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rabbit monoclonal anti mart1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti mart1 antibody
    In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted <t>MART1,</t> CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.
    Rabbit Monoclonal Anti Mart1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti mart1 antibody/product/Cell Signaling Technology Inc
    Average 93 stars, based on 9 article reviews
    rabbit monoclonal anti mart1 antibody - by Bioz Stars, 2026-02
    93/100 stars

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    1) Product Images from "A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens"

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2024.1444886

    In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.
    Figure Legend Snippet: In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

    Techniques Used: In Vitro, Biomarker Discovery, Activation Assay, Genetically Modified, Expressing, Cell Culture, Flow Cytometry, Co-Culture Assay, Standard Deviation

    Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.
    Figure Legend Snippet: Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.

    Techniques Used: Western Blot, Expressing, Comparison, Gene Expression, Activation Assay

    Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.
    Figure Legend Snippet: Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.

    Techniques Used: Co-Culture Assay, Fluorescence

    Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).
    Figure Legend Snippet: Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).

    Techniques Used: Expressing, Knockdown, Functional Assay, Biomarker Discovery, Disruption, Activation Assay, Standard Deviation

    Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.
    Figure Legend Snippet: Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.

    Techniques Used: Knockdown, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Comparison, Negative Control, Standard Deviation



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    In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted <t>MART1,</t> CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.
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    In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

    Journal: Frontiers in Immunology

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    doi: 10.3389/fimmu.2024.1444886

    Figure Lengend Snippet: In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

    Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

    Techniques: In Vitro, Biomarker Discovery, Activation Assay, Genetically Modified, Expressing, Cell Culture, Flow Cytometry, Co-Culture Assay, Standard Deviation

    Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.

    Journal: Frontiers in Immunology

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    doi: 10.3389/fimmu.2024.1444886

    Figure Lengend Snippet: Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.

    Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

    Techniques: Western Blot, Expressing, Comparison, Gene Expression, Activation Assay

    Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.

    Journal: Frontiers in Immunology

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    doi: 10.3389/fimmu.2024.1444886

    Figure Lengend Snippet: Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.

    Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

    Techniques: Co-Culture Assay, Fluorescence

    Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).

    Journal: Frontiers in Immunology

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    doi: 10.3389/fimmu.2024.1444886

    Figure Lengend Snippet: Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).

    Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

    Techniques: Expressing, Knockdown, Functional Assay, Biomarker Discovery, Disruption, Activation Assay, Standard Deviation

    Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.

    Journal: Frontiers in Immunology

    Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

    doi: 10.3389/fimmu.2024.1444886

    Figure Lengend Snippet: Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.

    Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

    Techniques: Knockdown, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Comparison, Negative Control, Standard Deviation