD40-MART-1 26–35 (27L) conjugate or MART-1 26–35 (27L) peptide. CD8 + T cells were stained with HLA-A*A0201-MART-1 26–35 tetramer. Representative flow cytometric data (left) and summarized data (right). Dots represent data generated with cells from individual healthy donors (n = 6). Data are presented as mean ± SD. Significance was determined using an ANOVA test. C . CD8 + T cells in A primed with Mo-DCs loaded with 1 μg/mL mAb-MART-1 26–35 (27L) were stained for granzyme B and perforin. D . A 5 h 51 Cr release assay using T2 cells loaded with 10 μM MART-1 26–35 peptide were used as target cells. CD8 + T cells primed with Mo-DCs loaded with 1 μg/mL αCD40-MART-1 26–35 (27L) or IgG4-MART-1 26–35 (27L) were used as effector cells. E . A 5 h 51 Cr release assay using MEL290 and control K562 cell lines as target cells. CD8 + T cells primed with Mo-DCs loaded with 1 μg/mL αCD40-MART-1 26–35 (27L) (left) or IgG4-MART-1 26–35 (27L) (right) were used as effector cells. Error bars in D and E indicate SD of triplicate assays. Significance was determined using an ANOVA test. Two independent experiments resulted in similar data. *, P

    Article Snippet: LIVE/DEAD fixable dead cell stain kit and mAbs to granzyme B were from Invitrogen.

    Techniques: Functional Assay, Purification, Cell Culture, Staining, Flow Cytometry, Generated, Release Assay

    LILRB1 and PD1 are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 and PD1 are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: FACS, Expressing, Isolation

    Anti-LILRB1 synergize with anti-PD1 to promote CD8 + T cell effector function. ( A ) Dot plots to show HLA-G and PDL1 expression on established tumor cells lines. ( B ) Representative FACS plots to show gating strategy for CCR7 − CD8 + T cell sorting from healthy donor PBMCs (left panel) and LILRB1 and PD1 expression on sorted CCR7 − CD8 + T cells (right panel). ( C ) Sorted CCR7 − CD8 + T cells were incubated with indicated tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results shown as mean ± SD from triplicated wells and are a representative from three independent experiments using two healthy donors as the source of CD8 + T cells. Numbers are maximum percentage of specific lysis. ( D – F ) BiTE molecule–mediated cytotoxicity of isolated CCR7 − CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (light gray bars), anti-PD1 blocking Ab (dark gray bars), combination of anti-LILRB1 and anti-PD1 (black bars), or isotype control Ab (open bars). T cell and tumor cells coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Percentage of specific lysis (D), T cell activation represented by CD69 upregulation measured by FACS (E), and perforin production by Luminex (F) was determined and plotted. Results shown as mean ± SD of triplicated wells. Data are a representative of three independent experiments using T cells isolated from two donors. ( G ) Quantitation of anti-LILRB1 plus anti-PD1 in promoting CCR7 − CD8 + T cell cytolytic activity. Each dot represents data obtained from an individual donor. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: Anti-LILRB1 synergize with anti-PD1 to promote CD8 + T cell effector function. ( A ) Dot plots to show HLA-G and PDL1 expression on established tumor cells lines. ( B ) Representative FACS plots to show gating strategy for CCR7 − CD8 + T cell sorting from healthy donor PBMCs (left panel) and LILRB1 and PD1 expression on sorted CCR7 − CD8 + T cells (right panel). ( C ) Sorted CCR7 − CD8 + T cells were incubated with indicated tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results shown as mean ± SD from triplicated wells and are a representative from three independent experiments using two healthy donors as the source of CD8 + T cells. Numbers are maximum percentage of specific lysis. ( D – F ) BiTE molecule–mediated cytotoxicity of isolated CCR7 − CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (light gray bars), anti-PD1 blocking Ab (dark gray bars), combination of anti-LILRB1 and anti-PD1 (black bars), or isotype control Ab (open bars). T cell and tumor cells coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Percentage of specific lysis (D), T cell activation represented by CD69 upregulation measured by FACS (E), and perforin production by Luminex (F) was determined and plotted. Results shown as mean ± SD of triplicated wells. Data are a representative of three independent experiments using T cells isolated from two donors. ( G ) Quantitation of anti-LILRB1 plus anti-PD1 in promoting CCR7 − CD8 + T cell cytolytic activity. Each dot represents data obtained from an individual donor. * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Expressing, FACS, Incubation, Lysis, Isolation, Blocking Assay, Construct, Activation Assay, Luminex, Quantitation Assay, Activity Assay

    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Expressing, FACS, Activation Assay, Quantitation Assay, Purification, Recombinant, Staining

    LILRB1 and PD1 are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 and PD1 are expressed by distinct CD8 + T cell subsets in tumor. ( A ) Representative FACS plot to show LILRB1 and PD1 expression on TCR-activated human CD8 + T cells. Percentage of cells in each quadrant is indicated. ( B and C ) Single-cell RNAseq swarm plots showing the expression of indicated genes in tumor-infiltrated CD8 + T cell clusters isolated from NSCLC (B) or hepatocellular carcinoma (C). ( D ) Boxplots of LILRB1 and PD1 gene expression in tumor-infiltrated CD8 + T cell clusters from NSCLC patients. Each dot represents average gene expression from a given patient. **** p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: FACS, Expressing, Isolation

    LILRB1 marks CD8 + cells with higher effector molecule expression. ( A ) Representative dot plots show LILRB1 coexpression with perforin (left panel) and granzyme B (GZMB, right panel) in gated CD8 + T cell by ex vivo FACS analysis. ( B ) Quantitation of perforin (left panel) and GZMB (right panel) expression in LILRB1 − CCR7 − and LILRB1 + CCR7 − CD8 + subsets from multiple donors ( n = 4). ( C and D ) Human CD8 + T cells from each donor were sorted into three subsets based on LILRB1 and CCR7 expression. Quantitation of effector molecules in supernatants were performed using a Luminex assay from resting (C) and TCR-activated (D) CD8 + T cell subsets after 48 h ( n = 6). ( E ) Single-cell RNAseq results show differential gene expression between LILRB1 + and LILRB1 − memory CD8 + T cells. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 marks CD8 + cells with higher effector molecule expression. ( A ) Representative dot plots show LILRB1 coexpression with perforin (left panel) and granzyme B (GZMB, right panel) in gated CD8 + T cell by ex vivo FACS analysis. ( B ) Quantitation of perforin (left panel) and GZMB (right panel) expression in LILRB1 − CCR7 − and LILRB1 + CCR7 − CD8 + subsets from multiple donors ( n = 4). ( C and D ) Human CD8 + T cells from each donor were sorted into three subsets based on LILRB1 and CCR7 expression. Quantitation of effector molecules in supernatants were performed using a Luminex assay from resting (C) and TCR-activated (D) CD8 + T cell subsets after 48 h ( n = 6). ( E ) Single-cell RNAseq results show differential gene expression between LILRB1 + and LILRB1 − memory CD8 + T cells. * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Expressing, Ex Vivo, FACS, Quantitation Assay, Luminex

    LILRB1 inhibits cytotoxic CD8 + T cell effector function in vitro. ( A ) Representative dot plot shows LILRB1 expression on expanded Mel-13 cytolytic CD8 + T cells. ( B ) In vitro Ag-specific CTL cytotoxicity assay with expanded Mel-13 cytolytic CD8 + T cells as effector cells and SK5 cells as target cells. Indicated ratio of effector cells and target cells were coincubated for 20 h in the presence of anti-LILRB1 blocking Ab (open squares) or isotype control Ab (filled circles). Results shown as mean ± SD of triplicate wells for each indicated ratio, and data are a representative of three independent experiments. ( C ) Representative histogram for HLA-G expression on SK2 cells (dotted line) and HLA-G–transfected SK2 cells (SK2.HLA-G, solid line) analyzed by FACS. Gray-filled histogram represents isotype control. ( D ) Representative FACS plots to show gating strategy for CD8 + T EMRA sorting from healthy donor PBMCs (left panel) and LILRB1 expression on sorted T EMRA (right panel). ( E ) BiTE molecule–mediated cytotoxicity of isolated CD8 + T EMRA to indicated target cells in the presence of anti-LILRB1 blocking Ab (filled bars) or isotype control Ab (open bars). T cell and tumor cell coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Results shown as mean ± SEM of T cells isolated from three donors ( n = 3). ( F ) Representative histograms for CD69 expression on T EMRA cocultured with SK2 cells (line histograms) or SK2.HLA-G cells (gray-filled histograms) for 45 h in the presence of 0.4 nM BiTE molecule (right panel) or without BiTE molecule (left panel). ( G ) Isolated CD8 + T EMRA were cocultured with indicated tumor cells for 18 (left panel) or 45 h (right panel) in the presence of 0.4 nM BiTE molecule. Percentage of CD69 + cells (mean ± SD) were determined by FACS analysis. Data are representative of three independent experiments with two individual donors as T cell source. ( H ) Isolated tumor-associated CD8 + T cells were added to SK2 or SK2.HLA-G cells and treated with a MART-1–specific BiTE molecule for 45 h. Expression of CD69 measured as mean florescence intensity (MFI) on CD8 + T cells was determined by FACS analysis. Each type of symbol represents data obtained from an individual donor with mean ± SEM shown. ( I ) BiTE molecule–mediated cytotoxicity of isolated tumor CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (gray-filled bars) or isotype control Ab (open bars). Each type of symbol represents data obtained from an individual donor with mean ± SEM shown. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 inhibits cytotoxic CD8 + T cell effector function in vitro. ( A ) Representative dot plot shows LILRB1 expression on expanded Mel-13 cytolytic CD8 + T cells. ( B ) In vitro Ag-specific CTL cytotoxicity assay with expanded Mel-13 cytolytic CD8 + T cells as effector cells and SK5 cells as target cells. Indicated ratio of effector cells and target cells were coincubated for 20 h in the presence of anti-LILRB1 blocking Ab (open squares) or isotype control Ab (filled circles). Results shown as mean ± SD of triplicate wells for each indicated ratio, and data are a representative of three independent experiments. ( C ) Representative histogram for HLA-G expression on SK2 cells (dotted line) and HLA-G–transfected SK2 cells (SK2.HLA-G, solid line) analyzed by FACS. Gray-filled histogram represents isotype control. ( D ) Representative FACS plots to show gating strategy for CD8 + T EMRA sorting from healthy donor PBMCs (left panel) and LILRB1 expression on sorted T EMRA (right panel). ( E ) BiTE molecule–mediated cytotoxicity of isolated CD8 + T EMRA to indicated target cells in the presence of anti-LILRB1 blocking Ab (filled bars) or isotype control Ab (open bars). T cell and tumor cell coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Results shown as mean ± SEM of T cells isolated from three donors ( n = 3). ( F ) Representative histograms for CD69 expression on T EMRA cocultured with SK2 cells (line histograms) or SK2.HLA-G cells (gray-filled histograms) for 45 h in the presence of 0.4 nM BiTE molecule (right panel) or without BiTE molecule (left panel). ( G ) Isolated CD8 + T EMRA were cocultured with indicated tumor cells for 18 (left panel) or 45 h (right panel) in the presence of 0.4 nM BiTE molecule. Percentage of CD69 + cells (mean ± SD) were determined by FACS analysis. Data are representative of three independent experiments with two individual donors as T cell source. ( H ) Isolated tumor-associated CD8 + T cells were added to SK2 or SK2.HLA-G cells and treated with a MART-1–specific BiTE molecule for 45 h. Expression of CD69 measured as mean florescence intensity (MFI) on CD8 + T cells was determined by FACS analysis. Each type of symbol represents data obtained from an individual donor with mean ± SEM shown. ( I ) BiTE molecule–mediated cytotoxicity of isolated tumor CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (gray-filled bars) or isotype control Ab (open bars). Each type of symbol represents data obtained from an individual donor with mean ± SEM shown. * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: In Vitro, Expressing, CTL Assay, Cytotoxicity Assay, Blocking Assay, Transfection, FACS, Isolation, Construct, Lysis

    Anti-LILRB1 synergize with anti-PD1 to promote CD8 + T cell effector function. ( A ) Dot plots to show HLA-G and PDL1 expression on established tumor cells lines. ( B ) Representative FACS plots to show gating strategy for CCR7 − CD8 + T cell sorting from healthy donor PBMCs (left panel) and LILRB1 and PD1 expression on sorted CCR7 − CD8 + T cells (right panel). ( C ) Sorted CCR7 − CD8 + T cells were incubated with indicated tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results shown as mean ± SD from triplicated wells and are a representative from three independent experiments using two healthy donors as the source of CD8 + T cells. Numbers are maximum percentage of specific lysis. ( D – F ) BiTE molecule–mediated cytotoxicity of isolated CCR7 − CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (light gray bars), anti-PD1 blocking Ab (dark gray bars), combination of anti-LILRB1 and anti-PD1 (black bars), or isotype control Ab (open bars). T cell and tumor cells coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Percentage of specific lysis (D), T cell activation represented by CD69 upregulation measured by FACS (E), and perforin production by Luminex (F) was determined and plotted. Results shown as mean ± SD of triplicated wells. Data are a representative of three independent experiments using T cells isolated from two donors. ( G ) Quantitation of anti-LILRB1 plus anti-PD1 in promoting CCR7 − CD8 + T cell cytolytic activity. Each dot represents data obtained from an individual donor. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: Anti-LILRB1 synergize with anti-PD1 to promote CD8 + T cell effector function. ( A ) Dot plots to show HLA-G and PDL1 expression on established tumor cells lines. ( B ) Representative FACS plots to show gating strategy for CCR7 − CD8 + T cell sorting from healthy donor PBMCs (left panel) and LILRB1 and PD1 expression on sorted CCR7 − CD8 + T cells (right panel). ( C ) Sorted CCR7 − CD8 + T cells were incubated with indicated tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results shown as mean ± SD from triplicated wells and are a representative from three independent experiments using two healthy donors as the source of CD8 + T cells. Numbers are maximum percentage of specific lysis. ( D – F ) BiTE molecule–mediated cytotoxicity of isolated CCR7 − CD8 + T cells to indicated target cells in the presence of anti-LILRB1 blocking Ab (light gray bars), anti-PD1 blocking Ab (dark gray bars), combination of anti-LILRB1 and anti-PD1 (black bars), or isotype control Ab (open bars). T cell and tumor cells coculture with no BiTE Ab construct was used as baseline for specific lysis calculation. Percentage of specific lysis (D), T cell activation represented by CD69 upregulation measured by FACS (E), and perforin production by Luminex (F) was determined and plotted. Results shown as mean ± SD of triplicated wells. Data are a representative of three independent experiments using T cells isolated from two donors. ( G ) Quantitation of anti-LILRB1 plus anti-PD1 in promoting CCR7 − CD8 + T cell cytolytic activity. Each dot represents data obtained from an individual donor. * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Expressing, FACS, Incubation, Lysis, Isolation, Blocking Assay, Construct, Activation Assay, Luminex, Quantitation Assay, Activity Assay

    LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: LILRB1 expression on CD8 + T cell surface is upregulated by effector cytokines and anti-PD1 blockade. ( A ) FACS analysis of LILRB1 (left panel) and PD1 (right panel) expression on the surface of CD8 + T cells upon TCR activation (solid lines). Gray-filled histograms are control CD8 + T cells without stimulation. ( B ) Quantitation of the results from (A) across multiple donors ( n = 5). ( C ) Purified human CD8 + T EM cells were activated with anti-CD3 plus anti-CD28 in the presence of 10 μg/ml recombinant human PDL1 (rhuPDL1, solid lines) or human IgG1 (control, dashed lines). After 48 h, LILRB1 (left panel) and PD1 (right panel) expression was determined by FACS analysis. Gray-filled histograms are isotype staining controls. ( D ) Quantitation results of (C) across multiple donors ( n = 3). Representative data shown as mean ± SD. ( E ) Human PBMCs were stimulated with rIL-2, IL-15, or TNF for 48 h (solid lines) and subjected to FACS analysis. Gray-filled histograms represent CD8 + T cells gated from unstimulated PBMCs. ( F ) Quantitation of (E) across multiple donors ( n = 5). * p

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Expressing, FACS, Activation Assay, Quantitation Assay, Purification, Recombinant, Staining

    CD8 + T EMRA show potent BiTE molecule–mediated tumor cell killing and preferentially express the LILRB1 inhibitory receptor. ( A ) Human CD8 + T cells from healthy donors were sorted into T N , T EM , and T EMRA subsets and incubated with SK2 tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results (mean ± SEM) are shown from four independent experiments using three healthy donors as the source of CD8 + T cells. ( B ) Volcano plot showing differentially expressed genes between CCR7 − and CCR7 + human CD8 + T cells with p value

    Journal: The Journal of Immunology Author Choice

    Article Title: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody–Induced Tumor Cell Killing by Effector CD8+ T Cells

    doi: 10.4049/jimmunol.1801472

    Figure Lengend Snippet: CD8 + T EMRA show potent BiTE molecule–mediated tumor cell killing and preferentially express the LILRB1 inhibitory receptor. ( A ) Human CD8 + T cells from healthy donors were sorted into T N , T EM , and T EMRA subsets and incubated with SK2 tumor cells in the presence of indicated amount of MART-1–specific BiTE molecule. Specific cytotoxicity was determined after 45 h. Results (mean ± SEM) are shown from four independent experiments using three healthy donors as the source of CD8 + T cells. ( B ) Volcano plot showing differentially expressed genes between CCR7 − and CCR7 + human CD8 + T cells with p value

    Article Snippet: When indicated, 25 μg/ml anti-LILRB1 (clone GHI/75; BioLegend) or 10 μg/ml of anti-PD1 (clone EH12.2H7; BioLegend) were added in the assay.

    Techniques: Incubation